CN105903036B

CN105903036B - application of miR-130a antisense nucleic acid and derivative thereof in Hippo-YAP signal pathway inhibitor

Info

Publication number: CN105903036B
Application number: CN201510416936.7A
Authority: CN
Inventors: 赵斌; 沈舒滢; 郭晓灿; 颜欢; 吉新彦
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2015-07-15
Filing date: 2015-07-15
Publication date: 2019-12-17
Anticipated expiration: 2035-07-15
Also published as: CN105903036A

Abstract

The invention provides application of miR-130a antisense nucleic acid and derivatives thereof in a Hippo-YAP signal pathway inhibitor, and provides application of the miR-130a antisense nucleic acid and derivatives thereof as a Hippo-YAP signal pathway inhibitor and application of the Hippo-YAP signal pathway inhibitor in preparation of medicines for treating or preventing overgrowth diseases related to YAP or TAZ activation, in particular application in preparation of anti-tumor medicines. The invention discloses that the function of YAP and TAZ is activated by directly reducing the expression level of VGLL4 protein through small RNA miR-130a for the first time, and proves that the activity of YAP and TAZ can be inhibited through inhibiting the combination of miR-130a and a target site through antisense nucleic acid, so that the growth and proliferation of cancer cells and the growth of tumors are inhibited, and the RNA miR-130a can be used for treating or preventing the cancers related to YAP or TAZ activation.

Description

Application of miR-130a antisense nucleic acid and derivative thereof in Hippo-YAP signal pathway inhibitor

Technical Field

The invention relates to the fields of molecular biology, cell biology and cell signal transduction, in particular to application of microRNA (ribonucleic acid), namely miR-130a antisense nucleic acid and a derivative thereof in a Hippo-YAP signal pathway inhibitor, and application of the miR-130a antisense nucleic acid and the derivative thereof in preventing or treating overgrowth diseases related to a Hippo-YAP signal pathway, for example, application of the miR-130a antisense nucleic acid and the derivative thereof in development of anticancer drugs.

Background

The Hippo signaling pathway is an evolutionarily highly conserved cellular signaling pathway. The main functions of the Hippo signaling pathway include regulation of organ size, tissue regeneration, and stem cell function, among others. The basis of cellular biology for achieving these functions is to regulate cell proliferation, apoptosis, and stem cell and precursor cell sternness maintenance and differentiation. The core of the Hippo signal path is a protein kinase chain, which consists of four proteins, Hippo (Hpo), Salvador (sav), Wts and Mats in Drosophila; the mammalian counterparts are Mst1/2, Sav1, Lats1/2 and Mob. Wherein Wts is the first discovered member of Hippo pathway, and Wts gene mutation is found to cause tissue overgrowth in Drosophila genetic screening experiments. Upstream of the four core members, there are many proteins that regulate them, Ex, Mer, Kibra, etc. originally found in drosophila, and more upstream are regulated by Fat, etc. Downstream of the core member, the Hippo signaling pathway down-regulates a transcriptional co-activator, Yki in Drosophila and YAP/TAZ in mammals. YAP (Yes-associated protein) proteins are effector proteins of the Hippo pathway, which play an important role in this pathway. TAZ is a homologous protein of YAP. Yki/YAP/TAZ is used as transcription co-activator, when the Hippo pathway is in inhibition state, it can enter nucleus to combine with transcription factor, to activate the expression of downstream gene; when the Hippo pathway is activated, Yki/YAP/TAZ is phosphorylated by Wts or Lats1/2, stays in cytoplasm in an inactive state and is finally ubiquitinated and degraded, thereby inhibiting the activity thereof.

studies have shown that YAP transgenes can lead to liver enlargement to one-fourth of the mouse weight in mice and liver restoration to the original Size after cessation of YAP-induced expression (citations: Jixin Dong, Georg Feldmann, JiainbinHuang, Shian Wu, Nailing Zhang, Sarah A. Commord, Mariana F. Gayyed, RobertA. Anders, Anirban Maitra, Duojia Pan, emulsification of a Universal Size-control Mechanism in Drosophila and Mammals. cell.2007Sep 21; 130(6): 1120-33). However, persistent expression of the YAP transgene resulted in liver cancer development. In human cells, YAP/TAZ is closely related to the activities of promoting the proliferation of tumor cells, inhibiting the apoptosis of tumor cells and regulating the dryness and differentiation of cells. From the cancer species, YAP has gene amplification in liver cancer, esophageal cancer, oral squamous cell carcinoma, intracranial ependymoma, medulloblastoma, and the like; TAZ is also gene amplified in esophageal cancer, breast cancer, etc. (citations: Zhao B, Li L, Lei Q, Guan KL,2010The Hippo-YAP breast approach size control and tomogenesis: an updated version. genes Dev 24: 862-874). From the protein expression level, YAP and TAZ show high protein expression and increased nuclear localization in lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, liver cancer and prostate cancer. YAP has been found to have an important function in the survival of beta-catenin dependent cancer cells during pancreatic cancer recurrence; TAZ was found to have a unique important function in maintaining the stem dryness of breast Cancer stem cells (citations: Moroishi T, Hansen CG, Guan KL. the empirical rols of YAP and TAZ in Cancer. Nature reviews Cancer 2015,15(2): 73-79.). In addition, many upstream genes of the Hippo signaling pathway exhibit inactivation phenomena such as down-regulation of expression to various degrees in cancer samples, for example RASSF1A is one of the most obvious genes inactivated by promoter methylation in human cancers. Of particular note are several classes: (ii) neurofibroma type 2. NF2 oncogene mutation is inactivated in this class of tumors. NF2 is the upstream gene of the Hippo pathway, its deletion causing YAP activation, whereas inactivation of one of the YAP alleles completely reversed the carcinogenesis of NF2 in a mouse model of NF2 mutation induced cancer, demonstrating the importance of YAP activity in this type of tumor (citations: Zhang N, Bai H, David KK, Dong J, Zheng Y, Cai J, et al. the Merlin/NF2tumor regulators through the YAP on protein to regulated tissue metabolism in mammals. Dev. Cell 2010,19(1): 27-38.). ② about 80% of cases in uveal melanoma contain activating mutations in GNAQ or GNA 11. This mutation leads to the activation of YAP and TAZ and the development of cancer, while inactivation of YAP inhibits the growth of tumor cells and tumor formation (cited documents: Yu FX, Luo J, Mo JS, Liu G, Kim YC, Meng Z, et al. Therefore, the YAP as a protooncogene plays an important role in the aspects of proliferation, apoptosis, dryness and the like of tumor cells, so that the inhibition of the YAP activity has a remarkable application value in the inhibition of tumors.

