KR101737706B1 - a tumor suppressor containing miR-5582-5p - Google Patents
a tumor suppressor containing miR-5582-5p Download PDFInfo
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- KR101737706B1 KR101737706B1 KR1020150179372A KR20150179372A KR101737706B1 KR 101737706 B1 KR101737706 B1 KR 101737706B1 KR 1020150179372 A KR1020150179372 A KR 1020150179372A KR 20150179372 A KR20150179372 A KR 20150179372A KR 101737706 B1 KR101737706 B1 KR 101737706B1
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Abstract
본 발명은 miR-5582-5p를 포함하는 항암제에 관한 것이다.
본 발명에서는 암 억제 기능을 갖는 새로운 miRNA인 miR-5582-5p를 증명하였다. miR-5582-5p는 암세포에서 세포사멸을 유도하고 세포주기를 정지시키지만, 정상세포에서는 이러한 변화를 보이지 않는다. miR-5582-5p의 타깃 단백질로서 GAB1, SHC1과 CDK2를 확인하였다.The present invention relates to an anticancer agent comprising miR-5582-5p.
In the present invention, miR-5582-5p, a novel miRNA having a cancer-suppressing function, has been demonstrated. miR-5582-5p induces apoptosis and stops the cell cycle in cancer cells, but does not show this change in normal cells. GAB1, SHC1 and CDK2 were identified as target proteins of miR-5582-5p.
Description
본 발명은 miR-5582-5p를 이용한 항암제에 관한 것이다.The present invention relates to an anticancer agent using miR-5582-5p.
본 발명에서는 암 억제 기능을 갖는 새로운 miRNA인 miR-5582-5p를 증명하였다. miR-5582-5p는 암세포에서 세포사멸을 유도하고 세포주기를 정지시키지만, 정상세포에서는 이러한 변화를 보이지 않는다. miR-5582-5p의 타깃 단백질로서 GAB1, SHC1과 CDK2를 확인하였다.In the present invention, miR-5582-5p, a novel miRNA having a cancer-suppressing function, has been demonstrated. miR-5582-5p induces apoptosis and stops the cell cycle in cancer cells, but does not show this change in normal cells. GAB1, SHC1 and CDK2 were identified as target proteins of miR-5582-5p.
miR-5582-5p의 기능과 유사하게, GAB1/SHC1 또는 CDK2를 각각 억제하면 세포사멸이 일어나거나 세포주기의 정지가 일어나는 것을 알 수 있었다. HCT116 세포를 이용한 마우스 이종이식모델에서 miR-5582-5p의 종양 내 주사 또는 Tet-miR-5582-5p에 의해 암세포의 성장이 저해되는 것을 확인하였다. 이러한 결과들은, 암 억제 기능을 갖는 새로운 miRNA인 miR-5582-5p의 암 치료제로서의 적용 가능성을 보여준다.Similar to the function of miR-5582-5p, inhibition of GAB1 / SHC1 or CDK2, respectively, leads to apoptosis or cell cycle arrest. It was confirmed that the growth of cancer cells was inhibited by intra-tumor injection of miR-5582-5p or Tet-miR-5582-5p in a mouse xenograft model using HCT116 cells. These results show the applicability of miR-5582-5p, a novel miRNA with cancer-inhibiting function, as a cancer treatment agent.
MicroRNAs (miRNAs)는 ~22 뉴클레오티드의 작은 RNA(Endogenous small non-coding RNAs)로 유전자 발현의 중요한 조절 인자로 많은 관심을 받아왔다. miRNA는 mRNA의 3′UTR sequence와 상보적으로 결합하여, 전사 후 단계에서 타깃 유전자 mRNA의 분해(degradation) 또는 번역 억제(translational repression)를 한다.MicroRNAs (miRNAs) have been attracting much attention as important regulators of gene expression with small RNAs of ~ 22 nucleotides (Endogenous small non-coding RNAs). miRNAs complementarily bind to the 3 'UTR sequence of mRNA, resulting in degradation or translational repression of target gene mRNA at post-transcriptional stages.
하나의 miRNA는 동시에 많은 타깃 유전자를 조절할 수 있기 때문에 전반적인 유전자 발현에 영향을 줄 수 있다. 많은 연구들에서 miRNA는 세포의 성장, 발달, 분열 및 사멸 등 생물학적 과정 대부분에 걸쳐 중요한 역할을 하고, 특히 암을 비롯한 여러 종류의 질병이 이와 연관되어 있다. 암에서는 많은 miRNA가 비정상적 발현을 보인다. 여러 miRNA가 다양한 종류의 암에서 중요한 유전자를 조절하거나 종양 형성 과정의 신호전달 경로에 관여하여 암 유발 또는 암 억제 유전자로서 기능을 한다.Because one miRNA can control many target genes at the same time, it can affect overall gene expression. In many studies, miRNA plays an important role throughout most of the biological processes, including cell growth, development, division and death, and is associated with many types of diseases, especially cancer. In cancer, many miRNAs exhibit abnormal expression. Several miRNAs function as cancer-inducing or cancer-suppressing genes by regulating genes important in a variety of cancers or by participating in the signaling pathway of tumorigenesis.
가장 잘 알려진 암 억제 miRNA로는 암 억제 유전자 TP53의 타깃인 miR-34 패밀리가 있다. miR-34는 MYC, BCL2, CDK4와 MET과 같은 다양한 암 유발 유전자를 타깃 하기 때문에 암 억제 과정에 있어 주요한 조절인자로 여겨지고, 그렇게 함으로써 다양한 암에서 항암의 역할을 한다. 또한, miR-29와 miR-193a-3p와 같은 몇몇의 miRNA는 p85α와 Mcl-1을 각기 저해함으로써 세포사멸을 유도하여 암 억제 기능을 한다고 알려져 있다. 반면에 일부 miRNA들은 다양한 암에서 높은 발현을 보이고, 이들은 암 억제 유전자를 타깃 함으로써 발암 효과를 가한다. 예를 들면, 대표적인 발암과정에 관여하는 miRNA인 miR-21은 phosphatase and tensin homolog (PTEN) 과 programmed cell death 4 (PDCD4)를 타깃 하고, miR-17-92 cluster는 BIM 과 PTEN을 타깃 한다.The most well-known cancer-suppressing miRNAs are the miR-34 family, the target of the tumor suppressor gene TP53. Because miR-34 targets a variety of cancer-causing genes such as MYC, BCL2, CDK4 and MET, it is considered to be a major regulator of cancer suppression and thus acts as an anti-cancer in a variety of cancers. In addition, several miRNAs such as miR-29 and miR-193a-3p are known to inhibit p85α and Mcl-1, respectively, thereby inducing apoptosis and inhibiting cancer. Some miRNAs, on the other hand, are highly expressed in a variety of cancers, and they have carcinogenic effects by targeting cancer suppressor genes. For example, miR-21, a miRNA involved in a typical carcinogenic process, targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), while miR-17-92 cluster targets BIM and PTEN.
암을 형성하거나 억제하는 데 관여하는 miRNA들의 이러한 특징은 암 치료를 위한 유용한 도구나 방법이 될 수 있다. 따라서, 현재 miRNA 모방체(mimics) 또는 저해제가 miRNA-based 치료제로서 암 치료에 임상적으로 이용 가능한지에 대한 연구가 진행되고 있다.This characteristic of miRNAs involved in forming or inhibiting cancer can be a useful tool or method for cancer treatment. Therefore, studies are currently under way to determine if miRNA mimics or inhibitors are clinically available for cancer treatment as miRNA-based therapies.
본 발명에서는 암세포에서 세포사멸 및 세포주기 정지를 유도하는 새로운 miRNA인 miR-5582-5p의 기능을 확인함으로써, miRNA 치료제 개발을 위한 암세포의 성장을 조절하는 새로운 miRNA를 밝혔다. 우리가 알고 있는 한 이것은 miR-5582-5p에 관한 첫 번째 연구이다. 따라서 본 발명은 강력한 암 억제 유전자인 miR-5582-5p의 새로운 항암 miRNA 치료제로서 높은 가능성을 제시한다.In the present invention, a novel miRNA that regulates the growth of cancer cells for development of miRNA therapeutic drug was identified by confirming the function of miR-5582-5p, a new miRNA that induces apoptosis and cell cycle arrest in cancer cells. As far as we know, this is the first study of miR-5582-5p. Therefore, the present invention presents a high possibility as a novel anti-cancer miRNA therapeutic agent of miR-5582-5p which is a strong cancer suppressor gene.
전술한 내용에서 이미 시사되었듯이, 본 발명의 목적은 새로운 항암 miRNA 치료제를 제공하는 데 있다.As already mentioned in the foregoing, it is an object of the present invention to provide a novel anticancer miRNA therapeutic.
본 발명은 miR-5582-5p를 포함하는 항암제인 것을 특징으로 한다.The present invention is characterized by being an anticancer agent comprising miR-5582-5p.
본 발명에 따른 miR-5582-5p는 암 억제 기능을 갖고 있어 이를 강력한 암 치료제로 활용할 수 있다.The miR-5582-5p according to the present invention has a cancer suppressing function and can be used as a powerful cancer treatment agent.
도면 1은 functional screening을 통한 성장 억제에 효과적인 miR-5582-5p의 선별실험에 관한 도면이고,
도면 2는 miR-5582-5p의 세포사멸 유도 및 세포주기 정지 실험에 관한 도면이며,
도면 3은 miR-5582-5p의 GAB1, SHC1와 CDK2의 직접적인 발현 억제 실험에 관한 도면이고,
도면 4는 GAB1/SHC1과 CDK2 억제를 통한 miR-5582-5p의 세포사멸과 세포주기 정지 효과 실험에 관한 도면이며,
도면 5는 암세포 보다 정상 세포에서 높은 miR-5582-5p의 발현과, 정상 세포에서는 세포사멸을 일으키지 않는 miR-5582-5p에 관한 실험 도면이고,
도면 6은 마우스에서 miR-5582-5p의 종양 성장 억제에 관한 실험 도면이며,
도면 7은 GAB1, SHC1, CDK2를 동시에 타겟 함으로써 암세포의 성장이나 세포주기의 진행을 억제하는 miR-5582-5p 암 억제 기능에 대한 간략한 모식도이다.
각 도면별로 좀 더 구체적인 설명을 기재한다.
<도면 1>
A. 합성된 267개의 miRNA pool에서의 1차 screening. HCT116 세포에서 각 miRNA를 과발현 시킨 뒤, 3일 후 MTS assay를 수행하여 생장률을 측정하였다. *P < 0.005 (n=3).
B. 2차 screening. 1차 screening을 통해 암세포의 성장을 유의하게 조절하는 29 개 miRNA를 선별하여 재 검증하였다.
C. 1차 와 2차 screening 을 통해 성장 억제 효과를 갖는 miRNA의 선정. HCT116 세포에서 각 miRNA를 과발현 시킨 뒤, 3일 후 MTS assay를 수행하여 생장률을 측정하였다. 오차 범위는 ± SEM. *P < 0.005 (n=3).
<도면 2>
A. miR-5582-5p의 성장 억제효과 검증. 대조군 또는 miR-5582-5p를 6-well에서 HCT11과SW480 각 세포에 과발현 시킨 후 24시간마다 세포의 수를 측정하였다. *P < 0.005 (n=3).
B. miR-5582-5p의 콜로니 형성능 억제 *P < 0.05 (n=3).
C. miR-5582-5p의 세포주기 정지. HCT116 세포에서 대조군 또는 miR-5582-5p를 과발현 하여 PI 염색을 한 후 유세포 분석기로 확인하였다.
D. miR-5582-5p의 세포사멸 유도. 대조군 또는 miR-5582-5p를 HCT11과SW480 각 세포에 과발현 시킨 후 3일 뒤 Annexin V-FITC/PI 염색하여 유세포 분석기로 확인하였다.
E. miR-5582-5p에 의한 세포 내 활성산소 변화 측정. 대조군 또는 miR-5582-5p를 HCT116세포에 과발현 한 후 DCF-DA 염색을 하여 유세포 분석기로 확인하였다.
F. miR-5582-5p의 TP53 비의존적 세포사멸 유도. 대조군 또는 miR-5582-5p 을 과발현 시킨 TP53-wild-type (TP53 +/+)과 TP53-null (TP53 -/-)HCT116세포를 Annexin V-FITC/PI 염색하여 측정하였다.
