微小RNA-30a-5p抑制胃癌增殖与侵袭的机制

The mechanism of microRNA-30a-5p inhibiting the proliferation and invasion of gastric cancer

目的探讨人微小RNA(has-miR)-30a-5p调控Frizzled 2(FZD2)基因激活Wnt/β-连环蛋白(β-catenin)信号通路对胃癌细胞增殖及侵袭的作用机制。方法体外培养胃癌MKN-45细胞;在胃癌细胞系中使用小干扰RNA(siRNA)转染建立微小RNA(miR)-30a-5p的敲除和过表达模型,胃癌MKN-45细胞分为3组:miR-30a-5p小干扰RNA(miR-30a-5p inhibitor)组,miR-30a-5p过表达RNA(miR-30a-5p mimics)组和空白对照组(negative control,NC),荧光定量聚合酶链反应(qPCR)、蛋白质印迹法(Western blot)检测miR-30a-5p的直接靶基因FZD2基因及Wnt/β-catenin信号通路下游关键基因c-myc的表达水平;双荧光素酶报告基因实验验证miR-30a-5p与直接靶基因FZD2的靶向调节关系;在培养的胃癌细胞MKN-45中加入Wnt/β-catenin信号通路抑制剂-银杏双黄酮,分为miR-30a-5p inhibitor组、miR-30a-5p inhibitor+银杏双黄酮组、miR-30a-5p Inhibitor NC组,采用细胞计数试剂盒(CCK-8)实验验证各组细胞的增殖能力,细胞迁移实验(Transwell)验证各组细胞的侵袭能力;通过免疫组织化学方法验证FZD2基因的蛋白表达水平。各组间比较采用t检验。结果qPCR实验结果显示FZD2 mRNA表达水平miR-30a-5p mimics组明显低于NC组(0.62±0.02比1.01±0.13,t=5.042,P<0.01),c-myc mRNA表达水平miR-30a-5p mimics组明显低于NC组(0.46±0.07比1.01±0.15,t=5.805,P<0.01),Western blot实验结果显示,FZD2蛋白表达水平miR-30a-5p inhibitor组明显高于NC组(1181347.00±39.27比206382.00±8.33,t=1080.000,P<0.01),miR-30a-5p mimics组明显低于NC组(996269.00±54.50比1168775.00±99.80,t=1104.000,P<0.01),c-myc蛋白表达水平miR-30a-5p inhibitor组明显高于NC组(3307840.00±81.00比253663.00±33.29,t=1072.000,P<0.01),miR-30a-5p mimics组明显低于NC组(138137.00±131.68比205780.00±77.69,t=766.300,P<0.01);双荧光素酶实验结果显示过表达miR-30a-5p后野生组中FZD2基因活性显著低于对照组(1.02±0.14比0.59±0.06,t=99.638,P<0.01);CCK-8结果显示转染48 h后miR-30a-5p inhibitor+银杏双黄酮组细胞增殖能力低于miR-30a-5p inhibitor组(93.09±4.18比146.42±5.80,t=12.920,P<0.01)。Transwell结果显示miR-30a-5p inhibitor+银杏双黄酮组穿膜细胞量低于miR-30a-5p inhibitor组[(63.67±9.61)个比(100.00±10.44)个,t=4.440,P<0.05]。免疫组织化学结果显示基因FZD2蛋白在miR-30a-5p低表达[miR-30a-5p/β-肌动蛋白(β-actin)<0.05]的胃癌组织中表达增强(评分≥5分),与对照组比较差异有统计学意义(P<0.01)。结论在胃癌中miR-30a-5p可直接靶向FZD2,通过抑制FZD2的表达,降低Wnt信号通路的活性,从而抑制胃癌的发生与发展。

Objective Exploring the mechanism of human microRNAs(has miR)-30a-5p regulating the Frizzled 2(FZD2)gene activation Wnt/β-catenin signaling pathway on the proliferation and invasion of gastric cancer cells.Methods In vitro culture of gastric cancer MKN-45 cells;A knockout and overexpression model of miR-30a-5p was established using small interfering RNA(siRNA)transfection in the studied gastric cancer cell line.Gastric cancer MKN-45 cells were divided into three groups:miR-30a-5p small interfering RNA(miR-30a-5p inhibitor)group,miR-30a-5p overexpressed RNA(miR-30a-5p mics)group,and blank control group(negative control,NC).Fluorescence quantitative polymerase chain reaction(qPCR)Western blotting was used to detect the expression levels of the direct target gene FZD2 gene of miR-30a-5p and the downstream key gene c-myc of the Wnt/β-catenin signaling pathway;Double Luciferase reporter gene experiment verified the targeted regulation relationship between miR-30a-5p and the direct target gene FZD2;The cultured gastric cancer cells MKN-45 were added with signal pathway inhibitor ginkgo biloba biflavone,which were divided into miR-30a-5p inhibitor group,miR-30a-5p inhibitor+ginkgo biflavone group,miR-30a-5p inhibitor NC group.Cell counting kit-8(CCK-8)test was used to verify the proliferation ability of cells in each group,and cell migration test was used to verify the invasion ability of cells in each group;Verify the protein expression level of FZD2 gene through immunohistochemical methods.The comparison between groups was conducted using t-test.Results The qPCR experiment results showed a significant decrease in FZD2 mRNA expression level in the miR-30a-5p mimics group compared to the NC group(0.62±0.02 vs.1.01±0.13,t=5.042,P<0.01),and a significant decrease in c-myc mRNA expression level in the miR-30a-5p mimics group compared to the NC group(0.46±0.07 vs.1.01±0.15,t=5.805,P<0.01).The Western blotting experiment results showed that,the expression level of FZD2 protein in the miR-30a-5p inhibitor group was significantly higher than that in the NC group(1181347.00±39.27 vs.206382.00±8.33,t=1080.000,P<0.01),and the miR-30a-5p mics group was significantly lower than that in the NC group(996269.00±54.50 vs.1168775.00±99.80,t=1104.000,P<0.01).The expression level of c-myc protein in the miR-30a-5p inhibitor group was significantly higher than that in the NC group(3307840.00±81.00 vs.253663.00±33.29,t=1072.000,P<0.01),The miR-30a-5p mimics group showed a significant decrease compared to the NC group(138137.00±131.68 vs.205780.00±77.69,t=766.300,P<0.01);The results of double Luciferase experiment showed that after overexpression of miR-30a-5p,the activity of FZD2 gene in the wild group was significantly lower than that in the control group(1.02±0.14 vs.0.59±0.06,t=99.638,P<0.01);The CCK-8 results showed that after 48 hours of transfection,the cell proliferation ability of the miR-30a-5p inhibitor+ginkgo biloba flavonoids group was lower than that of the miR-30a-5p inhibitor group(93.09±4.18 vs.146.42±5.80,t=12.920,P<0.01).The Transwell results showed that the number of transmembrane penetrating cells in the miR-30a-5p inhibitor+Ginkgo biloba biflavone group was lower than that in the miR-30a-5p inhibitor group(63.67±9.61 vs.100.00±10.44,t=4.440,P<0.05).The immunohistochemical results showed that the expression of FZD2 protein was enhanced in gastric cancer tissues with low expression of miR-30a-5p(miR-30a-5p/β-actin<0.05)(score≥5),with a statistically significant difference compared to the control group(P<0.01).Conclusion In gastric cancer,miR-30a-5p can directly target FZD2,reducing the activity of the Wnt signaling pathway by inhibiting the expression of FZD2,thereby inhibiting the occurrence and development of gastric cancer.