As described above, transcription co-activators (YAP/TAZ) are key effector molecules that function as Hippo-YAP signaling pathways to regulate cell proliferation, apoptosis, dryness, organ size, regeneration, etc., while TEAD family transcription factors are identified as important proteins that mediate the binding of YAP/TAZ to DNA and thus regulate gene expression. Targeting the binding of YAP and TEAD is an important pathway to inhibit YAP activity. A small molecule compound verteporfin can inhibit hepatomegaly by interfering the interaction of YAP and TEAD, and provides a theoretical basis for developing specific small molecule targeted anticancer drugs targeting a Hippo-YAP signal pathway (cited documents: Liu-Chittenen Y, Huang B, Shim JS, Chen Q, Lee SJ, Anders RA, ethylene, genetic and pharmacological diagnosis of the TEAD-YAP complex customized interaction of YAP. genes Dev 2012,26(12): 1300) 1305).

microRNAs (microRNAs, miRs) are a class of endogenous non-coding single-stranded small RNAs with the length of about 21-23 nucleotides, participate in various important physiological processes such as cell proliferation, differentiation, morphological change, apoptosis and the like, have tissue specificity in expression, and have dual functions of inhibiting and promoting cancers in different tissues, wherein the microRNAs with carcinogenic effects are called as "oncomics" and can be combined to target gene mRNAs, particularly to a 3' end untranslated region through base complementarity to cause the degradation of the target gene mRNAs or inhibit the translation of the target gene mRNAs; the microRNAs with the cancer metastasis promoting effect are called as "metastamis", and the microRNAs can directly act on tumor metastasis related genes so as to influence tumor migration and invasion capacity (cited documents: thinking, strong lingzhi, Kunming, miR-130 action research progress in malignant tumors, Chinese tumor 2014 02 th, P136).

in recent years, the development of modifications and delivery vehicles for microRNAs has greatly enhanced the utility of microRNAs as drug targets. For example, 2 '-O-methyl (2' -OMe), 2 '-O-methoxylethyl or 2' -fluoro modifications are used to increase stability; using a Locked Nucleic Acid (LNA), using a peptide nucleic acid pna (peptide nucleic acid); using inorganic or organic nanoparticle or liposome encapsulation as a delivery vehicle; using virus-based delivery systems, and the like. In summary, these methods are based on the use of nucleic acid or nucleic acid analog fragments that mimic the sequence of a microRNA or its antisense sequence, delivered into tumor cells in a manner that inhibits or de-inhibits the expression of the microRNA target gene, to achieve a cancer inhibitory effect. However, the existing inhibitors are very limited in types, and the cancer inhibition effect is lack of deep analysis and experimental verification and needs to be further explored.

The miR-130 family includes miR-130a and miR-130b, which are located on different chromosomes. miR-130a is located on chromosome 11q12.1 and is located in an intron of gene AP 000662.4; miR-130b is located on chromosome 22q11.21, on an exon of peptidyl prolyl isomerase (cyclophilin) -like 2 gene (ppil 2). The mature sequences of miR-130a and miR-130b are obtained by cutting from the 3' arm of the hairpin transcription precursor by Dicer enzyme, the mature sequence of miR-130a is CAGUGCAAUGUUAAAAGGGCAU, the mature sequence of miR-130b is CAGUGCAAUGAUGAAAGGGCAU, and the miRNA belongs to high homology. The pen workers conduct literature search on the existing related research of the miR-130 family, and the research results are summarized as follows:

(1) miR-130a and miR-130b belong to highly homologous miRNAs, but the expression in different tumors is obviously different, for example, miR-130a and miR-130b are both highly expressed in glioblastoma, miR-130a and miR-130b are highly expressed in esophageal cancer, and miR-130a and miR-130b are both highly expressed in bladder cancer (citations: Lujuan, Song Xin, research progress of relation between miR-130a/b and tumor, Ministry of university of Chongqing medicine 2014, Vol.39, No. 3, P274), the two are obviously different.

(2) miR-130a and miR-130b have different targeting effects on oncogenes in different tumors, or have a cancer promotion effect, or have a carcinogenic effect, and the action mechanisms of the miR-130a and the miR-130b are different.

(3) The target genes of miR-130a and miR-130b in the tumor are known at present and shown in Table 1 (cited literature: Ljuan, Songxin, research progress of relation of miR-130a/b and tumor, Chongqing university of medicine, Vol.39, No. 3, P275) and no literature reports the relevance of miR-130a and the target gene in Hippo-YAP signaling pathway.

TABLE 1 target genes of miR-130a and miR-130b in tumors

(4) YAP/TAZ is a key effector molecule for the Hippo-YAP signal path to play a role in regulating cell proliferation, apoptosis, migration and the like, and no literature reports the relevance of miR-130a and the inactivation or activation of YAP/TAZ.

(5) Modification of miR-130 family, especially miR-130a, or combination with a delivery carrier to develop a targeted inhibitor for a Hippo-YAP pathway is blank.

Disclosure of Invention

The invention aims to: based on the original discovery of the inventor that the microRNA miR-130a is a target gene activated and expressed by YAP, and the action mechanism of YAP/TAZ is activated by positive feedback, the application of miR-130a antisense nucleic acid and derivatives thereof in a Hippo-YAP signal channel inhibitor is provided, and the feasibility is provided for the development of specific targeted anticancer drugs taking the Hippo-YAP signal channel as a target spot.

In order to realize the purpose, the invention adopts the following technical scheme:

application of miR-130a antisense nucleic acid in Hippo-YAP signal pathway inhibitor.

Preferably, the application is that miR-130a antisense nucleic acid inhibits the activity of YAP or TAZ, inhibits the expression of a target gene of a Hippo-YAP signal channel, and inhibits the proliferation and metastasis of tumor cells.

More preferably, the miR-130a antisense nucleic acid inhibits the activity of YAP or TAZ, by increasing VGLL4 protein levels, inhibiting YAP or TAZ binding to TEAD family transcription factors.

preferably, the miR-130a antisense nucleic acid is a base sequence of SEQ NO1, SEQ NO 1: 5' AUGCCCUUUUAACAUUGCACUG 3’。

Application of derivatives of miR-130a antisense nucleic acid in Hippo-YAP signal pathway inhibitors.

Preferably, the miR-130a antisense nucleic acid derivative is a nucleic acid or a nucleic acid derivative fragment with the following characteristics: (1) binds to miR-130a by means of base pairing; (2) base pairing is complete base pairing of all bases, or base pairing of the underlined part in SEQ NO1, with the remainder base unpaired; the unpaired mode can be any base which is replaced by A, U, G, C.