<도면 3>
A. miR-5582-5p 의 GAB1, SHC1 과 CDK2의 발현 억제. 각 대장암 세포주에 miR-5582-5p를 과발현 한 후 48시간 뒤에 western blot를 수행하였다.
B. miR-5582-5p에 의한 GAB1, SHC1과 CDK2 의 발현 감소. HCT116 세포주에 대조군 또는 miR-5582-5p를 과발현 한 후 qRT-PCR을 사용하여 각 GAB1, SHC1과 CDK2의 mRNA 발현을 측정하였다. β-Actin 은 normalizer로 사용되었다. *P < 0.05, **P < 0.01 (n=3).
C. miR-5582-5p와 상보적으로 결합하는 GAB1, SHC1과 CDK2의 3'UTR의 sequence를 넣은 reporter 제작. 각 GAB1, SHC1과 CDK2의 결합부위 뉴클레오타이드를 변화시킨 부분은 회색으로 표시하였다. (3C 도면의 위에서부터 아래로 나열된 WT 3' UTR-1, miR-5582-5p, Mut 3' UTR-1, WT 3' UTR-2, Mut 3' UTR-2, WT 3' UTR, Mut 3' UTR, WT 3' UTR, Mut 3' UTR의 각 서열은 서열목록 1 내지 9로 첨부함.)
D. miR-5582-5p와 GAB1, SHC1와 CDK2의 3'UTR의 직접적인 결합 확인. HCT116 세포주에 MIR-5582-5p와 각 reporter를 함께 과발현 한 후 48시간 뒤에 luciferase 활성을 측정하였다. Firefly luciferase 활성은 Renilla luciferase 활성으로 normalize 하였다. *P < 0.05 (n=3).
<도면 4>
A. 세포 성장 신호 전달 경로에서 miR-5582-5p에 의한 AKT와 ERK 하위 경로 활성 억제. HCT-116 세포주에 대조군 또는 miR-5582-5p를 과발현 하여 serum 없는 상태로 24시간을 유지한 후 EGF (100 ng/mL)를 넣어준 뒤 강 시간에 세포를 얻어 western을 수행하였다.
B. HCT116 세포주에서 miR-5582-5p 또는 GAB1, SHC1 의 siRN를 과발현 한 후 48시간 뒤에 단백질 발현을 확인하였다.
C. GAB1과 SHC1의 동시 억제를 통한 세포사멸 유도. GAB1, SHC1을 각각 또는 siRNA를 과발현 하여 함께 억제한 후 3일 뒤에 유세포 분석기를 사용하여 분석하였다.
D. miR-5582-5p의 CDK2 억제효과에 있어 세포주기 관련 단백질 발현. HCT116 세포주에 miR-5582-5p, CDK2의 siRNA를 과발현 한 후 48시간 뒤 단백질 발현을 확인하였다.
E. siCDK2와 miR-5582-5p 과발현에 따른 세포주기 정지.
F. Tet-5582 세포주에서 doxycycline에 의한 miR-5582-5p의 발현 검증. 전체 RNA는 Tet-C와 Tet-5582세포에 doxycycline (500 ng/mL)을 처리 후 각 시간에 얻어 qRT-PCR로 발현량을 측정하였다.
G. miR-5582-5p 유도에 의한 세포사멸 관련 단백질 발현 변화. Doxycycline 처리 후 각 시간에 세포를 얻어 western blot을 수행하여 단백질 발현 변화를 보았다.
H. 유도된 miR-5582-5p에 의한 세포사멸. 세포사멸 분석은 doxycycline을 72시간 처리한 후에 수행하였다.
I. miR-5582-5p 유도에 의한 세포주기 관련 단백질 발현 변화.
J. 유도된 miR-5582-5p의 세포주기 정지 효과.
<도면 5>
A. 정상세포와 암세포에서의 miR-5582-5p 발현 비교. mature miR-5582-5p 와 primary miR-5582 (pri-5582)은 qRT-PCR을 수행하였다.
B. 정상 세포주 CCD-18co와 HPF의 성장을 억제하는 miR-5582-5p. 세포 수 측정은 대조군(α-cont) 또는 miR-5582-5p 억제제(α-5582-5p)를 과발현 한 후 정해진 시간에 각각 수행하였다. *P < 0.05 (n=3).
C. 정상 세포주에서 α-5582-5p 과발현에 의한 타겟 단백질 발현의 증가. 대조군(α-cont) 또는 miR-5582-5p 억제제 (α-5582-5p)를 과발현 한 후 48시간 뒤에 세포를 얻어 단백질 발현을 확인하였다.
D. 정상 세포주 CCD-18co 와 HPF에서는 세포 성장 억제 효과를 보이지 않는 miR-5582-5p. 대조군 또는 miR-5582-5p를 과발현 한 후 정해진 시간마다 세포 수를 측정하였다.*P < 0.05 (n=3).
E. 정상 세포주에서 miR-5582-5p에 의한 타겟 단백질 발현 확인. CCD-18co 와 HPF에 대조군 또는 miR-5582-5p를 과발현 한 후 48시간 뒤에 세포를 얻어 변화를 확인하였다.
F. 정상 세포주 CCD-18co와 HPF에서는 세포사멸을 야기하지 않는 miR-5582-5p. 대조군 또는 miR-5582-5를 과발현 한 후 3일 뒤 유세포 분석기를 사용하여 확인하였다.
<도면 6>
A-F. Doxycyclin에 의한 miR-5582-5p의 유도 발현은 마우스 이종이식 모델에서 종양의 성장을 억제한다.
A. 시간에 따른 종양 성장의 관찰. 마우스의 뒷다리에 Tet-C 혹은 Tet-5582 세포주를 각각 피하 접종하고 doxycycline (2 mg/ml)를 함유한 물을 먹인 군과 먹이지 않은 군 간에 시간에 따른 종양의 크기와 무게를 측정, 비교하였다. *P < 0.05 (n=6).
B. 마우스로부터 분리한 종양 사진.
C. 각 군 간의 종양 무게의 비교. 마우스에게서 떼어낸 종양의 무게를 측정하여 그래프로 나타내었다.
D. 종양 조직에서 수행한 면역화학염색 분석의 대표적인 그림.
E. Tet-5582 세포주를 접종한 마우스에서 분리한 종양의 miR-5582-5p 발현검증. 종양조직들에서 분리한 전체 RNA를 사용하여 qRT-PCR 로 분석하였다. *P < 0.005 (n=3).
F. Doxycycline으로 miR-5582-5p의 발현을 유도를 통한 종양 내 타겟 단백질의 발현 감소.
G-L. 마우스 이종이식 모델에서 miR-5582-5p의 종양 내 주사는 종양 성장을 억제한다.
G. 시간에 따른 종양의 성장. miR-5582-5p 또는 대조군 miRNA를 in vivo-jetPEI를 이용하여 종양 내 투여 후, 시간에 따른 종양의 크기를 측정, 비교하였다. *P < 0.01 (n=6).
H. miR-5582-5p 혹은 그것의 대조군을 투여한 종양의 대표적인 사진.
I. 종양의 무게 분석. miR-5582-5p (n=6) 혹은 대조군 (n=6)을 접종한 마우스에서 분리한 종양의 무게를 측정하여 그래프로 나타내었다. *P < 0.05.
J. 면역조직염색분석을 통한 종양 조직의 사진.
K. miR-5582-5p 혹은 대조군을 투여한 종양에서 miR-5582-5p의 발현량의 검증. *p < 0.005 (n=3).
L. miR-5582-5p를 투여한 종양의 타겟단백질의 발현 감소.1 is a diagram showing a screening experiment of miR-5582-5p effective for inhibiting growth through functional screening,
2 is a diagram showing the cell death induction and cell cycle termination experiments of miR-5582-5p,
FIG. 3 is a diagram showing an experiment for inhibiting the direct expression of GAB1, SHC1 and CDK2 of miR-5582-5p,
FIG. 4 is a graph showing the cell death and cell cycle arrest effect of miR-5582-5p through GAB1 / SHCl and CDK2 inhibition,
5 is an experimental view showing miR-5582-5p expression which is higher in normal cells than cancer cells and miR-5582-5p which does not cause apoptosis in normal cells,
Figure 6 is an experimental view of inhibition of tumor growth of miR-5582-5p in mice,
FIG. 7 is a simplified schematic diagram of the miR-5582-5p cancer suppressing function that inhibits the growth of cancer cells and the progression of the cell cycle by simultaneously targeting GAB1, SHC1, and CDK2.
A more specific description of each drawing should be provided.
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A. Primary screening in 267 miRNA pools synthesized. Each miRNA was overexpressed in HCT116 cells and the growth rate was measured 3 days later by MTS assay. * P < 0.005 (n = 3).
B. Secondary screening. Twenty-nine miRNAs, which regulate the growth of cancer cells significantly, were screened and re-verified.
C. Selection of miRNAs with growth inhibitory effect through primary and secondary screening. Each miRNA was overexpressed in HCT116 cells and the growth rate was measured 3 days later by MTS assay. The error range is ± SEM. * P < 0.005 (n = 3).
2,
A. Verification of growth inhibitory effect of miR-5582-5p. The control or miR-5582-5p cells were overexpressed in HCT11 and SW480 cells in 6-wells and the number of cells was measured every 24 hours. * P < 0.005 (n = 3).
B. Inhibition of colony forming ability of miR-5582-5p * P < 0.05 (n = 3).
C. Cell cycle arrest of miR-5582-5p. In HCT116 cells, control or miR-5582-5p was overexpressed and PI staining was performed and confirmed by flow cytometry.
D. induction of apoptosis of miR-5582-5p. The control or miR-5582-5p was overexpressed in HCT11 and SW480 cells, and stained with Annexin V-FITC / PI three days later and confirmed by flow cytometry.
E. Measurement of intracellular reactive oxygen species by miR-5582-5p. The control or miR-5582-5p was overexpressed in HCT116 cells and stained with DCF-DA and confirmed by flow cytometry.
F. Induction of TP53-independent cell death by miR-5582-5p. TP53-wild-type ( TP53 + / + ) and TP53-null ( TP53 - / - ) HCT116 cells overexpressing control or miR-5582-5p were stained with Annexin V-FITC / PI staining.
3,
A. Inhibition of GAB1, SHC1 and CDK2 Expression in miR-5582-5p. Each colon cancer cell line was overexpressed with miR-5582-5p, followed by
B. Reduced expression of GAB1, SHC1 and CDK2 by miR-5582-5p. Expression of mRNA of GAB1, SHC1 and CDK2 was measured using qRT-PCR after HCT116 cell line was overexpressed in control or miR-5582-5p. β-Actin was used as a normalizer. * P < 0.05, ** P < 0.01 (n = 3).
C. Reporter production of 3'UTR sequences of GAB1, SHC1 and CDK2 complementary to miR-5582-5p. The portion of each GAB1, SHC1 and CDK2 binding site nucleotide was changed to gray. (3T UTR-1, miR-5582-5p, Mut 3'UTR-1, WT 3'UTR-2, Mut 3'UTR-2, WT 3'UTR, Mut 3 'UTR, WT 3' UTR, and Mut 3 'UTR are attached to
D. Direct confirmation of miR-5582-5p and 3'UTR of GAB1, SHC1 and CDK2. The HCT116 cell line was overexpressed with MIR-5582-5p and each reporter, and luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized to Renilla luciferase activity. * P < 0.05 (n = 3).
4,
A. Inhibition of AKT and ERK subpathway activation by miR-5582-5p in the cell growth signaling pathway. EGF (100 ng / mL) was added to the HCT-116 cell line for 24 hours in the absence of serum or a control or overexpression of miR-5582-5p.
