miR-15b-3p靶向CMTM6对胃癌细胞上皮-间充质转化的影响

Effect of miR-15b-3p targeting CMTM6 on epithelial-mesenchymal transition in gastric cancer cells

目的探讨miR-15b-3p靶向趋化素样因子超家族6(CMTM6)对胃癌细胞上皮-间充质转化(EMT)的影响。方法采用实时荧光定量PCR法检测人胃癌细胞SGC-7901、BGC-823、MKN-1及人正常胃黏膜上皮细胞GES-1中miR-15b-3p和CMTM6表达水平。采用双荧光素酶报告基因实验检测miR-15b-3p和CMTM6的靶向关系。将BGC-823细胞随机分为对照组(不做处理)、miR-15b-3p抑制剂组(转染miR-15b-3p抑制剂)、miR-15b-3p抑制剂+shRNA-CMTM6组(转染miR-15b-3p抑制剂和shRNA-CMTM6)。分别采用MTT实验、细胞划痕愈合实验、Transwell实验检测细胞的增殖、迁移和侵袭能力。采用蛋白质印迹法检测EMT相关蛋白表达水平。结果与GES-1细胞相比,SGC-7901、BGC-823和MKN-1细胞中miR-15b-3p和CMTM6 mRNA的相对表达量均显著升高(P均<0.05)。双荧光素酶报告基因实验检测结果显示,与miR-NC+CMTM6-WT组相比,miR-15b-3p抑制剂+CMTM6-WT组细胞的相对荧光素酶活性显著降低(P<0.05)。与对照组相比,miR-15b-3p抑制剂组细胞中miR-15b-3p和CMTM6mRNA的相对表达量均显著降低(P均<0.05);与miR-15b-3p抑制剂组相比,miR-15b-3p抑制剂+shRNA-CMTM6组细胞中miR-15b-3p的相对表达量无显著变化(P>0.05),而CMTM6 mRNA的相对表达量显著降低(P<0.05)。与对照组相比,miR-15b-3p抑制剂组的细胞增殖抑制率升高,细胞划痕愈合率降低,细胞侵袭数目减少,N-cadherin和Vimentin蛋白表达水平均降低,E-cadherin蛋白表达水平升高,差异均具有统计学意义(P均<0.05)。与miR-15b-3p抑制剂组相比,miR-15b-3p抑制剂+shRNA-CMTM6组的细胞增殖抑制率升高,细胞划痕愈合率降低,细胞侵袭数目减少,N-cadherin和Vimentin蛋白表达水平均降低,E-cadherin蛋白表达水平升高,差异均具有统计学意义(P均<0.05)。结论miR-15b-3p表达下调可通过靶向CMTM6而抑制胃癌细胞的增殖、迁移、侵袭能力及EMT进程。

Objective This paper is to investigate the effect of miR-15b-3p targeting CKLF-like MARVEL transmembrane domain-containing protein 6(CMTM6)on epithelial-mesenchymal transformation(EMT)in gastric cancer cells.Methods The expression levels of miR-15b-3p and CMTM6 in human gastric cancer cells SGC-7901,BGC-823,MKN-1,and normal gastric mucosal epithelial cells GES-1 were detected by real-time fluorescence quantitative PCR.The dual luciferase reporter gene assay was used to detect the targeting relationship between miR-15b-3p and CMTM6.BGC-823 cells were randomly divided and assingned to the control group(no treatment),the miR-15b-3p inhibitor group(transfected with miR-15b-3p inhibitor),and the miR-15b-3p inhibitor+shRNA-CMTM6 group(transfected with miR-15b-3p inhibitor and shRNA-CMTM6).The MTT assay,scratch healing assay,and Transwell assay were used to detect cell proliferation,migration,and invasion.The expression level of EMT-related proteins was detected by Western blotting.Results Compared with GES-1 cells,the relative expression levels of miR-15b-3p and CMTM6 mRNA in SGC-7901,BGC-823,and MKN-1 cells are significantly increased(P<0.05).The results of double luciferase reporter gene assay show that compared with the miR-NC+CMTM6-WT group,the relative luciferase activity of the miR-15b-3p inhibitor+CMTM6-WT group is significantly decreased(P<0.05).Compared with the control group,the relative expression levels of miR-15b-3p and CMTM6 mRNA in the miR-15b-3p inhibitor group are significantly decreased(P<0.05).Compared with the miR-15b-3p inhibitor group,the relative expression level of miR-15b-3p in the miR-15b-3p inhibitor+shRNA-CMTM6 group has no significant changes(P>0.05),but the relative expression level of CMTM6 mRNA is significantly decreased(P<0.05).Compared with the control group,the cell proliferation inhibition rate in the miR-15b-3p inhibitor group is increased;the cell scratch healing rate,the number of cell invasion,and the protein expression levels of N-cadherin and Vimentin are decreased,but the protein expression level of E-cadherin is increased,with statistically significant differences(P<0.05).Compared with the miR-15b-3p inhibitor group,the cell proliferation inhibition rate in the miR-15b-3p inhibitor+shRNA-CMTM6 group is increased;the cell scratch healing rate,the number of cell invasion,and the protein expression levels of N-cadherin and Vimentin are decreased,but the protein expression level of E-cadherin is increased,with statistically significant differences(P<0.05).Conclusion Down-regulation of miR-15b-3p expression can inhibit the proliferation,migration,invasion ability and EMT level of gastric cancer cells by targeting CMTM6.