Suppressive Effect Of Hsa-miR-486-5p On The Proliferation And Invision Of Hepatocellular Carcinoma Cells And Its Mechanisms

Hepatocellular carcinoma (HCC) is the one type of malignant tumors with highmorbidity and mortality. Although the surgery and liver transplantation are ideal treatmentstrategies for HCC, their indications are very limited. Regular chemotherapy and targetedtherapy can kill HCC cells effectively, but there are also the side effects and drug resistance.Therefore, it is an urgent medical topic that further clarying the HCC mechanism anddeveloping of early diagnosis and new therapeutic drugs for HCC.MicroRNAs, involving post-transcriptional regulation, play a certain role in thepathophysiology of the process, including cancer. Hsa-miR-486-5p was first cloned fromthe fetal liver, but its role in hepatocellular carcinoma (HCC) has not been elucidated. Thisproject will clarify of the role of hsa-miR-486-5p in HCC and explore its mechanism, whichmay be helpful to develop new targets of HCC early diagnosis and therapy and provide theexperimental basis for further study.In this project, we firstly detected the expression of hsa-miR-486-5p and its host gene,Ank1, as well as other related microRNAs in HCC cell lines, tissues and serum. Secondly,we have constructed and packaged of several lentivirus vectors carrying hsa-miR-486-5pand other microRNA respectively; The HCC cell lines were transduced with lentivirusvectors and their proliferation, apoptosis, cycle, migration were detected in vitro; the rolesof hsa-miR-486-5p in HCC proliferation, invasion and metastasis in vivo were determined.Finally, we constructed and identificated the luciferase reporter vectors with target gene3'UTR and validated the target genes of hsa-miR-486-5p by dual luciferase activity report.To confirm the roles of hsa-miR-486-5p target genes in HCC proliferation and invasion, wedesigned and synthesised of target gene siRNA (knockdown target genes) to simulate ofhsa-miR-486-5p inhibition of target genes. We detected the role of the target gene siRNA onHCC cell lines and clearified the mechanism of hsa-miR-486-5p in HCC.The methods and results in this project were presentated according to the contentsabove which devided into3parts.1. MicroRNA expression by HCC cells.We detected the microRNAs expression profiles in different cell lines with microRNAarray chip; detected the has-miR-486-5p expression in hepatoma cell lines HepG2,SMMC-7721,97L,97H and LM3; detected the has-miR-486-5p expression in the cancertissues and adjacent tissues of HCC patients; detected the hsa-miR-486-5p expression in theserum of HCC patients and medical physical examination; detected the expression ofhsa-miR-486-5p host gene Ank1in the cancer tissues and adjacent tissues of HCC patients.MicroRNAarray chips suggest that there are84microRNAs including hsa-miR-486-5p,miR-106b-93-25, etc which expression higher more than2times LM3vs SMMC-7721cells. Hsa-miR-486-5p expression is significantly decreased in hepatoma cell lines HepG2andSMMC-7721,97L,97H, LM3; hsa-miR-106b-93-25, SMMC-7721,97L,97H, LM3celllines differentially expressed; hsa-miR-155expression in several hepatoma cell lines therewas no difference. Hsa-miR-486-5p expression in the majority of HCC cancerous tissues isdownregulated and hsa-miR-486-5p host gene the Ank1also decreased. There is no obviousrule of hsa-miR-106b-93-25, of hsa-miR-17-92and hsa-miR-155expression in HCC cancertissues and corresponding to the adjacent tissues. The expression of hsa-miR-486-5p ofHCC serum is lower than the level of expression of the serum of normal healthy volunteers.2. Hsa-miR-486-5p inhibits the proliferation and invision of HCC cells.We constructed and identificated of pHIV7lentiviral vectors using molecular cloning;packaged and purificated lentivirus using the second-generation lentiviral packaging system;determinated the lentiviral titer using flow cytometry; obtained the stable over expressionand low expression hsa-miR-486-5p in hepatoma cell lines by lentiviral infection and flowcytometry sorting. Then, we detected the proliferation of HCC cells infected withhsa-miR-486-5p and other microRNAs by the Dye670and CCK-8; detected the role ofapoptosis of hsa-miR-486-5p on the HCC cells by Annexin V and PI double labeling;detected the role of hsa-miR-486-5p and other microRNAs in HCC cell migration byscratching and transwell migration assay; detected the epidermis marker changes of HCCcells infected with hsa-miR-486-5p by Western blot and immunofluorescence confocalassay; detected the role of hsa-miR-486-5p in HCC in vivo with nude mice tumor-bearinganimal models and pathological analysis.Hsa-miR-486-5p and the corresponding control vector lentiviral titer is5×107pfu/ml;overexpression hsa-miR-486-5p inhibits the proliferation of HCC cells confirmed withDye670and CCK-8assay. The colony formation assay confirmed that hsa-miR-486-5p caninhibit HCC cell colony formation. Hsa-miR-486-5p can increase the sensitivity of thechemotherapy drug sorafenib to induce HCC cell apoptosis by application Annexin V andPI double labeling. The scratch test confirmed that hsa-miR-486-5p can inhibit the healingof the HCC cells scratches. Transwell migration assay confirmed that hsa-miR-486-5p invitro can inhibit HCC cell migration. Western blot and immunofluorescence confocal assaysuggested that hsa-miR-486-5p can increase the expression of epithelial marker such asE-cadherin, decrease the expression of mesenchymal marker such as N-cadherin. Nudemice tumor models and pathological analysis of hsa-miR-486-5p in vivo suggested thathsa-miR-486-5p can retardate LM3cells into the tumor; however, inhibition of tumorgrowth was not statistically significant and inhibition of tumor metastasis is not obvious.3. Identification of hsa-miR-486-5p target genes and their roles in proliferationand invasion of HCC cells.We use protein chip to screen of target genes of hsa-miR-486-5p; bioinformaticsanalysis to predicte the target genes according to the biology of HCC cells; use of molecularcloning methods to construct the target gene3'UTR luciferase reporter and the dual luciferase reporters gene assay to screen of target genes of hsa-miR-486-5p; detect the roleof target genes CITRON(CIT) and CLDN10on the biological characteristics of HCC usingsiRNA interference.The protein chip testing results intersecting with predicted target genes show that thedifferential genes are IR10RA,STAT6,APP,GRIP1,ESR2,CXADR,etc. Targetscanforecasting website predictes CIT, CLDN10, TWF1, AFF3, PLAGL2, SRF, DCBLD2andother target genes. Then we successfully constructed the target gene3'UTR luciferasereporter vectors; predicted target genes of CIT, CLDN10, TWF1and AFF3by the dualluciferase reporter experiments. Finally, we chose the CIT, CLDN10by literature andmRNA level. We confirmed that siRNA-CIT can decrease the proliferation of HCC cellsusing small RNA interference (analog of hsa-miR-486-5p the role); confirmed thatsiRNA-CLDN10can decrease HCC cell migration capacity using small RNA interference(simulation of hsa-miR-486-5p the role).In a conclusion, hsa-miR-486-5p is downregulated in HCC. Hsa-miR-486-5p inhibitsHCC proliferation and migration. Hsa-miR-486-5p maybe a novel HCC tumor suppressorgene and its target genes are CIT and CLDN10. In this project, we illustrated thepathogenesis of HCC more in-depth, and a new function of hsa-miR-486-5p.