CN102727909B - Novel method for inhibiting primary liver cancer growth and metastasis - Google Patents
Novel method for inhibiting primary liver cancer growth and metastasis Download PDFInfo
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- CN102727909B CN102727909B CN201210222212.5A CN201210222212A CN102727909B CN 102727909 B CN102727909 B CN 102727909B CN 201210222212 A CN201210222212 A CN 201210222212A CN 102727909 B CN102727909 B CN 102727909B
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Abstract
The invention provides a novel biotherapy method for inhibiting primary liver cancer growth and metastasis. According to the method, miRNA-885-5p is adopted as a target point, a synthesized miRNA-885-5p stimulant is injected intratumorally, such that the growth of liver tumor is inhibited.
Description
Technical field
The present invention relates to a kind of liver cancer treatment method, the method is carried out Hepatoma therapy using the miRNA-885-5p that can suppress liver cancer growth and transfer as target spot, realizes therapeutical effect by the synthetic miRNA-885-5p analogies of intratumor injection (miRNA-885-5p Agomir).
Background technology
Primary hepatoma is common malignant tumor of digestive tract, it is worldwide the malignant tumor being number five, annual new discovery malignant tumor patient approximately 6,350,000 examples in the world, wherein hepatocarcinoma accounts for greatly 260,000 examples, and in 260,000 examples, 42.5% occurs in the trend that China and its sickness rate are still cumulative year after year.Hepatocarcinoma finds to be generally late period, effectively Therapeutic Method is hepatectomy and liver transplantation, conventional chemotherapy, to liver cancer treatment effect not obvious, so the early diagnosis of hepatocarcinoma is particularly important, is explored effective hepatocarcinoma clinical diagnosis label and therapeutic strategy significant.
Along with the development of Protocols in Molecular Biology, people are more and more deep to the understanding of tumor development mechanism.The abnormal molecule and the gene that in occurring according to tumor, relate to, design and develop the medicine for specific molecular and gene target spot, selective killing tumor cell, and this method is called tumor molecular targeted therapy.In recent years, tumor molecular targeted therapy has become the developing direction that oncotherapy is new, and the studies and clinical application of tumor molecular targeted agents makes oncotherapy level rise to a new height.
MiRNA plays an important role in the bioprocesss such as Growth of Cells, growth, differentiation, death, and meanwhile, miRNA has almost participated in the process of each vital movement such as hemopoietic, insulin secretion, nervous system formation and human cancer cell's growth.Sequence analysis shows, have more than 1/3 human gene to be directly subject to the regulation and control of miRNA, and increasing experimental evidence shows that miRNA also has a lot of functions undiscovered.MiRNA has the effect of tumor-inhibiting factor and oncogene, is known as oncogenic miRNA, i.e. oncomiR.The imbalance of oncomiR is relevant with gene mutation or epigenetic variation (comprising deletion mutation, amplification sudden change, point mutation and DNA abnormal methylation etc.).Its expression is relevant to generation, development, diagnosis, the prognosis of mankind's Several Kinds of Malignancy.
MiR-885-5p is a kind of known microRNA, and this gene mapping of NCBI gene bank query display, in the 10411173-10411246 interval of No. 3 chromosomal p25.3 (minus strand), is found by cloning and sequencings such as Berezikov E.The relevant essential information of miR-885-5p is: Accession No:MIMAT0004947, basic sequence is 5 ' UCCAUUACACUACCUGCCUCU3 '.The research of miR-885-5p in tumor is also in the stage at the early-stage, and existing research shows that it plays the effect that suppresses tumor growth in neuroblastoma.In addition, research recently finds that miR-885-5p can suppress the transfer of glioma.Our early-stage Study discovery, the pathogenic process of miR-885-5p and hepatopathy is relevant.But its impact on primary liver cancer genesis and development have not been reported.
The present invention finds that miRNA-885-5p can be used as the target spot that suppresses liver cancer growth and transfer first, and the miRNA-885-5p analogies that activate miRNA-885-5p activity can be used for Hepatoma therapy.
Summary of the invention
The object of the invention is to solve current primary liver cancer difficulty, the problem that chemotherapy resistance is higher, a kind of new method of targeted therapy of liver cancer is provided, described method is using the miRNA-885-5p that can suppress liver cancer growth and transfer as target spot, utilize miRNA agonist to treat tumor, wherein miRNA agonist comprises miRNA-885-5p analogies.By the synthetic miRNA-885-5p analogies of intratumor injection, for example, realized the treatment of hepatocarcinoma by Rui Bo bio tech ltd, Guangzhou synthetic miRNA-885-5p analogies (miRNA-885-5p agomir).MiRNA agomir is the miRNA agonist of modifying through special chemical, and it enters miRISC complex by simulation endogenous miRNA and regulates the expression of said target mrna to play a role.Compared with common miRNA mimics, agomir has higher stability and miRNA facilitation effect in animal body.MiRNA agomir has Concentraton gradient dependency in vivo, and stability is high, is several weeks its action time.