B. Protein expression was confirmed 48 hours after overexpression of siRN of miR-5582-5p or GAB1, SHC1 in HCT116 cell line.
C. Induction of apoptosis by simultaneous inhibition of GAB1 and SHC1. GAB1, and SHC1, respectively, or siRNA were overexpressed and inhibited, and analyzed 3 days later using a flow cytometer.
D. Expression of cell cycle-related proteins in CDK2 inhibitory effect of miR-5582-5p. Expression of miR-5582-5p, CDK2 siRNA was overexpressed in HCT116 cell line for 48 hours.
E. Cell cycle arrest following siCDK2 and miR-5582-5p overexpression.
F. Expression of miR-5582-5p by doxycycline in Tet-5582 cell line. Total RNA was obtained from Tet-C and Tet-5582 cells with doxycycline (500 ng / mL) at each time point and the expression level was measured by qRT-PCR.
G. Changes in apoptosis-related protein expression by miR-5582-5p induction. Cells were harvested at each time point after treatment with doxycycline, and western blot was performed to observe changes in protein expression.
H. Cell death by miR-5582-5p induced. Cell death was performed after doxycycline treatment for 72 hours.
I. Changes in cell cycle-related protein expression by miR-5582-5p induction.
J. Cell cycle arrest effect of induced miR-5582-5p.
5,
A. Comparison of miR-5582-5p expression in normal and cancer cells. mature miR-5582-5p and primary miR-5582 (pri-5582) were subjected to qRT-PCR.
B. miR-5582-5p inhibiting growth of normal cell line CCD-18co and HPF. Cell counts were performed at defined times after overexpression of the control (α-cont) or miR-5582-5p inhibitor (α-5582-5p), respectively. * P < 0.05 (n = 3).
C. Increased expression of target protein by over-expression of α-5582-5p in normal cell lines. 48 hours after overexpression of the control (α-cont) or miR-5582-5p inhibitor (α-5582-5p), cells were obtained and protein expression was confirmed.
D. miR-5582-5p which does not inhibit cell growth in normal cell line CCD-18co and HPF. Cell numbers were measured at defined time intervals after overexpression of control or miR-5582-5p * P < 0.05 (n = 3).
E. Identification of target protein expression by miR-5582-5p in normal cell line. Cells were obtained 48 hours after overexpression of control or miR-5582-5p in CCD-18co and HPF to confirm the change.
F. miR-5582-5p, which does not cause apoptosis in normal cell line CCD-18co and HPF. Three days after overexpression of control or miR-5582-5 was confirmed using a flow cytometer.
6,
AF. Induction of miR-5582-5p by Doxycyclin inhibits tumor growth in a mouse xenograft model.
A. Observation of tumor growth over time. Tet-C or Tet-5582 cell lines were subcutaneously inoculated into the hind legs of the mice, and the size and weight of the tumors were measured and compared between the feeding and non-feeding groups containing doxycycline (2 mg / ml). * P < 0.05 (n = 6).
B. Tumor photograph taken from mouse.
C. Comparison of tumor weight between groups. The weight of the tumor removed from the mouse was measured and plotted.
D. Representative picture of immunochemical staining analysis performed on tumor tissue.
E. Verification of miR-5582-5p expression of tumors isolated from mice inoculated with Tet-5582 cell line. Total RNA isolated from tumor tissues was analyzed by qRT-PCR. * P < 0.005 (n = 3).
F. Decreased expression of target protein in tumor by inducing expression of miR-5582-5p with Doxycycline.
GL. Intratumoral injection of miR-5582-5p in a mouse xenograft model inhibits tumor growth.
G. Tumor growth over time. After miR-5582-5p or control miRNAs were administered intratumorally using in vivo-jet PEI, the tumor size was measured and compared over time. * P < 0.01 (n = 6).
H. Typical photograph of tumor treated with miR-5582-5p or its control.
I. Analysis of tumor weights. The tumor weighed from mice inoculated with miR-5582-5p (n = 6) or control (n = 6) was weighed and displayed as a graph. * P < 0.05.
J. Photograph of tumor tissue by immunohistochemical staining analysis.
Verification of the expression level of miR-5582-5p in K. miR-5582-5p or a control-treated tumor. * p < 0.005 (n = 3).
L. Reduced expression of target protein in tumors administered miR-5582-5p.
이하 본 발명을 실시예와 함께 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to examples.
<실험 재료 및 방법><Materials and Methods>
세포 배양 조건과 시약Cell culture conditions and reagents
인간 대장암 세포주인 HCT116, SW480, DLD-1, HCT-15와 비소폐암세포주인 A549과 H460, 대장 섬유 아세포인 CCD-18Co, 폐의 섬유 아세포인 HPF는 모두 ATCC (American Type Culture Collection) 에서 구입하고 FBS 10%, penicillin (100 U/mL)을 포함하는 RPMI 배지에 37℃, 5% CO2 환경에서 배양하였다. HCT116 (TP53+/+), HCT116 (TP53-/-) 세포주는 Dr. B Vogelstein (Johns Hopkins university, MD) 로부터 분양 받아 사용했다. 이 연구에 쓰인 항체인 GAB1, SHC1, CDL2, SOS1, BAX, BAK1, BCL2, E2F1, cyclin E, PCNA, 와 B-ACTIN 은 Santa Cruz Biotechnology사에서 구입하였고, RB1, phospho-RB1, AKT, phospho-AKT, ERK, phospho-ERK, XIAP, BCL2L1, GRB2는 Cell signaling Technology사에서 구입하여 사용하였다. The human colon cancer cell lines HCT116, SW480, DLD-1 and HCT-15 and the arsenic lung cancer cell lines A549 and H460, the large intestine fibroblast CCD-18Co and the lung fibroblast HPF were purchased from the American Type Culture Collection And cultured in RPMI medium containing 10% FBS and penicillin (100 U / mL) at 37 ° C in a 5% CO 2 environment. The HCT116 (TP53 + / +) and HCT116 (TP53 - / -) cell lines were obtained from Dr. B Vogelstein (Johns Hopkins University, MD). RB1, phospho-RB1, AKT, phospho-RB1, and B-ACTIN were purchased from Santa Cruz Biotechnology, Inc. GAB1, SHC1, CDL2, SOS1, BAX, BAK1, BCL2, E2F1, cyclin E, AKT, ERK, phospho-ERK, XIAP, BCL2L1 and GRB2 were purchased from Cell signaling Technology.
RNA 올리고뉴클레오타이드와 과발현 RNA oligonucleotides and over-expression
합성 miRNA mimic pool은 Genolution Pharmaceuticals (Seoul, Korea)에서 RNA duplex로 합성하였고, 이는 miRBase database에 등록된 sequence를 참고 하였다. version 16의 일부와 새로 등록된 miRNA인 version 17에 등록된 267개의 miRNA들을 사용하였다. miR-5582-5p에 대한 inhibitor 역시 Genolution Pharmaceuticals에서 2'-o-methyl 기를 붙인 단일 리보뉴클레오타이드로 합성하여 사용하였다. sequence는 5′-GCTATAACTTTAAGTGTGCCTA-3′이다(서열목록 10). GAB1, SHC1, CDK2를 타깃하는 siRNA 올리고 뉴클레오타이드, 그리고 음성 대조군으로 사용한 negative control siRNA 올리고 뉴클레오타이드는 Santa Cruz Biotechnoloty사에서 구입하였다. functional analysis를 수행하기 위해서, 세포에 모든 miRNA mimic 혹은 miR-5582-5p의 inhibitor, siRNA들을 각각 fianl 10 nM의 농도로 G-fectin (Genolution Pharmaceuticals)를 이용하여 과발현 하였다.The synthetic miRNA mimic pool was synthesized by RNA duplex in Genolution Pharmaceuticals (Seoul, Korea), which refers to the sequence registered in the miRBase database. version 16 and 267 miRNAs registered in version 17, a newly registered miRNA. The inhibitor for miR-5582-5p was also synthesized by Genolution Pharmaceuticals as a single ribonucleotide with a 2'-o-methyl group attached. sequence is 5'-GCTATAACTTTAAGTGTGCCTA-3 '(SEQ ID NO: 10). SiRNA oligonucleotides targeting GAB1, SHC1, and CDK2, and negative control siRNA oligonucleotides used as negative controls were purchased from Santa Cruz Biotechnoloty. To perform functional analysis, all miRNA mimic or miR-5582-5p inhibitors and siRNAs were overexpressed using G-fectin (Genolution Pharmaceuticals) at a concentration of 10 nM each of fianl.
세포성장의 결정Determination of cell growth
2.5 x 103개의 세포들을 96-well plate에, 깔고 miRNA들을 과발현 하였다. 96시간 후 MTS assay 를 수행하여 490 nm에서의 흡광도로 살아있는 세포의 수를 측정하였다. 세포 counting을 통한 세포성장률은 trypan blue 방법을 이용하였다. 이 모든 실험결과는 3번의 단독실험을 통해 결정 되었다.2.5 x 10 3 cells were overlaid on 96-well plates and overexpressed miRNAs. After 96 hours, MTS assay was performed to measure the number of living cells by absorbance at 490 nm. Cell growth rate through cell counting was determined by trypan blue method. All these experimental results were determined through three independent experiments.
soft agar 콜로니 형성능의 결정Determination of soft agar colony forming ability
대조군과 miR-5582-5p를 과발현 한 세포들을 TE를 이용하여 떼어낸 후, 1000개 세포를 최종 0.35%의 low-melting agarose 가 되도록 배지와 섞은 후 최종 0,5%의 low-melting agarose 가 깔린 60 mm dish에 overlay 하여 2주 후 콜로니 형성능을 관찰 하였다. 한 주에 한번씩 신선한 배지를 agarose 위에 첨가해주었다. 2주 후에 0.005%의 crystal violet으로 염색하였다. 염색된 콜로니 수를 세어 콜로니 형성능을 확인 하였다. Control cells and miR-5582-5p over-expressing cells were removed using TE, 1000 cells were mixed with the medium to be a final 0.35% low-melting agarose, and the final 0.5% low-melting agarose The colonies were observed after 2 weeks of overlaying on a 60 mm dish. Fresh medium was added to the agarose once a week. After 2 weeks, the cells were stained with 0.005% crystal violet. The number of stained colonies was counted to confirm colony forming ability.
세포 사멸 측정Cell death measurement
세포사멸은 annexin V-FITC/ Propidium iodide (PI) 를 함께 염색함으로써 유세포 분석기를 통해 측정하였다. 이종이식 종양 조직에서 사멸된 세포의 분석은 DeadEnd Fluorometric TUNEL system (Promega) 키트를 이용하여 terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) 방법을 통해 수행하였다.Apoptosis was measured by flow cytometry using annexin V-FITC / Propidium iodide (PI) staining. Analysis of dead cells in xenograft tumor tissues was performed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method using the DeadEnd Fluorometric TUNEL system (Promega) kit.
세포 주기 측정Cell cycle measurement
차가운 70% 에탄올에 1시간 동안 고정시킨 세포를 RNase A (10 ㎍/mL)을 37℃에서 30분간 처리하고 PBS를 이용하여 세척 하였다. 그 후 PI를 최종 농도 50 ㎍/mL로 처리하고 실온에서 1시간 동안 염색 하였다. 세포주기의 분포는 유세포 분석기를 이용하여 측정 하였다.Cells fixed in cold 70% ethanol for 1 hour were treated with RNase A (10 μg / mL) at 37 ° C for 30 minutes and washed with PBS. PI was then treated with a final concentration of 50 ug / mL and stained for 1 hour at room temperature. The distribution of the cell cycle was measured using a flow cytometer.
