Effect Of MicroRNA-192Gene Silencing On Proliferati Of A375Malignant Melanoma Cells

Background：MicroRNA, an endogenous control gene, Is a noncoding small moleculeRNA, which contains22nuleotides in eukaryotes, it plays a role in inhibit tingthe translation or degradation of target gene by combining the target gene. Atpresent, the study about how microRNA affect the tumor is a hot spot andexpected to be a target to diagnosis and treat tumor.Malignant melanoma(MM) is a high malignant tumor, Incidence rate ofmalignant tumors of the skin third (6.8%~20%), the higher malignacy, earlymetastasis, prognosis is poor, with high mortality. In this study, we analyseA375malignant melanoma cells using gene chip, that date showed thatmicroRNA-192gene was expressed highly in A375malignant melanoma cells.we studied how microRNA-192gene affect A375malign ant melanoma cellsin this experiment.Objective:To lay a foundation of mechanism how microRNA affect A375maligantmelanoma cells.Method:1Cell cultureA375malignant melanoma cells were cultured in DMEM with10%fetalbovine serum culture medium (containing penicillin100u/ml, streptomycin100u/ml,PH7.2),The conventional subculture was processed in5%CO2incubator that the temperature was37%.2To observe the differentiation and morphology of A375malignant melnomacells under inverted optical microscope.3To detect the difference of expression amount of microRNA-192bothtransfection and non-transfection by real-time quantitative PCR after micr- oRNA-192inhibitor transfect A375malignant melanoma cells for48hours.4To test how microRNA-192inhibitor which the dose was400nmol200nmol/l and100nmol/l respectively affect A375malignant melanoma cells on1sthday、2th day and3sth day respectively by MTT color-imetric assay.5To test the change of the early stage of A375malignant melanoma cellsapoptosis after being transfected on2th day by Anexin-V-PI double staini-ng.The experiment was set up into normal group, LipofectamineTM2000groupand the experimental group(200nmol/l).6Statistic analysis: the data was analyzed by statistical software SPSS13.0forwindows, Based on the One-way ANOVA with a=0.05as significantdifferences standards.Results:1Non-treatment A375malignant melanoma cells, which were spindle orpolygonal, distributed evenly and the size of A375malignant melanoma cellsis uniform. These cells proliferate well, which nuclear pyknosis was hard todetect.2About80percent to90percent of A375malignant melanoma cells transfectedby micro-192inhibitor were tested after being transfected for5-6h ours.3The expression of microRNA-192gene after microRNA-192inhibitortransfect A375malignant melanoma cells for48hours was significantlydecreased compared with the expression before A375malignant melanomacells were transfected. The date indicated that the inhibitor transfected A375malignant melanoma cells successfully and made microRNA-192gene silencepartially.4We detected how treatment factors affect proliferation of A375malignantmelanoma cells by MTT colorimetric detection. There was no significantdifference between the normal group and the LipofectamineTM2000group(P>0.05).There was significant differences between three experimental groupand normal group（P<0.05）.and also between three experimental group andLipofectamineTM2000group（P<0.05）.The inhibition off cell proliferationincreased when the concent ration of inhibitor increased(from100nmol/l 200nmol/l to400nmol/l).5To test the early apoptosis of A375malignant melanoma cells aftermicroRNA-192inhibitor transfected for48hours by Anexin-V-PI doublestaining, we found that the early apoptosis rate between this group and thecontrol group had a significant difference（p<0.05）.Conclusion:1Mir-192inhibitor inhibited the miR-192gene of A375malignant melanomacells significantly.2Mir-192inhibitor inhibited the cell proliferation of A375malignant melan-oma cells significantly.3A375malignant melanoma cells apoptosised early after miR-192inhibitortransfected A375malignant melanoma cells. miR-192inhibitor affected theproliferation of A375malignant melanoma cells. but the mechanism need tobe studied for further.