Experiment Study On MiR-769 Silencing Inhibits Proliferation Of Melanoma Cells

PurposeTo determine the expression level of miR-769 that was between malignant melanoma cells and normal human epidermal melanocytes.To screen of miR-769 targeted gene in melanoma cells.To analysis the impact on the proliferation of melanoma cell by regulating the expression level of miR-769,and to explore the mechanism of silencing MIR-769 expression on the inhibiting proliferation of malignant melanoma cells.Materials and Methods1.Subjects and treatment groups①24 cases of melanoma patients with pathological specimens,according to the pathological stage named in situ group(n = 10)and metastasis group(n = 14);pathological specimens of normal pigmented nevus,named for the group of pigmented nevus(24 cases).Seven kinds of melanoma cell lines(NHEM,SK-MEL-28,WM-115,UACC257,A375,A7 and MeWo)were called MM groups.Normal skin melanin cell lines groups were called PEM groups.②Melanoma A375 cells were divided into three groups(mimics group,inhibitor group and NC group)after transfected with miR-769 mimics、miR-769-inhibitor or the relative controls.③Wild-type recombinant plasmid GSK3β(WT group)and mut-type recombinant plasmid GSK3(3(MUT group)were synthesized.④Suppression of GSK3B by si-RNA GSK3p-siRNA#land GSK3p-siRNA#2 in cells after transfected with miR-769-in.2.To detect the miR-769 expression in Melanoma tissues and Melanoma cell lines We analyzed the expression of miR-769 in malignant melanoma tissues and tumor adjacent normal tissues.We analyzed the expression of miR-769 in different human melanoma cell lines and Normal skin melanin cell line PEM by qRT-PCR.3.The mechanism research of miR-769 in melanoma cells was detedted.GSK3β expression in mimics groups,inhibitor groups and NC group were detected by Western Blot assays.Luciferase activity in mimics groups,inhibitor groups,MUT groups and NC groups.Western blot assays and RT-PCR assays were used to detect cyclin D1 and c-myc expression.MTT cell proliferation assay,cell colony formation assay and BrdU cell proliferation assay was used to detect the role of miR-769 inmelanoma cells.4.GSK3B downregulation counteracted the proliferation arrest by miR-769-inRT-PCR,MTT cell proliferation assay,cell colony formation assay and BrdU cell proliferation assay was used in si-RNA groups and NC groups.5.Statistical AnalysisUsing SPSS21.0 statistical software for data analysis,make plans using Graphpad prism 5 software.Data was analysis by the two groups ratio chi-square test,t test and ANOVA.P<0.05 for the difference was significant.Result1.The expression of melanoma specimens and human melanoma cell lines of miR-769The miR-769 expression levels in 24 pairs melanoma tissue are relatively high than pigmented nevus tissues.The average expression level of miR-769 in seven kinds of melanoma cells(MM groups)is much higher than normal human epidermal melanocytes PEM(PEM groups).2.The effect of miR-769 in human melanoma cell proliferationResult of Western Blot indicated that compared to the control groups,miR-769 expression was no expressed in A375 cells after transfected with miR-769.Compared to PEM groups,miR-769 levels were significantly upregulated in MM groups.Result of Dual-Luciferase Reporter revealed that overexpression of miR-769 led to a remarkable decrease of luciferase activity of GSK3B 3’-UTR,whereas miR-769-in led to increased luciferase activity.However,luciferase activity was not affected by miR-769-mut.Using RT-PCR and Western bolting,compared to NC transfected cells,we observed that Cyclin D1 expression and MYC expression was up-regulated by miR-769,while Cyclin D1 expression and MYC expression was down-regulated after transfected with miR-769-in。Result of RT-PCR indicated that highest expression of miR-769 is in mimics group,followed by the NC group,inhibitor group expression level was minimum.The highest OD value by MTT assay was in mimics group,followed by the NC group,inhibitor group expression level was lowest.The highest clone formation rate by cell colony formation assay was in mimics group,followed by the NC group,inhibitor group expression level was lowest.BrdU cell proliferation experiment showed that the highest percentage of S phase cells was in mimics group,secondly the NC group,inhibitor groups is lowest.3.GSK3B downregulation counteracted the proliferation arrest by miR-769-in By Western Blot assay GSK3β silencing effect,results showed that compared to the negative control NC,turn transfection interfering RNA GSK3β-siRNAs cell lines,the protein bands hardly be described expression.Colony formation assay results showed compared to the negative control NC transfected interfering RNA GSK3β-siRNAs cell lines cell colony formation was significantly higher than NC group.Anchorage-independent growth analysis experiments showed that compared to the negative control NC,interfering RNA transfection GSK3β-siRNAs cell lines A375 human melanoma cell proliferation A375 cells formed colonies number more than 0.1mm diameter was significantly higher than NC group.By BrdU cell proliferation assay results found that compared to the negative control NC transfected interfering RNA GSK3β proportion-siRNAs cell line A375 human melanoma cells in s phase(DNA molecule incorporated BrdU cells)was significantly higher than NC group(P<0.001).Conclusion1.miR-769 down-regulated expression in melanoma cell lines and tissue samples,which is an important carcinogen related gene expression in melanoma.2.GSK3β is the target genes act with miR-769 in the melanoma cells,miR-769 is a negative regulator on the expression of GSK3β.3.Silencing miR-769 expression can promote the expression of GSK3β,and then inhibit the expression level of GSK3β pathway downstream related factor cyclin D1 and c-Myc mRNA,so that it can reduce the proliferation of melanoma cells obviously.4.miR-769-GSK3p feedback regulation system reveal the molecular mechanisms of the development of melanoma,by inhibiting the expression level of miR-769,miR-769 can be expected to be a potential therapeutic target in later clinical treatment to melanoma.