Preferably, the miR-130a antisense nucleic acid derivative is a nucleic acid fragment or peptide nucleic acid fragment having chemical modification, such as nucleotide derivatives of 2 ' -O-methyl, 2 ' -O-methoxy, 2 ' -fluoro, Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), etc.; the chemical modification is effective in promoting pharmacological properties such as stability and cell permeability.

preferably, the miR-130a antisense nucleic acid derivative can be constructed by the following steps:

(1) the primers for synthesizing the antisense nucleic acid encoding the three repeated miR-130a and the reverse primers thereof are as follows:

Forward direction:

5’GATCCATGCCCTTTTAACATTGCACTGCGATATGCCCTTTTAACATTGCACTGTCACATGCCCTTT TAACATTGCACTGTTTTTTG 3’

And (3) reversing:

5’AATTCAAAAAACAGTGCAATGTTAAAAGGGCATGTGACAGTGCAAT GTTAAAAGGGCATATCGCAGTGCAATGTTAAAAGGGCATG 3’；

(2) Mixing and heating the forward primer and the reverse primer in a buffer solution to 95 ℃, and annealing to room temperature to obtain an annealing product;

(3) digesting the lentivirus vector by using BamHI and EcoRI restriction endonucleases, and recovering;

(4) connecting the annealing product obtained in the step (2) with the vector subjected to enzyme digestion in the step (3) under the action of T4 ligase and converting the vector into escherichia coli;

(5) Culturing colibacillus, and extracting and amplifying plasmid to obtain the target product.

the invention also provides the application of the Hippo-YAP signal pathway inhibitor obtained according to the application in the preparation of a medicament for treating or preventing overgrowth diseases related to YAP or TAZ activation. As an effective component of the Hippo-YAP signal pathway inhibitor, the antisense RNA molecule of miR-130a shown in SEQ NO1 sequence or a derivative thereof allowable in biological activity can be used.

Preferably, the overgrowth diseases associated with YAP or TAZ activation include, but are not limited to, neurofibroma type 2, uveal melanoma, liver cancer, intestinal cancer, breast cancer, esophageal cancer, stomach cancer, prostate cancer, ovarian cancer, oral squamous cell carcinoma, brain cancer, pancreatic cancer, or lung cancer, and the like. Namely, the Hippo-YAP signal channel inhibitor is applied to preparing antitumor drugs.

The miR-130a antisense nucleic acid and the derivative thereof can be delivered into cultured cells or animal bodies to play a role in the form of chemical molecules synthesized in vitro under the action of a transfection reagent; it can also be achieved by delivering into the cell an expression plasmid containing the DNA sequence encoding the above-described RNA fragment (i.e., SEQ NO1), which can be a common expression plasmid, by transfection reagents. The plasmid may also be a lentiviral, retroviral, or adenoviral, adeno-associated viral plasmid, which is delivered into cells or animals by viral-mediated delivery through packaging of viral particles.

The miR-130a antisense nucleic acid derivative can be obtained on the market or prepared by synthesis. The mode of preparation is not limited, and for example, a single strand oligonucleotide can be synthesized by solid phase synthesis using a DNA synthesizer using a commercially available A, C, G, U monomer in accordance with standard procedures; commercially available 6-N-benzoyladenosine (A) can also be used^Bz),4-N-benzoylcytidine(C^Bz),2-N-isobutyrylguanosine(G^iBu) And the 5' -O- (4,4' -dimethylytrityl) -2 ' -O-methyl-3' -O- (2-cyanoethyl-N, N-diisopropyL) RNA phosphoramidite monomer of uridine (U), 2 ' -O-methyl oligonucleotide single strand was synthesized in solid phase by using a DNA synthesizer according to standard procedures; other commercially available nucleotide derivatives can also be used to synthesize modified oligonucleotide single strands by solid phase synthesis using a DNA synthesizer according to standard procedures; if cholesterol coupling is required, the synthesis can also be started from a controlled pore glass solid support with cholesterol hydroxyprolinol.

In the invention, the miR-130a antisense nucleic acid derivative, preferably the cavernous multi-copy antisense nucleic acid of miR-130a carried by the carrier is constructed in the way described above.

The new application provided by the invention has the following innovations:

The present invention is based on the discovery that micrornas (i.e., miR-130a) are direct target genes induced by YAP (see fig. 1), and that miR-130a further promotes the activity of YAP by directly inhibiting the translation of the competitive inhibitor molecule VGLL4 protein of YAP (see fig. 2). The antisense nucleic acid of miR-130a can inhibit the activity of YAP, inhibit cell proliferation, inhibit the transformation of non-cancer cells into cells with cancer cell activity, inhibit the formation of tumors by cancer cells in mice, and inhibit the organ size enlargement and tumorigenesis induced by abnormal activation of YAP.

Accordingly, the invention is directed to inhibiting YAP/TAZ activity by delivering an antisense nucleic acid of miR-130a or a derivative thereof into a cell and as a Hippo-YAP signaling pathway inhibitor for treating or preventing overgrowth diseases associated with YAP/TAZ activation, such as cancer; including but not limited to neurofibroma type 2, uveal melanoma, liver cancer, intestinal cancer, breast cancer, esophageal cancer, stomach cancer, prostate cancer, ovarian cancer, oral squamous cell carcinoma, brain cancer, pancreatic cancer, or lung cancer, among others.

It is to be noted that the miR-130a antisense nucleic acid used in the present invention can be a nucleic acid fragment completely complementary to mature miR-130 a; or a nucleic acid fragment containing a sequence complementary to the seed sequence of miR-130a (underlined part of SEQ NO 1); other derivatives that bind to miR-130a based on the base pairing principle and thus function to inhibit miR-130a, such as, but not limited to, Peptide Nucleic Acids (PNA), are also possible.

Drawings

FIG. 1 shows that YAP/TAZ directly induces the expression of miR-130 a.

wherein, 1A: YAP overexpression induces elevated miR-130a levels. Stable expression of either YAP wild-type or YAP 5 SA-activated mutants was mediated by lentiviral infection in MCF10A breast cells and HepG2 liver cancer cells. And carrying out microRNA extraction on the constructed stable cell strain by using an miRNA extraction kit, and detecting the expression level of miR-130a by using a specific Taqman miR-130a probe kit based on fluorescence quantitative PCR.

1B: YAP/TAZ knockdown reduces miR-130a expression levels. 5nM control or YAP/TAZ specific siRNA was transfected into MCF10A and HMLE cells, respectively, and miRNA was extracted with the kit 48h after transfection, and the detection method was the same as 1A.

1C: steady knockdown of TEAD decreased miR-130a levels. Lentivirally mediated TEAD1/3/4shRNA was used to interfere with the expression of TEAD1/3/4 in HepG2 and HMLE cells. And (3) extracting miRNA from the stable cell strain by using a kit, wherein the detection method is the same as 1A.

1D: YAP and TEAD bind directly to the miR-130a promoter. Collecting 1X10⁷HepG2 cells, lysed and chromatin immunoprecipitation (ChIP) with TEAD or YAP specific antibodies or control IgG, respectively. The product is used for performing protease decomposition on protein, phenol chloroform extraction on DNA, and the extracted DNA is used as a template for fluorescent quantitative PCR detection. P4 and P5 are two pairs of miR-130a proximal promoter region-specific primers.

1E: TEAD mediates the binding of YAP to the miR-130a promoter. YAP, TEAD2 and miR-130a promoter reporter plasmid were co-transfected in HEK293T cells. The promoter reporter plasmid of miR-130a is a wild type or a mutant of predicted TEAD binding sites 1, 2, 3 and 4. After 48h of culture, luciferase activity detection is carried out by a microplate reader. The results show that TEAD binding sites 1and 2 mediate the binding of YAP-TEAD and miR-130a promoter.

1F: MST1/2 conditional knockdown increased miR-130a expression levels. Conditional knockouts of MST1and MST2 in mouse liver were performed using albumin (alb) -Cre. Liver tissue samples are taken at two months of age, miRNA is extracted by the miRNA extraction kit, and the detection method is the same as 1A.

