| Hepatocellular carcinoma(HCC), one of the most deadly malignancies, is the most common primary liver cancer with higher mortality in the world. Each year there are more than 700,000 new cases and about 600,000 people died of it, and more than 50% of them occur in China. Despite of the implementation of screening programs for high-risk individuals, the majority of patients cannot be cured, and even for the terminal-cancer patients, the median overall survival span is less than 12 month. Since there are less typical clinical symptoms, it is difficult to make the early diagnosis and treatment of hepatocellular carcinoma. At the same time, hepatocellular carcinoma is of higher invasiveness, and the metastatic hepatic carcinoma is almost resistant to all the conventional chemotherapy drugs, so the survival time has been affected seriously. Lung is the part of the extra hepatic metastasis transferred from hepatocellular carcinoma, but it is still unclear for the causes and mechanism.Therefore, it will improve the treatment of hepatocellular carcinoma to find out the early metastasis markers of hepatocellular carcinoma and make research on the potential molecular mechanisms of metastasis.The Micro RNAs(mi RNAs) is a type of noncoding single-stranded RNA of the nucleotides with the length from 18 to 25, and now more than 2,500 mi RNAs are detected in plants, animals and viruses. After it is generated in cell nucleus, the Micro RNA is transferred to endochylema via nucleus. Then it is guided into RNA-induced silencing complex, and complement and base pair with target gene m RNA to suppress the degradation and translation of the target gene m RNA. The research in the past decades has showed that the micro RNA plays an important role intumor occurrence, tumor development and transfer process. Recently, there are more research on the role of mi R-195 in tumor occurrence and cancer therapy. Some research reports have proved that mi R-195 can greatly inhibit lung cancer from proliferating, migrating and invading by targeting myb. In colorectal cancer, mi R-195 represses cell proliferation, colony formation and invasion through targeting CARMA3. In recent studies, several target genes of mi R-195 associated with HCC have been identified, including Wnt3 a, Pcmt1, VAV2, CDC42 and VEGF.Angiogenesis refers to the development of new blood vessels coming from the existing blood capillaries or the veins behind the blood capillaries and it is a complicated and essential process for the growth, invasion, and metastasis of various malignant cancers. FGF2 and VEGFA, as the most important angiogenic factors,have become important therapeutic targets for the abnormal angiogenesis diseases like tumors, etc. FGF2 plays an important role in regulating angiogenesis, cell differentiation and cell migration, and has abnormal expression in many tumors such as melanoma, nasopharyngeal carcinoma and prostate cancer. VEGF can stimulate the proliferation of endothelial cells of the blood vessels, increase the vascular permeability, and promote the migration of cells. And it is overexpressed in breast cancer, lung cancer, esophageal cancer, stomach cancer and colorectal cancer. It is reported in some research that FGF2 and VEGFA are significantly elevated in HCC tissues. However, the mechanism is still unknown.In this experiment, it is found that mi R-195 is the mi RNA associated closely with HCC of lung metastases by analyzing mi RNA chips of pulmonary metastases cell lines. 92 cases of the expression of mi R-195 in the HCC tissues have been tested by Real-time PCR. The downstream target genes of mi R-195 have been analyzed by mircro RNA target gene prediction software, and the angiogenesis-related genes have been cross-over analyzed, then the mi R-195 target genes related to the angiogenesis have been identified. Then the gene experiment by means of the luciferase activity and enzyme-linked immunosorbent assay have been used to validate this hypothesis.Moreover, the technique of lentivirus infection has been adopted to build the mi R-195 stable transfection cell line and the Transwell assay is done to examine the effect of mi R-195 on cell migration and invasion ability. In conclusion, the findings in thispaper have replenished the comprehension of HCC metastasis and offered a new direction for the HCC therapy.Part One Screen the invasion and metastasis related mi RNAs inHCC and analyze the expression level and clinical significance ofmi R-195Methods1. To explore differentially expressed mi RNAs between lung metastasis HCC celllines and primary HCC cell lines by analyzing HCC mi RNA profile.2. Detecting the expression of mi R-195 in HCC tissues by real-time PCR.3. Analyzing the clinical significance of mi R-195 in HCC by statistical analysis.Results1. We identify 20 downregulated mi RNAs and 4 upregulated micro RNAs in lungmetastasis HCC cell lines compared with primary HCC cell lines.2. The mi R-195 has a low expression in HCC tumor tissues.3. The expression of mi R-195 is correlated with tumor size, portal vein, TNMstage and the 5-year overall survival of patients.1. Screen the potential target genes of mir-195 which is involved in angiogenesisby bioinformatics analysis.2. Predict the binding sites of mir-195 and the 3'UTR of FGF2 and VEGFA bybioinformatics analysis.3. To conform whether FGF2 and VEGFA are the target genes of mi R-195,luciferase reporter assay is performed.4. Constructing mi R-195 overexpressing cell lines by lentivirus infection.5. To confirm the regulation of FGF2 and VEGFA by mi R-195, ELISA assay isperformed.6. To investigate the function of mi R-195 in HCC, transwell assay is performed.Results1. Bioinformatic analysis confirms FGF2 and VEGFA are the target genes ofmi R-195.2. Luciferase report assay Conform that FGF2 and VEGFA are the target genes ofmi R-195.3. The construction of mi R-195 overexpressing cell lines is successful.4. Confirming the regulation of FGF2 and VEGFA by mi R-195 by ELISA assay.5. mi R-195 can inhibit the migration and invasion of BEL-7402 cell lines.Conclusions1. The expression of mi R-195 is significantly downregulated in HCC lungmetastases cell lines and HCC tissues.2. The expression of mi R-195 in HCC tissues is correlated with tumor size, portalvein, TNM stage and the 5-year overall survival, indicating that mi R-195canserve as a potential prognosis marker of HCC patients.3. mi R-195 can inhibit the migration and invasion of BEL-7402 cell lines bytargeting FGF2 and VEGFA.Part Two The molecular mechanisms of mir-195 in inhibiting theinvasion and metastasis of HCC Methods... |
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