The present invention has set up the subcutaneous xenogenesis of NOD/SCID mice and has become tumor model, by intratumor injection PBS, contrast agomir or miRNA-885-5p agomir, matched group and test group are set, the tumor volume of tracking measurement test group and matched group, and the situation of the neoplasm necrosis cell in matched group and test group is passed judgment on by the method for H & E dyeing.Experimental result shows that miRNA-885-5p agomir can suppress the growth of hepatoma carcinoma cell.
In addition, the present invention also has the hepatocarcinoma stable cell lines of Detectable effects by foundation and has the high expressed miR-885-5p hepatocarcinoma stable cell lines of Detectable effects, has further built NOD/SCID mice original position liver cancer model.After definite mice original position liver cancer model is successfully established, by the growing state of chemical luminescence imaging monitoring tumor focus in body, build up after 8 weeks at NOD/SCID mice original position liver cancer model, mice is put to death, lungs taking-up is fixedly made to paraffin section with paraformaldehyde, after dyeing with H & E, by microscopic examination lung tumors tuberosity, to determine that hepatoma carcinoma cell has or not transfer.Experimental group and the control sample of expressing miR-885-5p by mistake compare rear discovery, and growth in situ and pulmonary that high expressed miR-885-5p can suppress hepatoma carcinoma cell shift.
The invention still further relates to the purposes of miR-885-5p analogies in the medicine of preparing Hepatoma therapy; wherein said medicine is used for suppressing liver cancer growth or transfer; described medicine is except comprising miR-885-5p analogies; can also comprise pharmaceutically common pharmaceutical carrier, wherein said carrier is injection solvent, isoosmotic adjusting agent, pH adjusting agent, stabilizing agent, protective agent etc.Injection solvent is selected from water for injection, oil for injection, ethanol, glycerol, Polyethylene Glycol etc.; Described isoosmotic adjusting agent is selected from sodium chloride, potassium chloride, glucose, sodium bicarbonate, sodium lactate, glycerol etc., and sodium chloride concentration scope is generally 0.5-0.9%, and concentration of glucose scope is generally 4-5%, and glycerol concentration scope is generally 2.25% left and right; Described pH adjusting agent is selected from lactic acid, citric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, sodium carbonate, sodium bicarbonate etc., the concentration range of lactic acid is generally 0.1% left and right, the concentration of citric acid is generally 0.5% left and right, the concentration of sodium hydrogen phosphate and sodium dihydrogen phosphate is generally 1.7% and 0.71% left and right, and the concentration of sodium carbonate, sodium bicarbonate is generally 0.06% and 0.005% left and right; Described stabilizing agent is selected from creatinine, glycine, and the concentration of creatinine is generally 0.5-0.8%, and the concentration of glycine is generally 1.5-2.25%; Described protective agent is selected from lactose, sucrose, maltose, human albumin, and the concentration of lactose is generally 2-5%, and the concentration of sucrose is generally 2-5%, and the concentration of maltose is generally 2-5%, and human albumin's concentration is generally 0.2-2%.Described medicine can be prepared into common dosage form, such as injection, granule, tablet, capsule, aerosol etc., described injection can be solution-type, suspension type or emulsion-type, can pass through the mode administration of subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.Described analogies comprise double-stranded RNA, wherein the concrete sequence of the positive-sense strand of double-stranded RNA is: 5 ' UCCAUUACACUACCUGCCUCU3 ', the concrete sequence of antisense strand is: 5 ' AGAGGCAGGTAGTGTAATGGA3 ', described double-stranded RNA can be the RNA through modifying.
More specifically, the miRNA-885-5p analog using in the present invention is purchased from Rui Bo bio tech ltd, Guangzhou, and its concrete name is called miR-885-5p agomir.
Brief description of the drawings
After the subcutaneous one-tenth tumor of Fig. 1 .NOD/SCID mice, intratumor injection miR-885-5p analogies, suppress the growth of hepatocarcinoma as seen;
Fig. 2. tumor body is taken out, and fixing, after H & E dyeing, the tissue necrosis situation of injecting as seen miR-885-5p analogies group is more obvious than matched group;
Fig. 3 carries high expressed HCCLM3 cell biological luminous intensity and the proportional relation of cell number of luciferase;
The high expressed HCCLM3 cell in-situ that Fig. 4 carries luciferase becomes after tumor, spike situation in the body of NOD/SCID mice;
Fig. 5 original position becomes tumor model to build up after 8 weeks, and mice is put to death, and pulmonary is fixed to the H & E situation that dyes;
After the H & E of Fig. 6 pulmonary dyeing, pulmonary's tuberosity counting contrast;
Detailed description of the invention
Below in conjunction with accompanying drawing and detailed description of the invention, the present invention is described in further detail.
