微小RNA-30a-5p激活Wnt/β-连环蛋白信号通路对胃癌细胞增殖与侵袭的作用机制

Mechanism of miR-30a-5p activating Wnt/β-catenin signaling pathway on proliferation and invasionofgastriccancercells

目的探讨人微小RNA(has-miR)-30a-5p调控Wnt/β-连环蛋白(β-catenin)信号通路对胃癌细胞增殖及侵袭的影响及其作用机制。方法体外培养胃癌MKN45细胞;在所研究胃癌细胞系中使用小干扰RNA(siRNA)转染建立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-5pinhibitor)组,miR-30a-5p过表达RNA组和空白对照组(NC),荧光定量聚合酶链反应(qPCR)和蛋白质印迹法(Westernblot)检测Wnt/β-catenin信号通路相关标志物CyclinD1(CCND1)基因的表达水平;双荧光素酶报告基因实验验证miR-30a-5p与Wnt信号通路下游关键基因CCND1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-5pinhibitor组、miR-30a-5pinhibitor+银杏双黄酮组、miR-30a-5pInhibitorNC组及miR-30a-5pInhibitorNC+银杏双黄酮组,应用细胞计数试剂盒(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。各组间比较采用t检验。结果qPCR实验结果显示过表达miR-30a-5p能够显著抑制CCND1在mRNA的表达(0.58±0.05,t=11.39,P<0.01),Westernblot实验结果显示,CCND1蛋白水平,miR-30a-5pinhibitor组较NC组明显上升(284437±2127比215273±4097,t=25.95,P<0.01),miR-30a-5pmimics组较NC组明显下降(85586±309比197661±948,t=194.70,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WNT信号通路活性较对照组显著降低(0.39±0.03,t=13.96,P<0.01);CCK-8结果显示2d后miR-30a-5pinhibitor+银杏双黄酮组细胞抑制率明显高于miR-30a-5p inhibitor组[(93.09±4.18)%比(146.42±5.80)%,t=12.92,P<0.01];miR-30a-5p Inhibitor NC+银杏双黄酮组细胞抑制率明显高于miR-30a-5pInhibitorNC组[(53.80±3.54)%比(100.00±2.18)%,t=19.25,P<0.01]。Transwell结果显示miR-30a-5pinhibitor+银杏双黄酮组穿膜细胞量明显低于miR-30a-5pinhibitor组[(63.67±9.61)个比(100.00±10.44)个,t=4.44,P<0.05];miR-30a-5pInhibitorNC+银杏双黄酮组穿膜细胞量明显低于miR-30a-5pInhibitorNC组[(19.33±6.66)个比(45.00±4.58)个,t=5.50,P<0.01]。结论miR-30a-5p可以调控Wnt/β-catenin信号通路,过表达miR-30a-5p后可抑制Wnt/β-catenin信号通路降低胃癌细胞中CyclinD1(CCND1)的表达,进一步影响胃癌的增殖与侵袭。

Objective To explore the effect of human microRNAs(has miR)-30a-5p regulating Wnt/β-catenin signaling pathway on the proliferation and invasion of gastric cancer cells and its mechanism of action.Methods In vitro culture of gastric cancer MKN-45 cells was done.A knockout and overex-pression model of miR-30a-5p was established using 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)and Western blotting were used to detect the expression level of Cyclin D1(CCNDI)gene,a marker related to the Wnt/β-catenin signaling pathway.Double Luciferase reporter gene experiment verifies the targeted regulation relationship between miR-30a-5p and the key gene CCND1 downstream of Wnt/β-catenin signaling pathway.The cultured gastric cancer cell MKN-45 was added with Wnt/β-catenin signal pathway inhibitor ginkgo biloba biflavone,which was divided into miR-30a-5p inhibitor group,miR-30a-5p inhibitor+ginkgo biloba biflavone group,miR-30a-5p inhibitor NC group and miR-30a-5p inhibitor NC+ginkgo biloba biflavone group.Cell count kit(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.Comparison between groups was conducted using t-test.Results QPCR experiment results showed that overexpression of miR-30a-5p could significantly inhibit the expression of CCND1 in mRNA(0.58±0.05,t=11.39,P<0.O1).Western blotting experiment results showed that the level of CCND1 protein in miR-30a-5p inhibitor group was significantly higher than that in IC group(284437±2127 vs.215273±4097,t=25.95,P<0.01),and the level of CCND1 protein in miR-30a-5p mimics group was significantly lower than that in NC group(85586±309 vs.197661±948,t=194.70,P<0.01).The results of double luciferase experiment showed that the WNT signal pathway activity in the wild group was significantly lower than that in the control group after overexpression of miR-30a-5p(0.39±0.03,t=13.96,P<0.01).CCK-8 results showed that the cell inhibition rate in miR-30a-5p inhibitor+ginkgo biloba biflavone group was significantly higher than that in miR-30a-5p inhibitor group[(93.09±4.18)%vs.(146.42±5.80)%,t=12.92,P<0.01];The cell inhibition rate in inhibitor NC+Ginkgo biloba biflavone group was significantly higher than that in miR-30a-5p inhibitor NC group[(53.80±3.54)%vs.(100.00±2.18)%,t=i9.25,P<0.01].Transwell results showed that the number of transmembrane cells in miR-30a-5p inhibitor+ginkgo biloba biflavone group was significantly less than that in miR-30a-5p inhibitor group[(63.67±9.61)cells vs.(100.00±10.44)cells,t=4.44,P<0.05].The number of transmembrane cells in inhibitor NC+Ginkgo biloba biflavone group was significantly less than that in miR-30a-5p inhibitor NC group[(19.33±6.66)cells vs.(45.00±4.58)cells,t=5.50,P<0.01].Conclusion MiR-30a-5p can regulate Wnt/β-Catenin signaling pathway and overexpression of miR-30a-5p inhibits Wnt/β-Catenin signal pathway and reduces the expression of Cyclin D1 in gastric cancer cells.