1G: YAP-5SA induced liver neoplasia. Control or YAP 5SA PB transposase plasmids and PB transposase expression plasmids were delivered through the rat tail vein using hydrodynamic injection. Obvious tumors can be seen after taking the liver 100 days after injection. The arrow points to the tumor.

1H: miR-130a expression levels were elevated and VGLL4 protein levels were reduced in YAP-induced liver tumors. And (3) respectively collecting YAP-5SA induced tumor tissues and paracancerous tissue contrast, extracting miRNA by the miRNA kit, and detecting the miR-130a expression level by fluorescent quantitative PCR. Parallel samples were lysed with SDS lysate and cells were tested for protein expression levels of VGLL4 using immunoblot assays.

FIG. 2 shows that miR130a inhibits protein expression of VGLL 4.

Wherein, 2A: miR-130a regulates VGLL 43' UTR luciferase reporter gene activity. HEK293T cells were transfected with either wild type or mutant VGLL 43' UTR reporter genes, with co-transformation of miR-130a mimetics or inhibitors or placebo. Luciferase reporter experiments were performed 48 hours later and read with a microplate reader.

2B: miR-130a mimetibody inhibits VGLL4 protein levels. Transient control or miR-130a mimetibody was transferred in MCF10A, MGC-803, HepG2, a549 and HMLE cells and protein samples were collected 72h later for immunoblotting experiments.

2C: stable overexpression of miR-130a inhibits VGLL4 protein level. In HepG2 and HMLE cells, lentivirus stably transfects control no-load or miR-130a overexpression plasmids, constructs stable cell strains and collects protein samples for immunoblotting experiments.

2D: miR-130a inhibitors up-regulate VGLL4 protein levels. In MCF10A, MGC-803 and A549 cells, the control or miR-130a inhibitor was transiently transfected, and 72h later a protein sample was collected for immunoblotting.

2E: stable overexpression of miR-130a cavernosum up-regulates VGLL4 protein levels. In HepG2 and HMLE cells, lentivirus is stably transfected into a control empty vector or miR-130a sponge plasmid to construct a stable cell strain, and a protein sample is collected for an immunoblotting experiment.

FIG. 3 shows that miR-130a antisense nucleic acid molecule inhibits the activity of YAP and TAZ.

Wherein, 3A: the miR-130a antisense nucleic acid (inhibitor) inhibits the expression of the YAP target gene. In HepG2, HMLE and MGC-803 cells, miR-130a control and inhibitor are respectively transfected transiently, total RNA is extracted by Trizol, and mRNA expression levels of CTGF and Cyr61 genes are detected by fluorescent quantitative PCR.

3B: the miR-130a cavernosum inhibits the expression of YAP target genes. In HepG2, HMLE and MGC-803 cells, miR-130a control and cavernous body are stably transfected respectively, the constructed stable cells are lysed by Trizol to extract total RNA, and the detection method is the same as 3A.

3C: inhibition of YAP target gene by miR-130a depends on VGLL 4. HMLE cell transiently transfects control or VGLL4 interfering RNA, transfects control or miR-130a antisense nucleic acid after 24h, and extracts total RNA by Trizol lysis after continuously culturing for 48h, and the detection method is the same as 3A.

3D: the inhibition effect of miR-130a on the activity of the CTGF promoter of the YAP target gene depends on VGLL 4. In HEK293T cells stably expressing CTGF luciferase reporter gene, transient transfection of control or VGLL4 interfering RNA and simultaneous transfection of control or miR-130a antisense nucleic acid are carried out, luciferase reporter gene experiments are carried out after continuous culture for 48 hours, and reading is carried out by a microplate reader.

3E: the miR-130a cavernosum inhibits the binding of YAP to the target gene promoter region. Collecting 1X10⁷The HepG2 cells expressing the unloaded or miR-130a cavernous body are used for chromatin immunoprecipitation experiments, protein DNA compounds are precipitated by TEAD/YAP or a control IgG antibody respectively, DNA is extracted by phenol chloroform after protease decomposition of protein, and the enrichment level of the CTGF promoter is detected by fluorescent quantitative PCR.

FIG. 4 shows that miR-130a antisense nucleic acid molecule inhibits cell proliferation, organ size, and promotes apoptosis.

Wherein, 4A: the miR-130a cavernosum can inhibit the cell growth promoting effect of YAP 5 SA. Stably expressing unloaded YAP 5SA or co-expressing miR-130a cavernous body in HepG2 cells, planting the constructed Cell strain in a 96-well plate, wherein each well comprises 1000 cells, each time point comprises three multiple wells, and the Cell Titer Blue reagent staining method is adopted every other day for 0-9 days, and the reading of an enzyme-linked immunosorbent assay instrument is calculated to prepare a graph growth curve.

4B: inhibition of VGLL4 expression can block the inhibitory effect of miR-130a antisense nucleic acid on cell growth. Transient transfection of control and VGLL4 interfering RNA in HepG2 cells, 24h later transfection of control and miR-130a antisense nucleic acid, continuous culture for 48h after treatment, digestion and planting in a 96-well plate, and detection method is the same as 4A.

4C: the miR-130a inhibition can promote apoptosis caused by cell suspension. HepG2 cells are transiently transfected with control and miR-130a antisense nucleic acid, digestion is carried out after 24h for suspension culture, cells are collected for Annexin V staining after continuous culture for 48h, and then the apoptosis rate is detected on a flow cytometer.

4D: the miR-130a cavernosum inhibits liver enlargement caused by MST1/2 conditional knockout. Injection of Cre recombinase-expressing adenovirus and concurrent injection of control or miR-130a cavernous adenovirus in MST1/2 conditioned knockout mice, sacrifice after 9 days, weighing, and liver/body weight ratio calculations. P values were calculated according to T test.

4E: the miR-130a cavernosum inhibits hepatocyte proliferation caused by MST1/2 conditional knockout. Liver samples of the same 4D were frozen and immunostained for the proliferation marker Ki67, with green indicating a positive.

4F: the miR-130a cavernosum inhibits the increase of YAP target gene expression and the reduction of VGLL4 protein caused by MST1/2 knockout. The liver sample of the same 4D is cracked by Trizol to extract total RNA, the expression conditions of YAP target genes CTGF, Cyr61 and Inhba are detected by fluorescence quantitative PCR, meanwhile, protein samples are collected by SDS cracked tissues, and the change of the protein amount of VGLL4 is observed by immunoblotting test.

FIG. 5 shows that miR-130a antisense nucleic acid molecule inhibits cell cancer transformation and tumor growth.

Wherein, 5A: the miR-130a antisense nucleic acid inhibits cell anchorage-independent growth (cancer transformation) caused by YAP 5 SA. HepG2 cells expressed no-load or YAP 5SA plasmids, transfected with control or miR130a antisense nucleic acids, digested after 24h transfection, and 2.5X10⁴The test for the formation of soft agar colonies was carried out on 6-well plates, the concentration of agar in the upper layer was 0.33% and that in the lower layer was 0.6%, and the colonies were changed every 4 days, and after 20 days of incubation, the colonies were photographed with a microscope and stained with crystal violet staining solution, and the number of colonies was photographed and quantified with ImageJ.