Embodiment 1 miR-885-5p analogies suppress the growth of NOD/SCID mice subcutaneous tumors
(1) with the well-grown wild type people of trypsinization hepatocarcinoma SK-Hep-1 cell, wash and also use for one time DMEM minimal medium resuspended to 1 × 10 with PBS
8cell/4 milliliter;
(2) to the cell 200 μ l that handle well in 5-6 week NOD/SCID male mice right side back subcutaneous injection (1), the visible tumor body formation in visible right side of mice back afterwards in 10 days, the model of the subcutaneous one-tenth tumor of NOD/SCID mice builds up;
(3) intratumor injection miR-885-5p analogies 2nM/100 μ l, 2 times/week.Inject tracking amount tumor volume 6 times.Wherein miR-885-5p analogies are purchased from Rui Bo bio tech ltd, Guangzhou, its concrete name is called miR-885-5p agomir, wherein comprise double-stranded RNA, the concrete sequence of the positive-sense strand of double-stranded RNA is: 5 ' UCCAUUACACUACCUGCCUCU3 ', the concrete sequence of antisense strand is: 5 ' AGAGGCAGGTAGTGTAATGGA3 ', described double-stranded RNA is the more RNA of high stability that has through chemical modification.The blank group of intratumor injection PBS, the negative control group of contrast agomir are set simultaneously.Tracking results as shown in Figure 1.Tumor body is taken out, and fixing, after H & E dyeing, the tissue necrosis situation of test group of injecting as seen miR-885-5p analogies is obvious more serious than matched group.H & E coloration result as shown in Figure 2.
Embodiment 2 miR-885-5p suppress the transfer of hepatocarcinoma
(1) there is the foundation of the hepatocarcinoma stable cell lines of Detectable effects
Spread six orifice plates with 293T cell, density is 1.5 × 10
6cells/well, for transfection; With lipo-2000, the slow virus expression plasmid that contains luciferase gene and slow virus packaging plasmid (slow virus expression system is all purchased from biosettia biotech firm of the U.S.) are transfected into the 293T cell of bed board the previous day; Fresh DMEM culture medium is changed in transfection after 16 hours, within 24 hours, collect viral supernatant after changing liquid, is stored in-80 DEG C of refrigerators for subsequent use; Employment hepatocarcinoma HCCLM3 cell spreads six orifice plates, and density is 1 × 10
5cells/well, for viral infection; After viral supernatant room temperature thaws, mixing 2 milliliters does not add in HCCLM3 cell hole containing antibiotic full culture medium and 1 milliliter of viral supernatant, and add cationic polymer genophore polybrene 24 μ g/ holes (purchased from Sigma company), and 1600rpm, 37 DEG C are centrifugal 1 hour; After centrifugal end, renew 2 milliliters/hole of fresh full culture medium, continue to be placed in 37 DEG C of incubators and cultivate; In HCCLM3 cell after 48 hours backward above-mentioned viral infection, add Bsd to carry out medicine sieve and amount to 3 days, to obtain people's hepatocarcinoma HCCLM3 cell of stably express renilla luciferase, be called for short HCCLM3/luci.
(2) there is the foundation of the high expressed miR-885-5p hepatocarcinoma stable cell lines of Detectable effects:
Spread six orifice plates with 293T cell, density is 1.5 × 10
6cells/well, for transfection; With lipo-2000, the slow virus expression plasmid that contains miR-885-5p and slow virus packaging plasmid (slow virus expression system is all purchased from biosettia biotech firm of the U.S.) are transfected into the 293T cell of bed board the previous day; Fresh DMEM culture medium is changed in transfection after 16 hours, within 24 hours, collect viral supernatant after changing liquid, is stored in-80 DEG C of refrigerators for subsequent use; People's hepatocarcinoma HCCLM3 cell of the high expressed renilla luciferase of setting up with (1) spreads six orifice plates, and density is 1 × 10
5cells/well, for viral infection; After viral supernatant room temperature thaws, mix 2 milliliters and do not add in high expressed luciferase HCCLM3 cell hole containing antibiotic full culture medium and 1 milliliter of viral supernatant, and add polybrene 24 μ g/ holes, 1600rpm, 37 DEG C are centrifugal 1 hour; After centrifugal end, renew 2 milliliters/hole of fresh full culture medium, continue to be placed in 37 DEG C of incubators and cultivate; In HCCLM3 cell after 48 hours backward above-mentioned viral infection, add puromycin to carry out medicine sieve and amount to 3 days, to obtain stably express renilla luciferase and to cross people's hepatocarcinoma HCCLM3 cell of expressing miR-885-5p, be called for short HCCLM3/luci-miR-885-5p cell.By nucleic acid hybridization technique, the content of the miR-885-5p in cell is detected, testing result shows that in HCCLM3/luci-miR-885-5p cell, the expression of miR-885-5p obviously increases compared with not importing the HCCLM3/luci cell of miR-885-5p.
