Study The Effects Of OSW-1on MicroRNA-122Act On Gene Expression Of Hepatocellular Cell And WNT Signal Pathway

Objective:①To study the effect of microRNA-122on Hepatocellular Carcinoma Cell(HCC), build Human Hepatocellular Carcinoma Cell Models which can overexpress the microRNA-122.②To study the effect of OSW-1on microRNA’s processing and function in HCC.③To study the effect of OSW-1on Hepatocellular Carcinoma Cell Models which can overexpress the microRNA-122that is called Hep3B-miR-122by apoptosis, proliferation, Gene Expression Profile Analysis, Signal Pathway Analysis④To research on antitumor mechanism and biological functions of OSW-1and microRNA-122to determine the target gene.Methods:①Construct a recombinant pSIREN-miR-122plasmid vector that include hsa-mir-122’s subsequence successfully in vitro to transfect Hep3B. Select the monoclonal cell lines can overexpress the microRNA-122by qRT-PCR and Dual-Luciferase Reporter Assay System.②Hep3B is divided into four groups:(A) negative control group;(B) OSW-1group;(C) doxorubicin group;(D) OSW-1&doxorubicin group. Operate microarray analysis of microRNA expression.③Hep3B-miR-122is divided into two groups:(A) negative control group;(B) OSW-1group. Operate microarray analysis of human gene expression by GO Analysis and Pathway Analysis.④Cell growth curve of Hep3B and Hep3B-miR-122are detected with WST-8to determine the Proliferation rate and a repeated test demonstrate the effect of OSW-1on the two cell lines.⑤Divid the Hep3B and Hep3B-miR-122into four groups:(A) Hep3B group;(B) Hep3B-OSW1group;(C) Hep3B-miR-122group;(D) Hep3B-miR-122-OSW1group. Observe the morphologic changes by transmission electron microscope and laser confocal microscope.Results:①The recombinant pSIREN-miR-122plasmid vector was constructed in vitro. Hep3B-miR-122with stable microRNA-122overexpression are obtained successfully.②Hsa-mir-299-5p, has-miR-1908, has-miR-125b-1-3p, has-miR-187a-5p, has-miR-1275, hav1-miR-H6-3p, ebv-miR-BHRF1-2, has-miR-181a-5p, has-miR-210, has-mir-483-3P were up-regulated and hsa-miR-126-3p�'�hsa-miR-208a were down-regulated after OSW-1inducing. Has-miR-142-3p、miR-200c-3p were up-regulated by OSW-1with doxorubicin’s assistance. These microRNAs have a close contact with the proliferation, apoptosis, invasiveness and metastatic capacity of turner.③OSW-1could effect the gene expression on MAPK、WNT、Hedegehog、AXON、VEGF、FOCAL、Adherens Junction、P53signal pathway including oncogene and anti-oncogene such as Ras, c-JUN, c-Myc, CyclinD, Bcl-2, CytC, Caspase-3,8,9and so on.④The Cell growth curves were obviously different between the Hep3B group and Hep3B-miR-122group (P<0.01). MicroRNA and OSW-1down-regulated the curves in two groups.⑤Hep3B-miR-122showed high nuclear cytoplasmic and largened mitochondria which made the intact structure of mitochondrial crest was not seen. OSW-1made mitochondria pyknosis or vacuole accompanied with cell.Conclusion:①OSW-1regulated the express of several microRNAs which had relationship with proliferation, apoptosis, cellular adhesion, invasion and so on.②OSW-1suppressed proliferation and metastasis with promoting apoptosis in Hep3B and microRNA-122had better’promoting effect.③microRNA-122could inhibit Hep3B’s proliferation in vitro, promote apoptosis, largen mitochondria, increase sensitivity to the anticancer agents and made the intact structure of mitochondrial crest was not seen.