세포 내 활성산소 수준 측정Measurement of active oxygen levels in cells
세포 내 활성산소 수준은 DCF-DA (2′-7′-dichlorofluorescein diacetate, Molecular Probes)를 염색하여 측정 하였다. 대조군 혹은 miR-5582-5p를 과발현 하고 48 시간 후에, growth media 상태에 있는 세포에 DCF-DA를 최종 5 μM 의 농도로 처리하고 37℃에 1시간 배양하여 세포 내 활성산소를 염색하였다. 유세포분석기를 이용하여 세포의 형광 강도를 측정함으로써 세포 내 활성산소 수준을 측정 하였다.The level of active oxygen in the cells was determined by staining DCF-DA (2'-7'-dichlorofluorescein diacetate, Molecular Probes). After 48 hours of overexpression of the control or miR-5582-5p cells, DCF-DA was treated with DCF-DA at a final concentration of 5 μM and incubated at 37 ° C for 1 hour to stain intracellular reactive oxygen species. The intracellular levels of active oxygen were measured by measuring the fluorescence intensity of the cells using a flow cytometer.
Reporter assay (타깃 유전자의 검증)Reporter assay (verification of target gene)
타깃 유전자의 검증을 위해 reporter vector를 cloning 하였다. miR-5582-5p의 binding site를 포함하는 각각의 타깃 유전자의 3'UTR 부분을 pGL3UC 벡터에 cloning 하였다 (도. 3C). 각 타깃 유전자의 3'UTR을 증폭 하기 위한 Primer sequence는 표 1에 첨부하였다. mutant reporter vector를 제작하기 위해 miR-5582-5p의 결합 부위 중 3개의 뉴클레오타이드를 QuickChange II site-directed mutagenesis kit (Agilent Technologies, CA)을 이용하여 다른 뉴클레오타이드로 변형 시켰다. 이렇게 제작된 vector를 pRL-pRL-CMV-Renilla (Promega) plasmid (2 ng)와 각 miRNA와 함께 HCT116 세포에 Lipofectamine 2000 (Invitrogen, CA)를 사용하여 과발현 하고, 48 시간 후에 reporter 활성을 측정 하였다. Dual Luciferase Reporter Assay system (Promega) kit을 이용하여 Firefly luciferase 활성을 측정하였고, renilla luciferase activity로 normalize 하였다. 이는 triplicate로 실험 하였다.A reporter vector was cloned to verify the target gene. The 3'UTR portion of each target gene containing the binding site of miR-5582-5p was cloned into pGL3UC vector (Fig. 3C). The primer sequence for amplifying the 3'UTR of each target gene is shown in Table 1. Three nucleotides of the binding site of miR-5582-5p were transformed into other nucleotides using the QuickChange II site-directed mutagenesis kit (Agilent Technologies, CA) to generate a mutant reporter vector. These vectors were overexpressed in HCT116 cells using Lipofectamine 2000 (Invitrogen, CA) with pRL-pRL-CMV-Renilla (Promega) plasmid (2 ng) and each miRNA and reporter activity was measured after 48 hours. Firefly luciferase activity was measured using the Dual Luciferase Reporter Assay system (Promega) kit and normalized to renilla luciferase activity. This was experimented with triplicate.
(상기 표 1의 서열은 서열목록 11 내지 34로 첨부함.)(The sequence of Table 1 above is attached as SEQ ID NOS: 11-34).
RNA 분리와 qRT-PCR 분석RNA isolation and qRT-PCR analysis
세포나 조직에서의 RNA 분리는 배양 세포는 Trizol로, 얼렸던 종양조직은 RNeasy Mini kit (Qiagen, Germany)를 사용 하였다. 2 ㎍의 전체 RNA를 KAPA SYBR FAST one-Step qRT-PCR kit을 이용하여 다양한 유전자의 mRNA 발현량과 primary miRNA 전사체의 발현량을 확인하였다. mature miRNA의 발현양은 Mir-X miRNA qRT-PCR SYBR kit을 이용하여 확인 하였다. 이에 사용된 각 유전자에 특이전인 primer sequence 또한 표 1에 첨부하였고, 모든 유전자의 발현양은 β-actin과 U6로 normalize 하였다.RNA was isolated from the cells and tissues using Trizol as a culture medium and RNeasy Mini kit (Qiagen, Germany) as a frozen tumor. 2 μg of total RNA was quantitated using the KAPA SYBR FAST one-step qRT-PCR kit. The mRNA expression levels of various genes and the expression levels of primary miRNA transcripts were determined. The expression of mature miRNA was confirmed using Mir-X miRNA qRT-PCR SYBR kit. The primer sequence specific to each gene used was also attached to Table 1, and the expression level of all genes was normalized to β-actin and U6.
Western blotting (단백질발현 분석 )Western blotting (protein expression analysis)
준비된 과발현 세포를 SDS sample buffer에 끓이고 SDS-PAGE 실험을 수행하여 단백질을 분리하였다. 분리된 단백질을 nitrocellulose membranes (Whatman, PA)으로 옮겼다. 그 후, 옮겨진 membrane을 5% Skim milk solution (5% skim milk, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20)에 40 분간 처리한 후, 각 단백질에 특이적인 항체를 실온에서 2시간 혹은 4℃ 에서 overnight 하였다. 다음 membrane을 TBST에 세번 세척 한 후 HRP가 달린 2차 항체를 실온에서 1시간 처리하였다. 그 후에 chemiluminescence system (Amersham Pharmacia Biotech, NJ)을 이용하여 단백질 발현량을 측정하였다.Prepared overexpressed cells were boiled in SDS sample buffer and SDS-PAGE was performed to isolate the proteins. The separated proteins were transferred to nitrocellulose membranes (Whatman, PA). Then, the transferred membrane was treated with 5% skim milk solution (5% skim milk, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 40 minutes, For 2 hours or overnight at 4 ° C. The next membrane was washed three times with TBST and the secondary antibody with HRP was treated at room temperature for 1 hour. After that, the amount of protein expression was measured using a chemiluminescence system (Amersham Pharmacia Biotech, NJ).
miR-5582-5p 유도발현를 위한 세포주 제작Production of cell line for miR-5582-5p induction expression
miR-5582-5p의 조건유도를 위해 Mir-X inducible miRNA System (Clontech) kit를 사용하여 세포주를 제작하였다. 간단히 말하면, genomic DNA상에 miR-5582-5p의 precursor 부분(446bp)을 특이 프라이머를 이용하여 증폭 시켜 pmiR-mCherry vector에 cloning 하여, pTet-5582 vector를 제작하였다. Doxycyclin 혹은 Tetracycline 에 의해 특정 유전자가 발현되는 시스템을 가진 HCT-116 세포주는 pTet-on advanced plasmid 를 HCT-116 세포에 과발현하고 G418 (800 ㎍/mL) 농도로 selection 하여 제작 하였다. 이렇게 제작된 Tet-on advanced HCT116 세포에 pTet-5582 벡터 혹은 음성 대조군인 empty pmiR-mCherry vector와 hygromycin linear marker를 함께 과발현 시키고, 2주간의 hygromycin (200 ㎍/mL) selection을 통해 각각의 double stable 세포주를 제작 하였다. selection 뒤 각각의 클론은 Doxycycine 처리 하여 miR-5582-5p의 발현량을 확인하였다.Cell lines were prepared using the Mir-X inducible miRNA System (Clontech) kit for the induction of miR-5582-5p conditions. Briefly, the precursor part (446 bp) of miR-5582-5p was amplified on genomic DNA using a specific primer and cloned into pmiR-mCherry vector to construct pTet-5582 vector. An HCT-116 cell line with a specific gene expression by Doxycyclin or Tetracycline was over-expressed in HCT-116 cells and selected by G418 (800 ㎍ / mL) concentration. The pTet-5582 vector or the negative control pMR-mCherry vector and hygromycin linear marker were overexpressed in the prepared Tet-on advanced HCT116 cells, and the hygromycin (200 ㎍ / mL) selection for 2 weeks was used for each double stable cell line Respectively. After selection, each clone was treated with Doxycycine to confirm the expression level of miR-5582-5p.
면역조직화학염색법Immunohistochemical staining
면역조직화학염색은 Ready-to-use IHC/ICC kit (Biovision, CA)를 사용 하였다. 간단히 말하면, Xylen을 사용해 조직의 파라핀을 제거한 후, 알코올을 이용하여 재수화 한 뒤 실온에서 30 분간 GAB1 (1:100), CDK2 (1:100), 또는 PCNA (1:100)의 특이적 1차 항체를 처리하였다. PBS를 사용하여 세척 한 후, 이들을 One-Step HRP polymer 에 20분간 실온 처리한 뒤 TBST를 이용한 세척 이후 3,3′-diaminobenzidine (DAB)를 이용하여 염색을 관찰 하였다.Immunohistochemical staining was performed using Ready-to-use IHC / ICC kit (Biovision, CA). Briefly, tissue paraffin was removed using xylen, rehydrated with alcohol, and incubated for 30 min at room temperature with a specific 1 (100: 1) of GAB1 (1: 100), CDK2 The secondary antibody was treated. After washing with PBS, they were treated with One-Step HRP polymer for 20 minutes at room temperature. After washing with TBST, 3,3'-diaminobenzidine (DAB) was used for staining.
종양 이종이식 모델 실험Tumor xenograft model experiment
Orientbio에서 4주 된 BABL/c 누드 마우스를 구입했으며, 이 마우스들은 SPF 환경에서 키웠다. 5주가 된 마우스에 2x106개의 대장암 세포주 HCT116 세포를 마우스의 오른쪽 뒷다리에 피하 이식하였다 (체중 18-20g). 종양의 크기가 150 mm3이 되었을 때, in vivo-jetPEI (Polyplus Transfection, Illkirch, France)를 이용해 10 ㎍의 miR-5582-5p를 3일 간격으로 2번 종양 내 투여를 수행하였다 (10 ㎍/1.2 μL in vivo-jetPEI/50 μL D.W). Doxycycline에 의해 miR-5582-5p가 조건유도 되는 세포주의 경우, Tet-5582 세포주를 역시 마우스의 오른쪽 뒷다리에 피하이식 한 후 다음날부터 Doxycycline (SigmaAldrich, MO)을 2 mg/mL 이 되도록 10%의 수크로오즈가 함유된 물에 탄 후 마우스들에게 먹였다. 빛을 차단하여 Doxycycline 함유물의 활성 저해를 막았고, 이틀에 한 번씩 갈아주었다. 종양의 크기는 일주일에 두 번 volume (V) mm3 = (small diameter)2 x (largediameter) x (π/6). 공식에 따라 도출하였다. 모든 동물 실험은 Korea Institute of Radiological and Medical Sciences (Seoul, Korea)의 IACUC (the institutional animal care and use committee)의 승인 하에 진행하였다.In Orientbio, a 4-week-old BABL / c nude mouse was purchased and grown in an SPF environment. At 5 weeks of age, 2x10 < 6 > colon cancer cell line HCT116 cells were subcutaneously transplanted (weight 18-20 g) into the right hind leg of the mice. When the size of the tumor reached 150 mm 3 , 10 μg of miR-5582-5p was administered twice daily at 3-day intervals using in vivo-jetPEI (Polyplus Transfection, Illkirch, France) 1.2 μL in vivo-jet PEI / 50 μL DW). In the case of miR-5582-5p-induced cell lines, the Tet-5582 cell line was also subcutaneously transplanted into the right hindlimb of the mouse and the next day, 10% of the cells were treated with Doxycycline (Sigma Aldrich, MO) After feeding on water containing crossover, mice were fed. Light was blocked to prevent the inhibition of the activity of the Doxycycline-containing material, which was changed once a day. The size of the tumor is twice weekly volume (V) mm 3 = (small diameter) 2 x (largediameter) x (π / 6). According to the formula. All animal studies were conducted with the approval of the institutional animal care and use committee (IACUC) of the Korea Institute of Radiological and Medical Sciences (Seoul, Korea).