5B: the miR-130a cavernosum inhibits the tumor formation promoting effect of YAP S127A. HepG2 cells expressing YAP S127A infect a control or miR-130a cavernosum, are used for a nude mouse subcutaneous tumor forming experiment after being amplified, two injection points are arranged at the left and right of the lower limb of each mouse, each point is 200ul of serum-free culture medium cell suspension, and the cell concentration is 1 multiplied by 10⁷/ml。

5C: the miR-130a cavernosum reduces YAP S127A-induced tumor size. Experiment 5B, each tumor was weighed and then counted as a dot-plot.

Detailed Description

the following description will further explain the substance of the present invention by using the embodiments of the present invention with reference to the accompanying drawings, but the present invention is not limited thereto. Modifications of the invention which are in accordance with the spirit of the invention are within the scope of the invention.

Unless otherwise indicated, all experimental materials, reagents and equipment are commercially available from the examples of the present invention, and the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

Experimental materials:

miR-130a sense and antisense nucleic acids were purchased from Gima, Shanghai.

VGLL4 antibody was purchased from abcam.

Tubulin antibodies were purchased from sigma.

Scaramble siRNA and YAP/TAZ siRNA were purchased from Ribo, Guangzhou.

QPCR primers and CHIP primers were all purchased from Life.

The PE Annexin V apoptosis kit was purchased from BD.

Transfection reagents RNAIMAX and LIPO2000 were purchased from Invitrogen.

Cell Titer Blue kit was purchased from promega corporation.

Example 1:

The name of the experiment: both the miR-130a antisense nucleic acid and the plasmid encoding the cavernous multiple copy antisense nucleic acid inhibit YAP target gene expression and function via VGLL 4.

CTGF and Cyr61 are the most widely accepted YAP target genes. The real-time fluorescent quantitative PCR detection of the mRNA levels of CTGF and Cyr61 can reflect the influence of miR-130a antisense nucleic acid and cavernous body multicopy antisense nucleic acid plasmids on the transcription levels of two target genes, thereby reflecting the action of the antisense nucleic acid plasmids on the YAP activity.

In HepG2, HMLE and MGC-803 cells, miR-130a multicopy antisense nucleic acid plasmids are stably transfected or miR-130a antisense nucleic acids are transiently transfected respectively, and CTGF and Cyr61 gene expression analysis is carried out.

The experimental principle is as follows:

real-time fluorescent quantitative PCR is one of the most accurate methods for detecting the mRNA content of genes. The CTGF and Cyr61 target genes of the Hippo signal pathway oncogene YAP are also important components in the pathway, and the detection of the CTGF and Cyr61mRNA content of the Hippo pathway target genes by fluorescent quantitative PCR is one of important indexes reflecting YAP transcription activity.

The experimental method comprises the following steps:

(1) Cell culture: HepG2 cells were cultured in DMEM containing 10% fetal calf serum, HMLE cells in DMEM/F12 medium containing 10% horse serum and 10. mu.g/ml insulin, 100ng/ml cholera toxin, 20ng/ml EGF, 0.5. mu.g/ml hydrocortisone four factors, and MGC-803 cells in 1640 medium containing 10% fetal calf serum. Cells cannot be over dense, and can be passaged when 70-80%.

(2) Transient transfection treatment: cells were seeded into 6-well plates at 2 × 10 per well⁵And after the cell density reaches 60%, replacing each hole with fresh culture solution, simultaneously transfecting 20nM control or VGLL4 small interfering RNA by using RNAiMax according to a reagent specification, transfecting 20nM control or miR-130a antisense nucleic acid by using RNAiMax according to a reagent specification after 24h, and continuously culturing for 48h.

(3) Establishment of a stable expression cell line: preparing HEK293T cells with two eighty-full disks for each target cell, changing the cells into fresh culture solution, respectively transfecting 2ug of control no-load or miR-130a multicopy antisense nucleic acid plasmids by using Lipo2000 according to a reagent specification, simultaneously transfecting lentivirus packaging plasmids pxPAX21500ng and pMD2. G500 ng together, changing the culture solution after 24h, planting two disks of target cells to be infected simultaneously, continuously culturing for 24h, then carrying out first target cell infection, adding 3ml of virus-containing packaging cell culture medium into 3ml of fresh culture medium and 10 mu g/ml of polybrene during infection, adding the fresh culture medium and the 10 mu g/ml of polybrene onto the target cells to be infected, carrying out the operation once every 12h for 3 times, adding Puromycin after 36h of first infection for screening, and changing the culture solution after 48h.

(4) extraction of total RNA of cells: the medium was aspirated off, the cells were washed once with PBS, the residual medium was removed, PBS was then aspirated off, 1ml of Trizol was added to each well and the cells were lysed thoroughly by repeated pipetting several times, Trizol was transferred to a new rnase-free 1.5ml ep tube, total RNA was purified according to Trizol instructions, and the concentration was determined.

(5) Reverse transcription of mRNA into cDNA: 500ng of RNA was taken for each sample, and the resulting mixture was used as a 10ul system, and cDNA was obtained by reaction using a reverse transcription kit from Takara on a PCR instrument, and diluted 5-fold with water.

(6) Fluorescent quantitative PCR detection of mRNA levels: taking cDNA as a template, adding Hippo pathway YAP target gene CTGF, Cyr61, reference gene HPRT specific primers and super SYBR Green Mix of Biorad respectively, reacting on a Biorad fluorescent quantitative PCR instrument, and arranging three multiple holes for each sample. Primers were synthesized by Life, and purified by PAGE, and the sequences of the primers were as follows:

The reverse primer of HPRT: 5'-AGCCCTGGCGTCGTGATTA-3',

The reverse primer of HPRT: 5'-ACAATGTGATGGCCTCCCA-3',

CTGF forward primer 5'-CCAATGACAACGCCTCCTG-3'

CTGF reverse primer 5'-TGGTGCAGCCAGAAAGCTC-3'

Cyr61 Forward primer 5'-GTGGGTCTGTGACGAGGATAGTAC-3'

Cyr61 reverse primer 5'-AACAGGGAGCCGCTTCAGT-3'

(7) And (3) data analysis: the relative amount of mRNA was calculated by 2- Δ CT and a bar graph was drawn in excel with NC control group as 1.

The experimental results are as follows: see fig. 3A, B, C. Experimental results show that the miR-130a antisense nucleic acid or miR-130a cavernous body multicopy antisense nucleic acid plasmid can inhibit the expression of the YAP target gene of the Hippo signal channel. This inhibition can be blocked by knock-down VGLL 4.

Example 2 was carried out:

The name of the experiment: miR-130a antisense nucleic acid reduces YAP transcriptional activity.

