Study On The Role Of MiR-10b In Human Hepatocellular Carcinoma And Its Mechanism

【Backgrounds】 The occurrence of hepatocellular carcinoma(HCC) is a complex process, and it is difficult for early diagnosis. Moreover, it progresses rapidly, and the mortality is high. Thus, it is very important to clarify the pathogenesis of HCC. Chromosomal aberrations and gene abnormalities have become an important fields for cancer research. The imbalance of oncogenes and tumor suppressor genes is a key role in tumorigenesis. Therefore, some researches are trying to find new diagnostic markers and targets to explore the mechanism of invasion and metastasis in HCC. Micro RNA(mi RNA) is a class of non-coding small RNAs with 17-25 nucleotides, and inhibits gene expression at a posttranscriptional level. It regulates gene expression by binding with specific sites at the 3’-untranslated region(UTR) of target messenger RNA(m RNA), and inhibits the expression of gene either by decreasing m RNA or by degradating m RNA. mi RNA is expressed in a tissue-specific. Many studies have been reported about the relationship between mi RNA and HCC, and some mi RNA associated with the occurrence, development, invasion and migration of HCC have been demonstrated. Based on previous mi RNA array expression profiles and English literatures, we noticed mi R-10 b was up-regulated in HCC. However, the expression alteration in hepatocarcinogenesis have not been further studied. mi R-10 b is located in HOX gene cluster on chromosome 2, suggesting that it is closely related to tumor invasion and metastasis. According to the biological message software, we found a possible target gene CSMD1, which may be regulated by mi R-10 b. CSMD1 consists of 14 CUB domains and 28 Sushi domains. A single transmembrane helix is predicted between aa 3487–3509. An intracytoplasmic domain predicted between aa 3510 –3564 contains a likely phosphorylation site at tyrosine 3539 suggesting that the CSMD1 protein may be involved in signal transduction mechanisms. Studies have shown that CSMD1 is a new candidate tumor suppressor gene. The above views make us recgonize that there may be a certain relationship between the mi R-10 b and CSMD1 in HCC.【Aims】 To observe the expression level of mi R-10 b in HCC, and to elucidate the relationship between mi R-10 b and CSMD1 in HCC, and to provide a new candidate gene and target for the diagnosis and treatment of HCC.【Methods】 1. We observed the expression level of mi R-10 b in human HCC by q RT-PCR. 2. We observed the expression levels of mi R-10 b in HCC cell lines by q RT-PCR. Then, we examined the function of mi R-10 b in cell line by MTT assay, wound scratch assay, transwell assay, colony formation assay, soft agar colony formation assay and flow cytometry analysis. 3. We established animal HCC models, and observed the impact of agomir-10 b on HCCgrowth. 4. We confirmed the negative regulated relationship between mi R-10 b and CSMD1 by Dual-Fluorescence Report assay. And then, we observed the influence of overexpression of mi R-10 b on CSMD1 m RNA and CSMD1 protein by RT-q PCR, immunocytochemistry, respectively. 5. Finally, we observed the expression level of CSMD1 in human HCC by immunohistochemistry. Then, we examined the function of CSMD1 in cell line by MTT assay and transwell assay.【Results】 1. The expression level of mi R-10 b was up-regulated in HCC(-1.4590± 0.69542 vs.-1.7312 ± 0.62758, p < 0.01) and high expression of mi R-10 b was associated with age of patients and HBs Ag positivity. 2. The cellular function experiment demonstrated that overexpression of mi R-10 b promoted cell proliferation, migration, invasion and repressed cell apoptosis. 3. The animal experiment results demonstrated that HCC growth was obviously fast after agomir-10 b was injected in HCC tissues. 4. Dual-Fluorescence Report assay demonstrated mi R-10 b regulated CSMD1. In addition, the expression level of CSMD1 m RNA and CSMD1 protein were down-regulated after mi R-10 b was overexpressed. 5. The cellular function experiment demonstrated that silence of CSMD1 promoted cell proliferation, migration and invasion. CSMD1 expression was lower in HCC than normal counterparts.【Conclusions】 1. mi R-10 b was overexpressed in HCC tissues compared with their normal counterparts. Indeed, the level of mi R-10 b was positively correlated with age and HBV infection, suggesting that mi R-10 b may be associated with HCC. 2. mi R-10 b promoted cell proliferation, migration, invasion and repressed cell apoptosis, and mi R-10 b in vivo environment promoted tumor growth,indicated that mi R-10 b behaveas oncogene in HCC. 3. mi R-10 b can target CSMD1 directly by post-transcriptional regulation, indicated that CSMD1 behave as tumor suppressor gene in HCC. To sum up, the study first found that mi R-10 b and HCC were related, and the mechanism may be related to the negative regulation of CSMD1 gene expression. The results provided new ideas for the study of HCC etiology and molecular genetics, and provided a new potential molecular marker for the diagnosis of HCC.