<결과><Result>
functional screening을 통한 성장 억제에 효과적인 miR-5582-5p의 선별Selection of miR-5582-5p effective for growth inhibition through functional screening
암세포의 성장을 조절하는 새로운 miRNA를 찾기 위해서 miRBase 데이터베이스의 16 version 일부와 17 version에 등록된 267개의 miRNA pool을 합성하였다. 이는 연구를 시작할 당시 가장 최근 등록된 miRNA였다. miRNA pool의 각 miRNA를 대장암 세포주 HCT116에 과발현 하고, MTS assay를 수행하여 세포의 생장률을 측정하였다. 1차 screening을 통해 세포의 성장을 촉진하거나 억제하는 miRNA 10개를 선정하였고 이를 2차 screening을 하였다. (도 1 A and B). 1차와 2차 screening을 통해 성장을 저해하는 효과를 보이는 5개의 miRNA 최종 선별하였다. 선별된 5개의 miRNA 중, 가장 현저하게 HCT116 세포의 성장을 저해하는 miR-5582-5p를 선정하여 이 연구를 진행했으며, 이 외에 세포의 성장을 조절하는 것으로 선별된 다른 miRNA는 후속 연구에서 진행하기로 한다 (도 1C).To find new miRNAs that regulate the growth of cancer cells, we synthesized 16 versions of the miRBase database and 267 miRNA pools registered in 17 versions. This was the most recently registered miRNA at the time of the start of the study. Each miRNA in the miRNA pool was overexpressed in the colon cancer cell line HCT116 and the cell growth rate was measured by performing the MTS assay. Ten primary miRNAs were selected and screened for secondary cell growth by primary screening. (FIGS. 1A and B). Primary and secondary screening were used to select five miRNAs that had the effect of inhibiting growth. Of the five selected miRNAs, miR-5582-5p, which most significantly inhibited the growth of HCT116 cells, was selected and other miRNAs selected to modulate cell growth proceeded in subsequent studies (Fig. 1C).
암세포의 miR-5582-5p에 의한 세포사멸 유도 및 세포주기 정지Cell death induction and cell cycle arrest by miR-5582-5p of cancer cells
miR-5582-5p의 성장 억제 효과는 과발현 이후의 일정 간격 시간마다의 세포 수 측정을 통해 다시 한번 검증하였다. miR-5582-5p는 2개의 대장암 세포주 HCT116과 SW480의 성장을 현저히 억제하였다 (도 2A). miR-5582-5p에 의해 콜로니 형성 능력 또한 감소되었다 (도 2B). 세포 주기 분석을 수행하여 miR-5582-5p가 HCT116 세포주에서 G1기의 population을 증가시키고 S 기의 population을 감소시킴으로써 G1 arrest를 야기하는 것을 확인하였다 (도 2C). 다음으로 세포에 Annexin V/PI 염색을 한 후, 유세포 분석기을 사용하여 miR-5582-5p에 의한 세포 사멸 효과를 확인하였다. HCT116과 SW480 세포에서 miR-5582-5p에 세포사멸이 유의하게 증가하였다 (도 2D). 그러나 이때의 세포 내 활성산소 발현량은 차이가 없었다 (도 2E). 이는 miR-5582-5p가 유도하는 세포사멸이 세포 내 활성 산소와는 관련 없다는 것을 보여준다. TP53은 다양한 자극에 의한 세포사멸에 있어 주요한 역할을 하기 때문에, miR-5582-5p에 의한 세포사멸 또한 TP53 status에 따른 것인지 확인해 보았다. TP53-wild type (TP53+/+) 또는 TP53-null (TP53-/-) 의 유전적 형질을 갖는 HCT116 세포주에 miR-5582-5p를 과발현 시키고, 이들의 세포 사멸 비율을 측정한 결과 그 정도가 유사하였다 (도 2F). 이는 miR-5582-5p에 의한 세포사멸이 TP53 비의존적인 경로로 야기됨을 시사한다.The growth inhibitory effect of miR-5582-5p was once again verified by measuring the number of cells at regular intervals after overexpression. miR-5582-5p markedly inhibited the growth of two colon cancer cell lines HCT116 and SW480 (Fig. 2A). Colony forming ability was also decreased by miR-5582-5p (Fig. 2B). Cell cycle analysis was performed to confirm that miR-5582-5p caused a G1 arrest by increasing the population of the G1 group and decreasing the population of the S group in the HCT116 cell line (Fig. 2C). Next, the cells were stained with Annexin V / PI, and the apoptotic effect of miR-5582-5p was confirmed using a flow cytometer. Cell death was significantly increased in miR-5582-5p in HCT116 and SW480 cells (Fig. 2D). However, there was no difference in the amount of active oxygen in the cells at this time (Fig. 2E). This indicates that miR-5582-5p-induced apoptosis is not associated with intracellular reactive oxygen species. Since TP53 plays a major role in cell death by various stimuli, we have also examined whether miR-5582-5p-induced apoptosis is due to TP53 status. When miR-5582-5p was overexpressed in the HCT116 cell line with the genetic traits of TP53-wild type (TP53 + / +) or TP53-null (TP53 - / -) and their cell death rates were measured, (Fig. 2F). This suggests that miR-5582-5p-induced apoptosis is caused by the TP53 independent pathway.
miR-5582-5p의 GAB1, SHC1와 CDK2의 음성적 조절Negative regulation of GAB1, SHC1 and CDK2 in miR-5582-5p
miR-5582-5p의 타깃 유전자를 찾기 위해 miRNA 타깃 예측 프로그램인 TargetScan (http://www.targetscan.org/), miRDB (http://mirdb.org/miRDB/) 와 DIANA-microT 3.0 (http://diana.cslab.ece.ntua.gr/)를 사용하였다. 이를 통해 Receptor tyrosine kinases (RTKs)의 Pro-mitogenic/-survival 경로 조절에 관련된 Grb2-associated binding protein 1 (GAB1), Src homology 2 domain containing 1 (SHC1), growth factor receptor-bound protein 2 (GRB2), guanine nucleotide exchange factor SOS1, 그리고 세포주기 진행에 있어 중요한 역할을 하는 cyclin-dependent kinase 2 (CDK2)와 같은 몇 개의 유전자를 타깃으로 추정하였다. 따라서 먼저 우리는 몇 개의 대장암 세포주에서 miR-5582-5p에 의해 타깃 단백질의 발현이 감소하는지 확인하였다.TargetScan (http://www.targetscan.org/), miRDB (http://mirdb.org/miRDB/) and DIANA-microT 3.0 (http : //diana.cslab.ece.ntua.gr/). (GAB1),
그 결과, 모든 세포주에서 miR-5582-5p에 의해 GAB1, SHC1 그리고 CDK2는 유의하게 변화 하였지만, SOS1은 변화하지 않았다 (도 3A). 반면 GRB2의 발현은 세포주 마다 다른 경향을 나타냈다. GRB2는 HCT116 과 HCT15 세포주 에서는 감소되었으나 SW480 과 DLD-1 세포주 에서는 증가하였다. 이는 miR-5582-5p에 의한 GRB2의 조절은 직접적인 타깃팅을 통한 저해 보다는 세포 내 환경에 따라 간접적인 메커니즘을 통해 조절된다고 여겨지고, 이로 하여금 GRB2를 직접적인 타깃 단백질 후보에서 제외하였다. 단백질 발현과 마찬가지로, HCT116 세포에서 miR-5582-5p를 과발현 하였을 때 GAB1, SHC1과 CDK2의 mRNA 발현량 또한 감소하였다 (도 3B).As a result, GAB1, SHC1 and CDK2 were significantly changed by miR-5582-5p in all cell lines, but SOS1 did not change (Fig. 3A). On the other hand, the expression of GRB2 showed a tendency different for each cell line. GRB2 was decreased in HCT116 and HCT15 cell lines but increased in SW480 and DLD-1 cell lines. This suggests that regulation of GRB2 by miR-5582-5p is thought to be mediated through an indirect mechanism depending on the intracellular environment rather than by direct targeting, thereby eliminating GRB2 from direct target protein candidates. Similar to protein expression, mRNA expression of GAB1, SHC1 and CDK2 was also decreased when miR-5582-5p was overexpressed in HCT116 cells (Fig. 3B).
따라서 우리는 miR-5582-5p의 직접적인 타깃 유전자가 GAB1, SHC1 그리고 CDK2인지 확인하기 위해서 reporter assay를 수행하였다. miR-5582-5p의 seed region와 상보적으로 결합할 것으로 예측되는 각 mRNA의 3'UTR 을 찾았다. 그 부위들이 miR-5582-5p에 의한 직접적인 타깃 되는지 알아보기 위해, 변형된 (modified) pGL3 reporter vector의 firefly luciferase open reading frame의 downstream에 miR-5582-5p의 seed region에 상보적인 sequence를 포함한 각각의 타깃 단백질 3'UTR 부분을 cloning 하여 reporter vector를 만들었다 (도 3C).Therefore, we performed a reporter assay to determine whether the direct target gene of miR-5582-5p is GAB1, SHC1 and CDK2. We found the 3'UTR of each mRNA that would be complementary to the miR-5582-5p seed region. To determine if the sites were directly targeted by miR-5582-5p, we used the sequence of the complementary sequence in the miR-5582-5p seed region downstream of the firefly luciferase open reading frame of the modified pGL3 reporter vector A 3 'UTR portion of the target protein was cloned into a reporter vector (FIG. 3C).
각 타깃 단백질에 대한 miR-5582-5p의 타깃-특이성을 알아보기 위해 seed sequence에 결합하는 부위에서 3개의 nucleotides가 mutation 된 mutant reporter를 만들었다. 이렇게 제작된 reporter들과 miR-5582-5p를 함께 HCT116 세포주에 과발현 시킨 후 luciferase 활성을 측정한 결과 예측대로 모든 wild type (3'UTR) reporter들은 miR-5582-5p에 의해 luciferase 활성이 감소하였다. (도 3D). 반대로 모든 mutant 타입 3'UTR reporter들은 miR-5582-5p에 의해 luciferase 활성 변화가 없었다. 이는 miR-5582-5p에 의해 직접적으로 GAB1, SHC1, CDK2의 발현이 억제 되는 것을 의미한다. 도 3C는 miR-5582-5p가 각 타깃 단백질의 발현을 조절 하기 위해 결합하는 특이적 sequence 와 region을 모식도로 나타냈다.To investigate the target-specificity of miR-5582-5p for each target protein, a mutant reporter was constructed in which three nucleotides were mutated at the binding site of the seed sequence. The luciferase activity of miR-5582-5p and miR-5582-5p were overexpressed in the HCT116 cell line. As predicted, all wild-type (3'UTR) reporters were reduced in luciferase activity by miR-5582-5p. (Fig. 3D). In contrast, all mutant-type 3'UTR reporters did not show any change in luciferase activity by miR-5582-5p. This means that the expression of GAB1, SHC1, and CDK2 is directly inhibited by miR-5582-5p. FIG. 3C schematically shows a specific sequence and region in which miR-5582-5p binds to control the expression of each target protein.
GAB1/SHC1과 CDK2 억제를 통한 miR-5582-5p의 세포사멸과 세포주기 정지 효과Cell death and cell cycle arrest effect of miR-5582-5p through GAB1 / SHC1 and CDK2 inhibition
Ras-MAP kinase 활성에 GAB1 과 SHC1이 주요한 역할을 하고 활성화된 RTKs 의 downstream 이 PI3K-Akt/PKB 경로이기 때문에, 우리는 성장인자 EGF 처리 후 ERK와 AKT의 활성에 대한 miR-5582-5p의 효과를 알아보았다.Since GAB1 and SHC1 play a major role in Ras-MAP kinase activity and the downstream of activated RTKs is the PI3K-Akt / PKB pathway, we observed the effect of miR-5582-5p on ERK and AKT activity after growth factor EGF treatment .