YAP acts as a transcriptional co-activator that binds directly to the target gene promoter and activates target gene expression. This activity can be detected using the level of reporter gene expression driven by the promoter of the target gene. CTGF acts as a target gene of YAP, and a reporter gene of a promoter thereof is a recognized and widely used marker of YAP activity. Therefore, whether the miR-130a can influence the transcriptional activity of the YAP can be judged by detecting the influence of the miR-130a on the reporter gene.

The experimental principle is as follows:

the luciferase reporter gene is a common experimental method for detecting the transcription activity, luciferin can emit fluorescence under the catalysis of luciferase, and the fluorescence intensity detected by a microplate reader represents the strength of the transcription activity.

In HEK293 cells, a plasmid expressing beta-galactosidase is transiently transfected, and continuously expressed beta-galactosidase is used as an internal reference, which can hydrolyze a colorless substrate ONPG to generate a yellow product, and the activity of the beta-galactosidase can be obtained by measuring the absorbance. The YAP protein highly expressed in the Myc-YAP/TEAD/CTGF promoter-luciferase activity detection system acts on a CTGF promoter through the endogenously expressed TEAD protein to start the expression of luciferase, thereby showing a high-intensity fluorescent signal. When the YAP activity is changed, the fluorescence intensity is changed correspondingly.

the experimental method comprises the following steps:

(1) Cell culture: HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum and transfected when the cell confluence reached 40-50%.

(2) Transient transfection treatment: transfer cells to 12-well plates, 2 × 10 per well⁵Cells, half an hour prior to transfection were replaced with fresh medium and transfected with beta-galactosidase expressing plasmid, control or miR-130a antisense nucleic acid and control or VGLL4 small interfering RNA as indicated in the figure. Luciferase activity was detected 48h after transfection. The medium was first removed, 300ul of cell lysate (125mM Tris-H3PO4(pH 7.8),10mM EDTA,10mM DTT, 50% Glycerol, 5% Trition X-100,5mg/ml BSA) was added, lysed thoroughly for five minutes on a shaker, centrifuged in a plate centrifuge to remove cell pellet, 20ul of cell lysate supernatant was mixed with 20ul of luciferase substrate, and luciferase activity was read immediately. In addition, 20ul of cell lysate was mixed with 150ul of beta galactosidase substrate ONPG, incubated at 37 ℃ for 15 minutes in an incubator, and then the absorbance at 420nm was read.

the experimental results are as follows: as shown in fig. 3D. Experimental results show that miR-130a antisense nucleic acid (miR-130a inhibitor) inhibits the transcriptional activity of a Hippo signal pathway YAP, and the CTGF promoter activity is reduced. And when VGLL4 is knocked down, the miR-130a antisense nucleic acid does not play an inhibition role.

Example 3:

The name of the experiment: the miR-130a cavernous body multicopy antisense nucleic acid inhibits the combination of YAP and a promoter region of CTGF of a target gene thereof.

YAP can be combined with transcription factor TEAD in nucleus, and can play the role of co-transcription factor to promote downstream gene expression. The chromatin immunoprecipitation experiment can verify the combination of the transcription regulatory protein and the gene promoter, so that the combination strength of the YAP protein and the CTGF promoter region can be detected by the method to directly reflect the transcription activity of the YAP.

The experimental principle is as follows:

Treatment of cells with a crosslinking agent can cause the DNA and protein bound thereto to be crosslinked together. The DNA is then fragmented by sonication and specific antibodies are used to precipitate the specific proteins, along with the DNA to which they bind. These DNAs can be detected and quantified by PCR reaction after the decrosslinking. This method reflects the sequence and amount of DNA that binds to a particular protein.

The experimental method comprises the following steps:

(1) Cell culture: HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and passaged when the cell density reached 70-80%.

(2) Establishment of stable cells was the same as in step (3) of example 1.

(3) Chromatin immunoprecipitation, 1X10 preparation per sample⁷cells, first crosslinked, were removed from the petri dish, added 37% formaldehyde to a final concentration of 1% in 15ml of broth, incubated for 12min at room temperature on a horizontal shaker, added 2.5M Glycine to a final concentration of 0.125M, and incubated for 5min at room temperature on a horizontal shaker to terminate crosslinking. The cells were then washed twice with pre-cooled PBS and collected in 1.5ml EP tubes with PBS (with the protease inhibitors cocktail and PMSF) and centrifuged at 1000rpm for 3min at 4 ℃. Subsequently, the cells were lysed, the supernatant removed, and the pellet resuspended in 800ul cell lysis buffer (supplemented with protease inhibitors cocktail and PMSF) and incubated on a 4 ℃ shaker for 10 min. And (3) performing nuclear lysis again, centrifuging at the rotation speed of 5000rpm at 4 ℃ for 5min, removing the supernatant, adding 300ul of nuclear lysis solution for resuspension, and standing at 4 ℃ for 20 min. 300ul of dilution buffer (dilution buffer) was added to each EP tube for sonication. Setting ultrasonic instrument (QSonica model Q125)Probe CL-18) power 35%, on 2s, off4s, for a total of 7 minutes. After all samples are subjected to ultrasonic treatment, the samples are set to rotate at 12000rpm and centrifuged at 4 ℃ for 10min, and supernatant is taken and diluted by diluent by 5 times to ensure that the final concentration of SDS is 0.1%. Protein G beads were then measured as 60. mu.l of 50% beads slurry per sample. The supernatant was washed twice with dilution buffer (dilution buffer), 5000g, centrifuged for 1min, and the supernatant was removed in the last step, and dilution buffer (dilution buffer) of the same volume as beads was added. Pre-cleaning the sample: each sample was added to 60ul of beads suspension prepared above and incubated for 1h at 4 degrees rotation. Centrifugation was carried out at 5000g for 1min at 4 ℃ and beads were discarded, the supernatant was taken and incubated overnight (12-16h) with 5ug of antibody, one of which was added control IgG as a control. Adding ssDNA (200ug/ml final), BSA (500ug/ml final) and 40ul of blocked beads suspension into each tube, incubating for 2-4h at four degrees, centrifuging at 3000rpm for 2min, removing supernatant, washing the beads with 1ml of low-salt buffer solution, high-salt buffer solution, Licl buffer solution and TE buffer solution (twice), shaking the shaking table at 4 ℃ for 10min, removing supernatant at last, adding 150ul of eluent into each tube, eluting for 15min, centrifuging at 5000rpm for 3min, transferring to a new EP tube, eluting again, combining the eluates, adding RNaseA (10mg/ml) and 5M NaCl to a final concentration of 0.3M, and incubating at 65 degrees for 4h to relieve crosslinking. Then adding proteinase K, incubating at 42 ℃ for 2h, then performing phenol chloroform extraction or purification column extraction on DNA, and using the obtained DNA as a template to detect gene enrichment by QRT-PCR. CTGF promoter forward primer: GGA GTG GTG CGA AGA GGA TA, respectively; CTGF promoter reverse primer GCC AAT GAG CTG AAT GGA GT.

The experimental results are as follows: as shown in fig. 3E, in contrast to HepG2 cells stably expressing unloaded stably with miR-130a sponge multi-copy antisense nucleic acid plasmid, the binding of TEAD to the CTGF promoter region was not substantially affected and the binding of YAP to the CTGF promoter region was reduced, i.e., miR-130a multi-copy antisense nucleic acid plasmid inhibited the binding of YAP to the promoter region of its target gene CTGF.

