Effect Of Silencing Protease-activated Receptor2on Cell Proliferation Invasion And Metastasis Of Hepatocellular Carcinoma SMMC-7721cells

Objective: The hepatocellular carcinoma (HCC) is one of most commonmalignant tumors in our country, which leads to high mortality rate andrecurrence rate after surgery or involvement treatment; however thepharmacological treatment is not effective. High risk of metastasis is theessential reason that causes HCC's bad prognosis. Therefore, selecting theinvasion and metastasis pathway of HCC and identifying key genes in thisprocess will be beneficial to provide potent prognostic biomarker andmolecular targeted drugs against HCC. Proteinase-actived receptors2(PAR-2)is a G protein-coupled seven-trans-membrane-domain receptor, the trypsin andtryptase are natural agonists of this receptor, artificial synthesis of smallmolecule polypeptide just like SLIGKV-NH2can also activate this receptor.Our previous research demonstrated that PAR-2express in hepatocarcinomacell line HepG2, SMMC-7721. PAR-2can be activated by trypsin andSLIGKV-NH2in HepG2, thereby promoting the proliferation and induce DNAsynthesis and cell division of HepG2cells. RNA interference is a periodof double-stranded small RNA molecules into cells, so that thosewith the homologous sequence of the gene expression was inhibited. RNAitechnology has been increasingly used to study cancer risk factors and toexplore its effective gene effective gene therapy. RNAi technology to inhibitexpression of oncogenes and tumor suppressor genes in tumor cells, toanalyze the phenotypic changes, which will help reveal the molecularmechanisms of tumor development, signs and therapeutic targets for tumordiagnosis and treatment.Purpose: The purpose of this test in vitro culture of human hepatomaSMMC-7721cells and the establishment of PAR-2gene silencing in stable cell lines,PAR-2short hairpin RNAs (short hairpin RNA shRNA) in humanhepatocellular carcinoma SMMC-7721cell proliferation andinhibition of migration.Methods:1Three PAR-2shRNAs were designed and synthesized in vitro and inserted toexpression vector pGFP-V-RS(shRNA-PAR-2-1shRNA-PAR-2-2shRNA-PAR-2-3),and transfected into SMMC-7721cells and the cells stablyexpressing the shRNA were selected by puromycin．The inhibitory effect ofshRNA on PAR-2gene expression was measured by Realtime PCR to selectthe best shRNA．And detected the efficiency of PAR-2gene inte-rference.2The proliferation and cycle of shRNA-PAR-2SMMC-7721cells wasexamined by Flow cytometry and MTT assay.3The adhesive potential was determined by cell adhesion assay on matrigel.4The invasion and migration capability were examined by Transwell chamberwith or without matrigel.Results:1The plasmid expression vectors were checked by enzyme digestion andsequencing, and they were successfully constructed. shRNA-PAR-2-1couldsignificantly knock down the PAR-2genes expression of SMMC-7721cellscompared to the control group. Real-time PCR result showed that theknockdown effect of PAR-2mRNA expression level decreased about99%ofshRNA-PAR-2-1. shRNA-PAR-2-2group and shRNA-PAR-2-3group ofPAR-2mRNA expression level decreased about90%than the normalcell group. Out-of-order group PAR-2-mRNA expression level no decreasedthan the normal cell group.2Flow cytometry showed S phase arresting after transfecting withshRNA-PAR-2-1SMMC-7721cells.The cells of S phase in the control groupwith24,48, and72-hour was (17.7±2.91)%,(16.8±2.48)%,(18.2±2.86)%.The cells of S phase in the transfected group was (30.60±2.73)%,(20.2±1.54)%and (30.5±2.85)%. 3The adhesion of transfected group of SMMC-7721cells was significantlyreduced detected by adhesion assay.4The cell invasion and migration changes examined by Trans-well chamber assay. In the invasion experiments, ShRNA-PAR-2-1decreasedthe number of SMMC-7721cells that damage matrigel through the Polyca-rbonate membrane than that of control groups. Statistically, the difference issignificant. In the migration experiments, ShRNA-PAR-2-1significantlydecrease the number of SMMC-7721cells resulted from deformation ofmovement through the Polycarbonate membrane than the control groups.5The proliferation of shRNA-PAR-2-1SMMC-7721cell assayed by MTT.It showed silenced of PAR-2can inhibit the proliferation of SMMC-7721cells than the control group, transfection reagent and negative plasmid transf-ected SMMC-7721cell growth inhibition was not obvious.Conclusions:1Successfully constructed a targeting PAR-2gene silenced plasmidexpression vector and shRNA-PAR-2-1can effectively decreased the PAR-2mRNA expression.2shRNA-PAR-2-1SMMC-7721cells arrested in S phase, therebyaffecting cell proliferation.3The invasion and migration was decreased of shRNA-PAR-2-1SMMC-7721cells.4The adhesion was decreased of shRNA-PAR-2-1SMMC-7721cells.5The cell proliferation rate slows down of shRNA-PAR-2-1SMMC-7721.