HCT116 세포에 Serum starvation 직후 EGF를 처리하였을 때, miR-5582-5p의 과발현 조건에서 ERK와 AKT의 인산화가 감소되었다 (도 4A). 이는 miR-5582-5p에 의해 survival 경로가 저해되는 것을 보여 준다. 암세포의 생장을 억제하는 miR-5582-5p의 역할이 타깃 유전자의 저해를 통한 경로인지 확인하기 위해서, 타깃 유전자를 인위적으로 억제하여 miR-5582-5p의 효과와 유사한지 알아보았다. 먼저 siRNA를 사용하여 GAB1 와 SHC1를 함께 억제하자 miR-5582-5p에 의한 결과와 같이 세포사멸이 강력하게 유도되었다. 반면에 GAB1, SHC1을 각각 억제 하였을 때에는 세포사멸이 약하게 일어났다 (도 4B). miR-5582-5p의 과발현은 ERK와 AKT의 인산화 감소와 이것들의 하위경로인 anti-apoptotic 단백질 XIAP, BCL2, 와 BCL2L1 발현이 감소시켰다. (도 4C). 그러나 ERK 또는 AKT 에 의해 억제되어 있는, pro-apoptotic 단백질 BAX와 BAK1은 증가하였다. miR-5582-5p의 효과와 같이 GAB1과 SHC1를 저해 하였을 때 ERK와 AKT의 인산화가 감소되었고, 이에 따라 anti-apoptotic 단백질 발현은 감소하고, pro-apoptotic 단백질 발현은 증가하는 것을 확인하였다. 이러한 결과들로 GAB1과 SHC1의 동시 억제에 의한 ERK와 AKT경로 저해는 세포사멸을 야기하기에 충분하고, miR-5582-5p에 의한 세포사멸의 대부분은 GAB1 와 SHC1의 억제를 통해 일어난다는 것을 알 수 있다. miR-5582-5p의 과발현 조건과 같이, siRNA를 이용하여 CDK2를 억제한 결과 G1 population이 증가하는 세포주기 분포의 변화를 가져 왔다 (도 4D). miR-5582-5p 또는 siCDK2의 과발현에 의해 phosphorylated retinoblastoma 1 (p-RB1)와 PCNA 는 감소하였고, E2F1 와 cyclin E 발현은 변화가 없었다 (도 4E). 이를 통해 CDK2가 miR-5582-5p의 직접적인 타깃으로써 세포주기 정지를 조절하는 것을 알 수 있다.When HCT116 cells were treated with EGF immediately after serum starvation, phosphorylation of ERK and AKT was decreased in the over-expression condition of miR-5582-5p (FIG. 4A). This shows that the survival pathway is inhibited by miR-5582-5p. To determine whether the role of miR-5582-5p, which inhibits the growth of cancer cells, is through the inhibition of the target gene, we artificially suppressed the target gene and examined whether it is similar to the effect of miR-5582-5p. First, inhibition of GAB1 and SHC1 using siRNA strongly induced apoptosis as shown by miR-5582-5p. On the other hand, when GAB1 and SHC1 were inhibited, apoptosis was weak (Fig. 4B). Overexpression of miR-5582-5p reduced the phosphorylation of ERK and AKT and their anti-apoptotic proteins, XIAP, BCL2, and BCL2L1. (Fig. 4C). However, the pro-apoptotic proteins BAX and BAK1, which are inhibited by ERK or AKT, have increased. Similar to the effect of miR-5582-5p, the inhibition of GAB1 and SHC1 resulted in decreased phosphorylation of ERK and AKT, thereby decreasing anti-apoptotic protein expression and pro-apoptotic protein expression. These results suggest that inhibition of ERK and AKT pathway by simultaneous inhibition of GAB1 and SHC1 is sufficient to induce apoptosis and that most of the apoptosis by miR-5582-5p occurs via inhibition of GAB1 and SHC1 . Inhibition of CDK2 using siRNA, as in miR-5582-5p overexpression conditions, resulted in a change in the cell cycle distribution in which the G1 population increased (Fig. 4D). Overexpression of miR-5582-5p or siCDK2 reduced phosphorylated retinoblastoma 1 (p-RB1) and PCNA, and E2F1 and cyclin E expression did not change (Fig. 4E). It can be seen that CDK2 regulates cell cycle arrest as a direct target of miR-5582-5p.
miR-5582-5p의 기능을 더욱 자세히 연구하기 위해, Tetracycline에 의해 miR-5582의 발현이 유도 되는 Tet-5582 세포주를 만들었다. Tet-5582 세포주에 Doxycycline을 처리 한 뒤에 72시간 후 miR-5582-5p의 발현량 변화를 측정한 결과 대조군에 비해 8배 높아지는 것을 확인 하였다. (도 4 F). Doxycycline에 의해 Tet-5582 세포주에서 세포사멸이 유도 됨을 확인함으로써 miR-5582-5p의 세포사멸 유도 능을 다시 한번 검증하였다 (도 4 G). Doxycycline에 의해 유도된 miR-5582-5p 때문에 일어난 세포 사멸은 이전에 세포 수준에서 확인한 바와 같이 GAB1, SHC1 그리고 GRB2 발현 억제를 유도 하는 동시에 XIAP, BCL2L1의 감소, BAX 발현을 증가시킨다 (도 4H). Tet-5582 세포에 Doxycycline를 처리함으로써 유도된 miR-5582-5p의 발현은 또한 CDK2, RB1의 인산화, PCNA 발현 감소를 가져와 G1기를 증가 시키는 것으로 확인한 바 (도 4 I,J), miR-5582-5p에 의한 세포 주기 억제는 CDK2를 타깃함으로써 일어나는 것임을 알 수 있었다.To further study the function of miR-5582-5p, Tet-5582 cell line was constructed in which miR-5582 expression was induced by Tetracycline. The expression level of miR-5582-5p after 72 hours of Doxycycline treatment in Tet-5582 cell line was 8 times higher than that of the control group. (Fig. 4F). The ability of miR-5582-5p to induce apoptosis was again verified by confirming that Doxycycline induces apoptosis in the Tet-5582 cell line (Fig. 4G). The apoptosis induced by Doxycycline-induced miR-5582-5p induces the inhibition of GAB1, SHC1 and GRB2 expression as well as the decrease of XIAP, BCL2L1 and BAX expression as previously confirmed at the cellular level (Fig. 4H). The expression of miR-5582-5p induced by Doxycycline treatment on Tet-5582 cells was also confirmed to increase the G1 phase by inducing phosphorylation of CDK2, RB1 and reduction of PCNA expression (Fig. 4 I, J), miR-5582- 5p-induced cell cycle arrest was caused by targeting of CDK2.
miR-5582-5p은 암세포보다 정상세포에서 발현이 많으며, 정상세포에서는 세포사멸을 유도하지 않음miR-5582-5p is more expressed in normal cells than cancer cells and does not induce apoptosis in normal cells
세포사멸을 야기하고 세포주기 정지를 통해 암세포의 성장을 저해하는 miR-5582-5p의 암 억제 유전자로의 가능성을 더욱 알아보고자 하였다. 첫째로, 기원이 같은 조직의 암세포와 정상세포에서 miR-5582-5p의 발현을 비교했다. 대장 유래 정상세포 CCD-18-co에서 대장암 세포주인 HCT116과 SW480보다 유의하게 높은 miR-5582-5p의 발현을 보였다. human pulmonary fibroblasts (HPFs) 또한 폐암세포주인 A549와 H460보다 높은 발현을 나타냈다 (도 5A, top). miR-5582-5p의 초기전사체인 pri-miR-5582 또한 정상세포에서 더욱 발현이 높은 양상을 나타냈다. (도 5A, bottom). 이를 통해 암세포에서 miR-5582-5p의 발현은 전사 단계 에서 부터 억제 되어 있다는 것을 유추할 수 있다.To investigate the possibility of miR-5582-5p as a cancer-suppressing gene, which causes apoptosis and inhibits the growth of cancer cells through cell cycle arrest. First, we compared miR-5582-5p expression in cancer cells and normal cells of the same origin. The expression of miR-5582-5p was significantly higher in the colon-derived normal cell CCD-18-co than in the colon cancer cell lines HCT116 and SW480. Human pulmonary fibroblasts (HPFs) also showed higher expression than lung cancer cell lines A549 and H460 (Fig. 5A, top). pri-miR-5582, an early transcription factor for miR-5582-5p, also showed a higher expression pattern in normal cells. (Fig. 5A, bottom). This suggests that the expression of miR-5582-5p in cancer cells is suppressed from the transcriptional stage.
이어서 miR-5582-5p의 저해제가 miR-5582-5p의 발현이 높은 정상 세포주의 성장에 어떠한 영향을 미치는지 알아 보았다. miR-5582-5p 저해제를 과발현 하였을 때 CCD-18Co 와 HPF 두 세포 모두에서 세포의 성장이 유의하게 증가하였고 (도 5B), (이는 정상세포에서는 높은 miR-5582-5p의 발현 때문에 대조군 비해 균형잡힌 성장을 보이는데, miR-5582-5p의 발현을 억제 하였을 때에는 대조군에 비해 확실하게 세포의 생장이 높아지는 것으로 보인다.) miR-5582-5p 저해제의 과발현에 의해 두 세포주의 GAB1과 SHC1 발현이 모두 증가하였지만, 반면 CDK2는 뚜렷한 증가는 없었다 (도 5C).We next examined the effect of inhibitors of miR-5582-5p on the growth of miR-5582-5p-expressing normal cell lines. When the miR-5582-5p inhibitor was overexpressed, the cell growth was significantly increased in both CCD-18Co and HPF cells (Fig. 5B), indicating that miR-5582-5p expression in normal cells was balanced The inhibitory effect of miR-5582-5p inhibitor on the expression of GAB1 and SHC1 in both cell lines was increased by the overexpression of miR-5582-5p inhibitor , Whereas there was no significant increase in CDK2 (Figure 5C).
이어서 miR-5582-5p가 정상세포의 성장에도 효과가 있는지 확인하였다. miR-5582-5p를 과발현에 의해 CCD-18Co와 HPF 세포의 성장이 억제되지는 않았다 (도 5D). 이미 적은 양인 타깃 단백질의 발현은 miR-5582-5p에 의해 추가 감소 되었다 (도 5E). 또한 정상세포 CCD-18Co와 HPF에서는 miR-5582-5p의 과발현에 의한 세포사멸이 일어나지 않았다 (도 5F). 이 결과들은, 암 억제 유전자로서 miR-5582-5p의 세포사멸 야기와 세포 생장 억제가 암세포 특이적이라는 중요한 이점을 제시한다.Next, it was confirmed whether miR-5582-5p was effective for the growth of normal cells. Overexpression of miR-5582-5p did not inhibit the growth of CCD-18Co and HPF cells (Fig. 5D). Expression of the target protein, which was already low, was further reduced by miR-5582-5p (Fig. 5E). In addition, cell death by overexpression of miR-5582-5p did not occur in normal cell CCD-18Co and HPF (Fig. 5F). These results suggest that miR-5582-5p, a cancer suppressor gene, is an important cancer cell specific inhibitor of cell death and cell growth inhibition.
마우스에서 miR-5582-5p의 종양 성장 억제 Inhibition of tumor growth of miR-5582-5p in mouse
생체 내에서 miR-5582-5p의 암 억제 효과를 확인하기 위해, 두 가지 다른 방법으로 암세포의 마우스 이종이식 모델에서 miR-5582-5p의 발현을 증가시켰다. 첫 번째 방법은 Doxycycline에 의해 miR-5582-5p의 발현이 유도되는 세포주를 주사하여 이종이식 모델을 만들었다, 이를 Tet-5582 그 대조군을 Tet-C로 명명하였다. Tet-5582 세포를 주사하고 Dox를 먹인 마우스가 Dox를 먹이지 않은 마우스에 비하여 암세포의 성장이 유의하게 감소했다 (도 6A-C). 반면에 Tet-C 세포를 주사한 군은 Dox를 먹인 여부와 상관 없이 종양의 크기가 차이 없었다. 면역조직화학분석법을 수행한 결과 Tet-5582 세포를 주사 한 후 Dox를 먹인 군이 대조군에 비해서 PCNA 발현이 감소되어 있고, TUNEL 염색 비율이 높았고 (도 6D), 이는 이 군에서 종양의 성장이 저해되고 세포사멸이 유도되었다는 바를 보여준다.To confirm the inhibitory effect of miR-5582-5p in vivo in cancer, two different methods increased the expression of miR-5582-5p in a mouse xenograft model of cancer cells. In the first method, a cell line in which miR-5582-5p expression was induced by Doxycycline was injected into a xenotransplantation model. Tet-5582 was designated as a control and Tet-C was designated as a control. Tet-5582 cells were injected and Dox-fed mice significantly decreased cancer cell growth compared to mice that did not feed Dox (Fig. 6A-C). On the other hand, Tet-C cell injected group did not differ in tumor size regardless of whether Dox was fed or not. Immunohistochemical analysis showed that PCNA expression was decreased and TUNEL staining rate was higher in the Dox-fed group after injection of Tet-5582 cells (FIG. 6D) than in the control group (FIG. 6D) And cell death was induced.