Example 4:

The name of the experiment: the miR-130a cavernous body multicopy antisense nucleic acid inhibits the promoting effect of YAP 5SA on cell growth.

YAP 5SA is a mutant insensitive to a Hippo signal channel, has the effect of promoting cell growth, and the miRNA-130a multicopy antisense nucleic acid plasmid can inhibit the activity of YAP, so that the effect and the action mechanism of the miRNA-130a multicopy antisense nucleic acid plasmid on the cell growth can be researched. A commonly used method for detecting Cell viability is to use Cell Titer Blue reagent.

the experimental principle is as follows:

Cell Titer-Blue Cell activity detection Cell activity was detected by a homogeneous, fluorescent method. The principle is that living cells are able to convert a redox dye (Resazurin) into a fluorescent end product (resorufin). Since non-viable cells do not have a capacity for rejuvenating agents because they are unable to generate a fluorescent signal. Homogeneous assays are performed by adding the signal-producing reagent directly to the serum-containing cell culture medium, incubating, and recording the data using a 96-well fluorometer (recommended) or spectrophotometer.

The experimental method comprises the following steps:

(1) cell culture: HepG2 cells were cultured in DMEM medium containing 10% fresh fetal bovine serum.

(2) Establishment of stable cells was the same as in step (3) of example 1.

(3) Cells were transiently transfected as in step (2) of example 1.

(4) and (3) cell viability detection: after digesting the transiently transfected cells or the constructed stable Cell strain, inoculating 1000 cells per well into a 96-well plate, setting 0 (Cell adherence stable), 6 detection time points of 1, 3, 5, 7 and 9 days, setting two multiple wells for each time point, and adding a Cell Titer-Blue reagent for activity detection. The method comprises the steps of directly adding 20ul of Cell Titer-Blue Cell activity detection reagent of Promega company into a Cell culture medium of 96 holes to be detected at each detection point, incubating for 4 hours in an incubator at 37 ℃ in a dark place, taking out 100ul of the culture medium after reaction to a non-transparent 96-well plate, setting 540 exciting light/590 emitting light, and reading fluorescence intensity as Cell activity data.

The experimental results are as follows: as shown in fig. 4A, B. In HepG2 cells, YAP 5SA can be promoted by stable overexpression, and the miR-130a co-expression multi-copy antisense nucleic acid plasmid can inhibit the increase of the cell growth rate to a certain extent. The independent transient expression of the miR-130a antisense nucleic acid can inhibit cell proliferation, and the knocking-down of VGLL4 restores the growth rate of the cells on the basis.

example 5:

The name of the experiment: the miR-130a antisense nucleic acid can promote apoptosis caused by cell suspension.

the anti-cell suspension apoptosis experiment can reflect the survival ability of cancer cells in the process of metastasis, and some cancer cells have stronger anti-suspension apoptosis ability. It is known that YAP can obviously promote the survival of cells in suspension culture, and miR-130a antisense nucleic acid can inhibit the activity of YAP and should inhibit the survival of cells in suspension culture.

The experimental principle is as follows:

Apoptosis plays an important role in embryonic development, tissue repair and the like of organisms. Uncontrolled regulation of apoptosis can lead to a variety of clinical conditions. Apoptosis has a series of changes in cell morphology and biochemistry including the appearance of chromatin condensation, DNA degradation, apoptotic body formation, and the like. In normal living cells, Phosphatidylserine (PS) is located inside the cell membrane, but in apoptotic cells, PS flips from the inside of the cell membrane to the surface of the cell membrane, exposing the extracellular environment. Annexin-V is a Ca2+ dependent phospholipid binding protein with molecular weight of 35-36KD, and can be combined with PS with high affinity. Therefore, Annexin-V is labeled by fluorescein (such as FITC and PE) or Biotin (Biotin), and the labeled Annexin-V is used as a probe to detect the occurrence of apoptosis by using a flow cytometer or a fluorescence microscope. Normal epithelial cells undergo apoptosis in a suspension culture state, while cancer cells with metastatic ability have a strong ability to resist apoptosis.

The experimental method comprises the following steps:

(1) cell treatment: after cell counting, according to the growth rate and characteristics of HepG2 cells, planting a proper amount of the cells in a six-well plate, changing a fresh culture medium when the cell density reaches 60-70%, transfecting 20nM control or miR-130a antisense nucleic acid in each well by using an RNAiMax transfection reagent, culturing the two treated cells into the six-well plate with ultra-low adhesiveness for culturing 48H after 24H of transfection, and taking the consistent cell amount in each well.

(2) Harvesting machineCell collection: digesting the cells with pancreatin, centrifuging at 1000rpm and 4 deg.C for 5min after digestion is terminated, collecting cell precipitate, discarding supernatant, and adjusting cell concentration to 2X10⁶And/ml, adding 200ul of the suspension into 1ml of precooled PBS, shaking to suspend the cells, centrifuging at 1000rpm and 4 ℃ for 3 minutes, discarding the supernatant, repeatedly adding PBS for washing and discarding the supernatant.

(3) Cell staining: adding 300ul of labeling buffer solution into each cell sediment sample, re-suspending, adding 5ul of Annexin V/FITC dye probe, mixing gently, incubating for 15 minutes at room temperature in the dark, immediately detecting on a machine (flow cytometer), and paying attention to setting a blank control and a negative control without the staining probe.

The experimental results are as follows: as shown in FIG. 4C, a significant portion of the control HepG2 cells were not apoptotic in suspension culture (46%), whereas the apoptosis rate was significantly increased after transfection of the miR-130a antisense nucleic acid (73.9%).

Example 6

The name of the experiment: the miR-130a cavernous body multicopy antisense nucleic acid adenovirus inhibits MST1/2 conditional knockout-induced hepatocyte proliferation, liver enlargement and YAP target gene expression.

MST conditional knockouts

The experimental principle is as follows: mst1and Mst2 are two homologous protein kinases, core proteins in the Hippo signaling pathway that inhibit YAP. The MST1/2 knockout can activate YAP activity and promote the expression of downstream target genes, thereby enlarging the liver. This physiological function is dependent on YAP activity. The miR-130a cavernous body multicopy antisense nucleic acid adenovirus is injected on the basis of MST1/2 condition knockout, and the effect of miR-130a inhibition on tissue growth under in vivo physiological conditions can be further proved by observing hepatocyte proliferation condition, liver size and YAP target gene expression.

The experimental method comprises the following steps:

Mst1/2 conditional knockout mice have been reported (Song H, Mak KK, Topol L, Yun K, Hu J, Garrett L, et al.Mammarian Mst1and Mst2kinase plant entry roles in organisiza control and tumor deletion. Proc Natl Acad Sci U2010, 107(4): 1431-. Mice with homozygous conditional knockouts of Mst2 and Mst1 knockouts or wild-type genotypes were used. By means of mouse tail silencePulse injection of 5X10⁸Adenovirus with Cre recombinase and 5X10⁹Control or adenovirus expressing miR-130a cavernous body multicopy antisense nucleic acid. After 9 days, the mice were sacrificed and livers were removed for analysis, including liver to weight ratio analysis, immunofluorescent staining analysis of frozen sections and extraction of RNA or total protein for gene and protein expression analysis.