(3) foundation of NOD/SCID mice original position liver cancer model:
Use respectively trypsinization well-grown people's hepatocarcinoma HCCLM3/luci or HCCLM3/luci-miR-885-5p cell, wash and also use for one time DMEM minimal medium resuspended to 3.75 × 10 with PBS
7cell/1.2 milliliter; To the above-mentioned cell of handling well the 200 μ l of 5-6 week NOD/SCID male mice right side back subcutaneous injection, the visible tumor body formation in visible right side of mice back afterwards in 10 days, the model of the subcutaneous one-tenth tumor of NOD/SCID mice builds up; It is that 1mm left and right size puncture needle is implanted in the male NOD/SCID Mouse Liver in other 4-6 week that the taking-up of tumor body is cut into diameter.Crossing expression miR-885-5p and negative control NOD/SCID mice original position liver cancer model forms.
(4) chemical luminescence imaging monitoring tumor focus growing state in body:
To (3) middle NOD/SCID mouse peritoneal injection LUC Photinus pyralis LUC Photinus pyralis FL substrate D-Luciferin (150mg/kg), after 5 minutes, utilize Xenogen IVIS Lumina II living imaging system to detect chemiluminescence signal, time of exposure is according to circumstances made as 5-30 second; Testing result as shown in Figure 3,4.Can find out from the result of Fig. 4 demonstration, compared with negative control group, the growth of crossing tumor focus in the experimental group of expressing miR-885-5p obviously weakens.
(5) H & E staining examine NOD/SCID rat liver cancer is to lung transfer case:
NOD/SCID mice original position liver cancer model built up after 8 weeks, and mice is put to death, and lungs taking-up is fixedly made to paraffin section with paraformaldehyde, after H & E dyeing, and microscopic examination lung tumors tuberosity.H & E staining procedure is specific as follows:
Claims (4)
1.miRNA-885-5p the purposes of analogies in the medicine for the preparation of Hepatoma therapy, it is characterized in that described miRNA-885-5p analogies are miRNA-885-5p agomir, miRNA-885-5p agomir comprises double-stranded RNA, wherein the concrete sequence of the positive-sense strand of double-stranded RNA is: 5 ' UCCAUUACACUACCUGCCUCU3 ', and the concrete sequence of antisense strand is: 5 ' AGAGGCAGGTAGTGTAATGGA3 ';
Described medicine is for suppressing liver cancer growth or suppressing hepatocarcinoma and shift.
2. purposes according to claim 1, is characterized in that described medicine also comprises pharmaceutically acceptable carrier, and wherein said carrier is injection solvent, isoosmotic adjusting agent, pH adjusting agent, stabilizing agent, protective agent.
3. purposes according to claim 1, is characterized in that described medicine is injection, and described injection is solution-type, suspension type or emulsion-type.
4. purposes according to claim 3, is characterized in that described medicine can pass through the mode administration of subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.
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| CN105148274B (en) * | 2015-08-31 | 2018-03-23 | 北京泱深生物信息技术有限公司 | Applications of the 5p of miRNA 885 in acute myeloid leukemia diagnosis and treatment |
| CN105521490B (en) * | 2015-12-24 | 2018-08-17 | 北京致成生物医学科技有限公司 | Applications of the miRNA-888-3p in Degenerative disc disease diagnosis and treatment |
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| CN101921863A (en) * | 2010-09-01 | 2010-12-22 | 中国人民解放军总医院 | MiRNA expression model for diagnosing hepatic diseases independently |
| WO2011029903A1 (en) * | 2009-09-10 | 2011-03-17 | Flemming Velin | Method for the preparation of micro-rna and its therapeutic application |
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| WO2011029903A1 (en) * | 2009-09-10 | 2011-03-17 | Flemming Velin | Method for the preparation of micro-rna and its therapeutic application |
| CN101921863A (en) * | 2010-09-01 | 2010-12-22 | 中国人民解放军总医院 | MiRNA expression model for diagnosing hepatic diseases independently |
Non-Patent Citations (2)
| Title |
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| microRNA 在恶性肿瘤临床应用中的研究进展;马强等;《现代肿瘤医学》;20120430;第20 卷(第4 期);835-838 * |
| 马强等.microRNA 在恶性肿瘤临床应用中的研究进展.《现代肿瘤医学》.2012,第20 卷(第4 期),835-838. |
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