마우스에서 분리한 종양조직에서 miR-5582-5p와 타깃 단백질을 정량 하였고, 이를 통해 Tet-5582 세포를 이종 이식한 후 Dox를 먹인 군에서 miR-5582-5p 발현 증가를 검증하였고, 타깃 단백질의 발현이 유의하게 감소한 것을 확인하였다 (도 6E and F). 또 다른 방법으로 miRNA delivery 시스템을 이용하였다. HCT116 세포주를 주사하여 종양을 만들고, miR-5582-5p와 대조군 miRNA 를 각각 in vivo 전용 과발현 agent를 사용하여 complex를 만들어 주사하였다. 3일 간격으로 세 번의 주사 후에 관찰한 결과 miR-5582-5p를 과발현 시킨 군이 대조군에 비해 종양의 성장이 유의하게 감소한 것을 확인하였다 (도 6G-I). 이에 부합하게 miR-5582-5p 과발현군에서 PCNA의 발현은 감소되었고, TUNEL 염색된 세포의 비율이 증가되었다 (도 6J). miR-5582-5p와 타깃 단백질은 miRNA의 마지막 주사 후 20일이 경과 후, 마우스에서 분리한 종양 조직에서 측정하였다. miR-5582-5p의 발현은 대조군에 비해 70 배가 증가하는 현저한 차이를 보였고, 이와 유의하게 타깃 단백질의 발현은 감소하였다 (도 6K and L), 이를 통해, miR-5582-5p가 생체 내 에서도 암 억제 유전자로서 효과적인 기능을 하고 있음을 명백히 검증하였고, 이는 항암치료제로서 높은 가능성을 보여준다. MiR-5582-5p and target protein were quantitated in tumor tissues isolated from mouse, and the expression of miR-5582-5p was examined in Dox-fed group after xenotransplantation of Tet-5582 cells. Expression of target protein (Fig. 6E and F). The miRNA delivery system was used as an alternative. The HCT116 cell line was injected into the tumor, and miR-5582-5p and control miRNAs were injected into the complex using in vivo over-expressing agents. After three injections at 3-day intervals, tumor growth was significantly reduced in the group overexpressing miR-5582-5p compared to the control group (Fig. 6G-I). Correspondingly, the expression of PCNA was decreased in the overexpressing group of miR-5582-5p, and the proportion of TUNEL-stained cells was increased (Fig. 6J). miR-5582-5p and target protein were measured in tumor tissues isolated from the
<고찰><Review>
최근 연구에 따르면 다양한 암에서 수많은 miRNA들이 비정상적으로 발현 되어 있고, 이것들은 암 유발 혹은 암 억제 유전자로서 암의 발달과정에 매우 중요한 역할을 하고 있다. 본 발명에서는 암세포의 세포사멸과 세포주기억제를 유도하는 기능을 갖는 새로운 암 억제 miRNA인 miR-5582-5p를 발굴하였다. 암세포의 성장을 조절할 수 있는 miRNA를 찾기 위해 기존에 연구된 바 없으면서 암세포의 성장에 영향을 주는 miRNA의 발굴을 위해 연구가 시작될 당시 최근 등록된 267개의 miRNA로 구성된 miRNA pool을 합성하여 사용하였다. 암세포에 각 miRNA mimic을 과발현 시킨 후 functional screening을 수행 하였다.Recent studies have shown that a number of miRNAs are abnormally expressed in a variety of cancers, and they play a very important role in the development of cancer as a cancer-inducing or cancer-suppressing gene. In the present invention, miR-5582-5p, a novel cancer-suppressing miRNA having the function of inducing apoptosis and cell cycle inhibition of cancer cells, was unearthed. To investigate miRNAs that can regulate the growth of cancer cells, miRNA pools of recently registered 267 miRNAs were synthesized and used at the beginning of the study to discover miRNAs that affect the growth of cancer cells without any previous studies. Functional screening was performed after overexpression of each miRNA mimic in cancer cells.
결과에 따르면, 많은 miRNA들이 세포성장을 유의하게 촉진 또는 억제 하는 것을 확인할 수 있었고, 동시에 이러한 functional screening이 효과적인 방법이라는 것도 알 수 있었다. 이를 통해 대장암 세포주인 HCT116의 성장을 가장 효과적으로 저해하는 miR-5582-5p를 선택 하여 이 연구를 수행 하였다.(그 외에 암세포의 성장을 조절하는 후보로 선정된 miRNA들 또한 다른 연구에서 진행하고 있다.) miR-5582-5p의 세포사멸 유도와 성장 억제 능력에 중요한 역할을 하는 여러 가지 타깃 유전자를 확인하였다. 확인한 유전자 중 GAB1과 SHC1은 EGFR과 같은 RTK들의 하위 신호전달 경로의 매개를 위해 세포질 쪽에 형성되는 복합체의 구성단백질이다. 이 단백질들은 활성화 된 RTK와 결합하여 Ras-MAP kinase와 PI3K-Akt/PKB pathway를 활성화 함으로써 세포분열을 촉진하는 중요한 역할을 한다. 비록 SHC1은 그것의 isoform들 중 p66이 산화촉진 단백질로서 세포사멸을 촉진한다고 종종 언급 되었지만 많은 논문에서 GAB1 과 SHC1이 다양한 암들에서 발암과정에 중요한 역할을 한다고 연구되어 있다. 최근 들어 GAB1 혹은 SHC1을 직접적으로 타깃으로 하는 miRNA들이 보고 되어오고 있다. miR-150은 급성 림프성 백혈병에서 타깃 유전자인 GAB1 억제하여 B-cell receptor signaling을 저해한다. miR-409-3p 또한 GAB1 발현을 저해 함으로써 대장암 전이 억제에 부분적으로 기여한다고 보고되었다. miR-365는 췌장암에서 gemcitabine에 대한 저항성을 유도 하는 SHC1과 BAX를 동시에 타깃 유전자로 갖는다. 직접적인 타깃팅을 통해 GAB1과 SHC1를 동시에 저해하는 것은 miR-5582-5p에 의해 유도 되는 세포사멸에 중요한 기여를 하는 것으로 보인다. GAB1과 SHC1을 동시에 저해 했을 때, anti-apoptotic 단백질 XIAP와 BCL2L1을 감소시키고, pro-apoptotic 단백질 BAK1의 발현을 증가시키는 것과 같은 분자적 현상 뿐만 아니라, miR-5582-5p에 의한 세포사멸 기능을 더욱 잘 모방한다 (도 4B). GAB1 또는 SHC1을 단독으로 저해하는 것에 비해서 miR-5582-5p에 의한 두 가지 발암단백질의 동시 억제는 보다 효율적으로 암 유발 신호전달을 저해 할 수 있고, 세포사멸 또한 더욱 증가 되었다. 이는 암 억제 miRNA로서 큰 이점이다. GAB1과 SHC1과 함께 복합체를 이루는 GRB2 역시 miR-5582-5p의 타깃 단백질 목록에 포함되어 있지만 miR-5582-5p에 의한 GRB2의 발현은 세포주 의존적 이었다 (도 3A). 이러한 결과는 GRB2가 miR-5582-5p의 직접적 타깃단백질로서의 가능성이 낮지만, GRB2의 발현변화가 환경에 의존적으로 RTK signaling을 저해하는 활성을 증가 혹은 감소시킴으로써 miR-5582-5p의 기능에 기여할 것이다.The results showed that many miRNAs significantly promoted or inhibited cell growth, and at the same time, this functional screening was an effective method. This study was carried out by selecting miR-5582-5p, which most effectively inhibits the growth of HCT116, a colon cancer cell line. (Other miRNAs selected as candidates for regulating the growth of cancer cells are also being studied in other studies .) Several target genes have been identified that play an important role in inducing apoptosis and inhibiting growth of miR-5582-5p. Among the identified genes, GAB1 and SHC1 are constitutive proteins of the complex formed on the cytoplasmic side to mediate the downstream signaling pathways of RTKs such as EGFR. These proteins play an important role in promoting cell division by activating Ras-MAP kinase and PI3K-Akt / PKB pathway in combination with activated RTK. Although SHC1 is often referred to as promoting apoptosis as an oxidation promoting protein among its isoforms, many studies have suggested that GAB1 and SHC1 play an important role in carcinogenesis in various cancers. Recently, miRNAs that directly target GAB1 or SHC1 have been reported. miR-150 inhibits B-cell receptor signaling by inhibiting the target gene GAB1 in acute lymphoblastic leukemia. miR-409-3p was also reported to partially inhibit the metastasis of colorectal cancer by inhibiting GAB1 expression. miR-365 has both SHC1 and BAX as target genes that induce resistance to gemcitabine in pancreatic cancer. Simultaneously inhibiting GAB1 and SHC1 through direct targeting appears to play an important role in miR-5582-5p-induced apoptosis. Inhibition of GAB1 and SHC1 simultaneously inhibits the apoptotic proteins XIAP and BCL2L1 as well as molecular events such as increasing the expression of the pro-apoptotic protein BAK1, as well as cell death by miR-5582-5p (Fig. 4B). Compared to inhibiting GAB1 or SHC1 alone, simultaneous inhibition of two cancer proteins by miR-5582-5p could more effectively inhibit cancer-induced signaling and further increase cell death. This is a great advantage as a cancer-inhibiting miRNA. GRB2 complexed with GAB1 and SHC1 was also included in the target protein list of miR-5582-5p, but expression of GRB2 by miR-5582-5p was cell-line dependent (FIG. 3A). These results suggest that although GRB2 is unlikely to be a direct target protein of miR-5582-5p, changes in the expression of GRB2 will contribute to the function of miR-5582-5p by increasing or decreasing the activity of inhibiting RTK signaling in an environmentally dependent manner .