The experimental results are as follows: as shown in FIG. 4D, E, F, MST1/2 conditional knockdown resulted in increased liver specific gravity, whereas miR-130a cavernous multicopy antisense nucleic acid adenovirus reduced liver body-weight ratio after its action in MST conditional knockdown mice. Ki67 staining results show that the proliferation marker Ki67 of miR-130a multicopy antisense nucleic acid adenovirus injection group is normal compared with a control group. And fluorescence quantitative PCR and immunoblotting results prove that the miR-130a multicopy antisense nucleic acid adenovirus injection group reduces the expression of YAP target genes and increases the expression of VGLL4 protein.

example 7:

The name of the experiment: the miR-130a antisense nucleic acid inhibits cell anchorage-independent growth caused by YAP 5 SA.

YAP 5SA overexpression promotes cancer transformation of cells, enables the cells to obtain the capability of anchorage independent growth, and can form clones on soft agar. Studying the effect of miR-130a antisense nucleic acid under this condition can show the ability to inhibit cellular cancer transformation.

The experimental principle is as follows:

Anchorage-independent growth is a prominent feature of cancer cells. Normal cells cannot grow in soft agar, while cancer cells acquire the ability to grow in soft agar, thereby forming macroscopic colonies. The clonality rate reflects the degree of cancer transformation of the cell population.

The experimental method comprises the following steps:

(2) Establishment of stable cells was the same as in step (3) of example 1.

(3) Cells were transiently transfected as in step (2) of example 1.

(4) Clone formation experiments: the medium was heated to 37 ℃ and 42 ℃ before use, and 3% agarose was prepared in sterile PBS, autoclaved and placed at 60 ℃ until use. 3% agarose was diluted to 0.6% concentration with medium and 2ml was added to each well of a six-well plate to allow it to solidify. Digesting cells, preparing a cell density of 25000/ml by using a culture medium, diluting agarose to 0.5% by using the culture medium, mixing 1ml of cell suspension per 2ml, uniformly mixing 3ml of the cell suspension, planting the cell suspension in each hole, setting multiple holes, setting the final concentration of the upper layer glue to be 0.33%, adding 1ml of the culture medium after the upper layer glue is solidified, placing the cell suspension in a cell culture box for culture, observing the clone formation condition after 20 days, photographing and counting by using crystal violet staining.

The experimental results are as follows: as shown in FIG. 5A, HepG2 itself could not form cell clone, but has strong clone forming ability after YAP 5SA is expressed, the clone number is obviously increased, and on the basis, the clone forming ability of transient transfection miR-130a antisense nucleic acid YAP 5SA is inhibited to a certain extent.

Example 8:

Name: the miR-130a cavernous body multicopy antisense nucleic acid inhibits YAP S127A-induced tumor formation.

the nude mouse subcutaneous tumor formation experiment can verify the tumor formation ability of the tumor cells in an in vivo environment, and YAP-S127A overexpression can promote cell tumor formation. On the basis, the effect of miR-130a multicopy antisense nucleic acid can reflect the effect of inhibition of miR-130a on YAP-induced tumor formation.

The experimental principle is as follows:

Since most tumor studies use human cells, due to the presence of xeno-rejection, immunodeficient mice are required as carriers for transplantation tumor models, and the biological changes are determined by injecting tumor cells into the mice, making them tumor, and observing the growth of fluid.

The experimental method comprises the following steps:

(2) Establishment of stable cells was the same as in step (3) of example 1.

(3) subcutaneous tumorigenesis of nude mice: after expansion of the cells, the cells were collected by digestion and placed at 1X10 in PBS⁷per ml cell suspensionNude mice were injected with a certain amount of cells, each nude mouse leg was divided into left and right injection points, and 200ul of cell suspension was injected at each point. And (4) feeding the nude mice until the tumor bodies can be seen by naked eyes (three weeks), taking the tumors, measuring the sizes of the tumor bodies, weighing, photographing and recording the processing results.

The experimental results are as follows: as shown in FIG. 5B and C, after the miR-130a cavernous body multicopy antisense nucleic acid plasmid and YAP-S127A are co-expressed, the size and weight of a tumor are obviously changed in a control YAP-S127A single overexpression group, and the tumor volume and weight of the co-expression group are small and light, which indicates that the miR-130a multicopy antisense nucleic acid plasmid can inhibit the tumorigenic capacity of YAP to a certain extent.

Claims

Application of miR-130a antisense nucleic acid in preparation of Hippo-YAP signal pathway inhibitor.
2. The use of claim 1, wherein the miR-130a antisense nucleic acid inhibits the activity of YAP or TAZ, inhibits the expression of Hippo-YAP signaling pathway target genes, and inhibits tumor cell proliferation and metastasis.
3. The use of claim 2, wherein the miR-130a antisense nucleic acid inhibits the activity of YAP or TAZ by increasing VGLL4 protein levels, inhibiting YAP or TAZ binding to TEAD family transcription factors.
4. The use of claim 1, wherein the miR-130a antisense nucleic acid is the sequence of SEQ NO1, SEQ NO 1: 5' AUGCCCUUUUAACAUUGCACUG3’。
5. The use of any one of claims 1 to 4 wherein the inhibitor of the Hippo-YAP signaling pathway is used to treat or prevent an overgrowth disease associated with YAP or TAZ activation.
6. The use according to claim 5 wherein the overgrowth diseases associated with YAP or TAZ activation are selected from neurofibromatosis type 2, uveal melanoma, liver cancer, intestinal cancer, breast cancer, esophageal cancer, stomach cancer, prostate cancer, ovarian cancer, oral squamous cell carcinoma, brain cancer, pancreatic cancer or lung cancer.
The application of the derivative of the miR-130a antisense nucleic acid in preparing a Hippo-YAP signal pathway inhibitor is characterized in that the miR-130a antisense nucleic acid derivative is a nucleic acid or a nucleic acid derivative fragment with the following characteristics: (1) binds to miR-130a by means of base pairing; (2) the base pairing pattern is base pairing of the underlined part of SEQ NO 1and base pairing of the non-underlined part; or the like, or, alternatively,

The miR-130a antisense nucleic acid derivative is a nucleic acid segment or a peptide nucleic acid segment with chemical modification.
8. The use of claim 7 wherein the Hippo-YAP signaling pathway inhibitor is used to treat or prevent an overgrowth disease associated with YAP or TAZ activation.
9. The use according to claim 8 wherein the overgrowth diseases associated with YAP or TAZ activation are selected from neurofibromatosis type 2, uveal melanoma, liver cancer, intestinal cancer, breast cancer, esophageal cancer, stomach cancer, prostate cancer, ovarian cancer, oral squamous cell carcinoma, brain cancer, pancreatic cancer or lung cancer.