또한 miR-5582-5p는 CDK2를 직접적으로 타깃팅 함으로써 세포 주기를 정지 시켰다. G1기의 checkpoint 조절 단백자인 CDK2는 세포 주기 진행을 조절하는데 중요한 역할을 하고 이것은 여러 miRNA들에 의해 억제된다고 연구 되어 있다. 예를 들어, miR-885-5p는 CDK2를 억제하고 TP53의 활성을 통해 신경아세포의 세포 증식과 생존을 저해한다. 암 억제 miRNA로 아주 잘 알려진 miR-29b 또한 CDK2를 저해하여 장상피 세포의 성장을 조절한다. CDK2를 억제하여 유도 되는 세포주기의 정지는 GAB1과 SHC1의 발현 억제를 통해 이루어지는 세포사멸과 더해져 암 세포의 성장을 억제 하는 miR-5582-5p의 효과적인 활성에 기여한다. 암 억제 유전자로서 miR-5582-5p의 장점은 암세포 특이적인 효과를 들 수 있다. miR-5582-5p의 발현은 정상세포인 CCD-18co와 HPF가 암세포 보다 높으며 그것의 타깃 단백질은 암세포보다 정상세포에서 낮은 발현을 보인다. 암세포에서와는 달리, 정상세포에서의 miR-5582-5p 과발현은 정상세포의 성장이나 세포사멸에 영향을 주지 않았지만 miR-5582-5p의 억제는 정상세포의 성장을 용이하게 하였다 (도 5). 암세포의 성장을 위한 GAB1과 SHC1의 과발현과 관계있는 RTK signaling의 지속적인 활성은 miR-5582-5p의 효과에 기여할 수 있고, RTK signaling이 결여된 정상세포에서는 miR-5582-5p가 많이 존재함으로써 계속적인 생존에 기여한다고 설명할 수 있다. 하지만 이러한 가정이 맞는지, 혹은 암세포에 특이적인 miR-5582-5p 역할에 기여하는 또 다른 요인이 있는지는 지금으로서는 분명하지 않다. 암세포 특이적으로 진행되는 miR-5582-5p의 메커니즘은 추후에 체계적으로 분석할 필요가 있고, 이러한 메커니즘을 이해하는 것은 정상세포에는 최소한의 손상만으로 암세포를 치료할 수 있는 더욱더 정교한 전략을 세우는 데에 아주 효과적일 것이다. MiR-5582-5p also terminated the cell cycle by directly targeting CDK2. CDK2, a checkpoint regulator of G1, plays a crucial role in regulating cell cycle progression and has been studied to be inhibited by several miRNAs. For example, miR-885-5p inhibits CDK2 and inhibits cell proliferation and survival of neuroblastoma through activation of TP53. MiR-29b, a well-known cancer suppressor miRNA, also inhibits CDK2 and regulates the growth of epithelial cells. The cell cycle arrest induced by CDK2 inhibition contributes to the effective activity of miR-5582-5p, which inhibits the growth of cancer cells, in addition to apoptosis through inhibition of GAB1 and SHC1 expression. The advantage of miR-5582-5p as a cancer suppressor gene is the cancer cell specific effect. The expression of miR-5582-5p is higher than that of normal cells, CCD-18co and HPF, and its target protein shows lower expression in normal cells than cancer cells. Unlike cancer cells, miR-5582-5p overexpression in normal cells did not affect normal cell growth or apoptosis, but inhibition of miR-5582-5p facilitated normal cell growth (Fig. 5). The persistent activity of RTK signaling, which is related to overexpression of GAB1 and SHC1 for cancer cell growth, may contribute to the effect of miR-5582-5p and the presence of miR-5582-5p in normal cells lacking RTK signaling Can contribute to survival. However, it is not clear at this time whether this assumption is true or whether there are other factors contributing to the cancer cell-specific miR-5582-5p role. The mechanisms of cancer cell-specific miR-5582-5p need to be systematically analyzed in the future, and understanding these mechanisms is very important for establishing a more sophisticated strategy for treating cancer cells with minimal damage to normal cells It will be effective.
치료제로서의 miRNA의 다양한 이점들 때문에 많은 연구들에서 암 치료를 위한 miRNA 치료법의 발달에 중점을 두고 연구한다. 암 억제 역할을 하는 miRNA 합성 mimic의 과발현 혹은 암 유발 기능을 갖는 miRNA의 저해제를 사용하는 것이 항암 miRNA 치료제를 개발하는 주요 접근 방법이다. 이것의 아주 좋은 예로는 간암 치료법의 발달을 위한 임상 혹은 임상 전 단계의 miR-34 mimic과 miR-21의 저해제가 있다. 이 연구를 통해 miR-5582-5p 또한 miRNA 치료제의 발전을 위한 좋은 후보가 될 것으로 기대한다. 우리는 마우스 이종이식 모델에 miR-5582-5p 과발현 세포주 혹은 종양 내 직접 주사를 통한 miR-5582-5p의 과발현, 이 두 가지 방법을 사용하여 miR-5582-5p의 항암 효과를 증명하였다. 그 결과 세포 내에서의 효과와 마찬가지로 생체 내에서도 miR-5582-5p가 암의 성장과 타깃 단백질 발현을 효과적으로 억제한다는 것을 유의하게 증명하였다. 이러한 결과들은 환자에서 종양 억제제로서 miR-5582-5p의 잠재력을 잘 보여준다 (도 6). 암의 억제를 위해 TP53의 활성이 필요한 miR-34 와는 반대로 miR-5582-5p는 세포 사멸 유도에서 TP53의 활성을 필요로 하지는 않았다 (도 2F). 암환자에게서 TP53은 종종 비활성화 되어있거나 발현 되지 않는 점을 고려 하였을 때, TP53에 대해 비 의존적인 것은 miR-5582-5p의 훗날 임상 적용 조건에 있어 좋은 장점이 된다.Because of the various advantages of miRNA as a therapeutic, many studies focus on the development of miRNA therapeutics for cancer treatment. MiRNAs that inhibit cancer The use of miRNA inhibitors with overexpression or cancer-inducing function of synthetic mimic is a key approach to developing anticancer miRNA therapeutics. A very good example of this is the clinical or preclinical miR-34 mimic and miR-21 inhibitors for the development of liver cancer therapies. Through this study, miR-5582-5p is also expected to be a good candidate for the development of miRNA therapeutics. We demonstrated the anticancer effect of miR-5582-5p using either of these two methods, overexpression of miR-5582-5p through miR-5582-5p over-expressing cell line or direct injection into tumor xenograft model. As a result, it has been demonstrated that miR-5582-5p effectively inhibits cancer growth and target protein expression in vivo as well as in cells. These results demonstrate the potential of miR-5582-5p as a tumor suppressor in patients (Figure 6). In contrast to miR-34, which requires TP53 activity to inhibit cancer, miR-5582-5p did not require TP53 activity in inducing apoptosis (Fig. 2F). Considering that TP53 is often inactivated or not expressed in cancer patients, the independence of TP53 is a good advantage for future clinical applications of miR-5582-5p.
이 연구를 통해 새로운 암 억제 miRNA로서 miR-5582-5p를 발굴하였다. miR-5582-5p의 메커니즘은 도 7에 묘사 하였다. 간단히 말하자면, miR-5582-5p는 GAB1과 SHC1의 억제와 CDK2의 억제를 통해 암의 세포 사멸과 세포주기 정지를 야기했다. 이를 세포 수준과 마우스를 이용한 생체 내 실험을 통해 항암 효과를 검증 하였다. 우리가 아는 한 이것은 miR-5582-5p의 기능을 증명한 첫 번째 연구이다. miR-5582-5p는 TP53의 활성에는 비의존적이면서 암세포에만 특이적으로 다양한 관련 단백질을 동시에 저해하기 때문에 강력한 성장 억제 활성을 가진 항암 miRNA 치료제로서 장점을 갖는다. 이 연구를 통해 miR-5582-5p는 새로운 항암 miRNA 치료법의 발전에 이용될 수 있는 강력한 암억제 miRNA라 할 수 있다.This study uncovered miR-5582-5p as a novel cancer-inhibiting miRNA. The mechanism of miR-5582-5p is depicted in FIG. In brief, miR-5582-5p caused cancer cell death and cell cycle arrest through inhibition of GAB1 and SHC1 and inhibition of CDK2. The anti - cancer effect was verified by in vivo experiment using cell level and mouse. As far as we know, this is the first study to demonstrate the function of miR-5582-5p. miR-5582-5p has an advantage as an anticancer miRNA therapeutic agent having strong growth inhibitory activity because it inhibits various related proteins simultaneously, independent of the activity of TP53 and specific to cancer cells only. Through this study, miR-5582-5p is a powerful cancer-inhibiting miRNA that can be used to develop new anti-cancer miRNA therapeutics.
<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> a tumor suppressor containing miR-5582-5p <130> ula15-12 <160> 34 <170> KopatentIn 2.0 <210> 1 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 1 cuggcccauu ggccauagua cugugccua 29 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 2 uaggcacacu uaaaguuaua gc 22 <210> 3 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 3 cuggcccauu ggccauagua cugaagcua 29 <210> 4 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 4 augaaaucua aaauccugaa augugccua 29 <210> 5 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 5 augaaaucua aaauccugaa augaagcua 29 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 6 cccccauguu uaaacuuugu gccuu 25 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 7 cccccauguu uaaacuuuga agcuu 25 <210> 8 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 8 guuuguagcu cauuaaaaaa augugccua 29 <210> 9 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> artificial sequence <400> 9 guuuguagcu cauuaaaaaa augaagcua 29 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 10 gctataactt taagtgtgcc ta 22 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 11 gcctctagag agaaaggagt gcccacag 28 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 12 ccgaattcat tcatcctcca agtaac 26 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 13 gcctctagaa ggtgagtgct tgtcatg 27 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 14 ccgaattcct ctaagggttg catatc 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 15 gcctctagat aggagttaga agttagg 27 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 16 ccgaattctt ttataaaact aggcac 26 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 17 acggatccgg tcatccgctc tgtgaacc 28 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 18 actctagaca ttgtcattgg tagctgag 28 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 19 ccattggcca tagtactgaa gctaatcaat gtaatagg 38 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 20 cctattacat tgattagctt cagtactatg gccaatgg 38 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 21 ctaaaatcct gaaatgaagc taaactatca aaaca 35 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 22 tgttttgata gtttagcttc atttcaggat tttag 35 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 23 ccatgtttaa actttgaagc tttgaccatc tcttag 36 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 24 ctaagagatg gtcaaagctt caaagtttaa acatgg 36 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 25 gctcattaaa aaaatgaagc tagttttata aaa 33 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 26 ttttataaaa ctagcttcat ttttttaatg agc 33 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 27 aagactacct gttgctcatc aactgt 26 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 28 ggacgttatc attggagtct gtttc 25 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 29 tggatgcctc tgctctcact g 21 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 30 gaggacccga tgagaatggc 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 31 cacttgggag ctacattgcc tg 22 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 32 gtggtggagg tggcatctgt t 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 33 cgtaagtcaa cttcctaggc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 34 ggctaaaata cctttggtcc 20 <110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> a tumor suppressor containing miR-5582-5p <130> ula15-12 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 1 cuggcccauu ggccauagua cugugccua 29 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 2 uaggcacacu uaaaguuaua gc 22 <210> 3 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 3 cuggcccauu ggccauagua cugaagcua 29 <210> 4 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 4 augaaaucua aaauccugaa augugccua 29 <210> 5 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 5 augaaaucuaa aaauccugaa augaagcua 29 <210> 6 <211> 25 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 6 cccccauguu uaaacuuugu gccuu 25 <210> 7 <211> 25 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 7 cccccauguu uaaacuuuga agcuu 25 <210> 8 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 8 guuuguagcu cauuaaaaaa augugccua 29 <210> 9 <211> 29 <212> RNA <213> Artificial Sequence <220> Artificial sequence <400> 9 guuuguagcu cauuaaaaaa augaagcua 29 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 10 gctataactt taagtgtgcc ta 22 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 11 gcctctagag agaaaggagt gcccacag 28 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 12 ccgaattcat tcatcctcca agtaac 26 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 13 gcctctagaa ggtgagtgct tgtcatg 27 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 14 ccgaattcct ctaagggttg catatc 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 15 gcctctagat aggagttaga agttagg 27 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 16 ccgaattctt ttataaaact aggcac 26 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 17 acggatccgg tcatccgctc tgtgaacc 28 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 18 actctagaca ttgtcattgg tagctgag 28 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 19 ccattggcca tagtactgaa gctaatcaat gtaatagg 38 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 20 cctattacat tgattagctt cagtactatg gccaatgg 38 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 21 ctaaaatcct gaaatgaagc taaactatca aaaca 35 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 22 tgttttgata gtttagcttc atttcaggat tttag 35 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 23 ccatgtttaa actttgaagc tttgaccatc tcttag 36 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 24 ctaagagatg gtcaaagctt caaagtttaa acatgg 36 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 25 gctcattaaa aaaatgaagc tagttttata aaa 33 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 26 ttttataaaa ctagcttcat ttttttaatg agc 33 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 27 aagactacct gttgctcatc aactgt 26 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 28 ggacgttatc attggagtct gtttc 25 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 29 tggatgcctc tgctctcact g 21 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 30 gaggacccga tgagaatggc 20 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 31 cacttgggag ctacattgcc tg 22 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 32 gtggtggagg tggcatctgt t 21 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 33 cgtaagtcaa cttcctaggc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 34 ggctaaaata cctttggtcc 20
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