微小RNA-203靶向RGS17蛋白对食管癌细胞增殖、周期和迁移的影响

Effects of microRNA-203 targeting RGS17 protein on proliferation,cell cycle and migration of esophageal cancer cells

目的探讨微小RNA(miR)-203靶向RGS17蛋白对食管癌细胞增殖、周期和迁移的影响。方法采用荧光定量聚合酶链反应(PCR)分析正常食管上皮细胞HEEC、食管癌细胞TE-8、EC9706和EC-11中miR-203表达水平。将对数生长期食管癌细胞EC9706分为对照组和miR-203组,分别采用对照组和miR-203过表达慢病毒感染,筛选稳定细胞系。采用细胞计数试剂盒(CCK-8)和克隆形成实验分析两组细胞增殖能力;采用流式细胞术分析两组细胞凋亡和周期变化;采用Transwell实验分析两组细胞迁移能力;采用生物信息学和双荧光素酶报告基因分析miR-203靶基因;采用蛋白免疫印迹分析靶基因及相关蛋白表达水平。计量资料比较采用t检验。结果正常食管上皮HEEC细胞miR-203表达水平(1.19±0.17)明显高于食管癌TE-8、EC9706和EC-11细胞miR-203表达水平(0.56±0.11、0.39±0.09、0.47±0.10),差异有统计学意义(t=3.719、4.498、3.909,P<0.05)。对照组细胞miR-203表达水平(1.07±0.12)明显低于miR-203组细胞miR-203表达水平(3.41±0.19),差异有统计学意义(t=5.576,P<0.05)。对照组细胞吸光度(A)值(1.95±0.19)明显高于miR-203细胞A值(1.36±0.16),差异有统计学意义(t=3.274,P<0.05)。对照组细胞克隆形成率[(69.94±6.51)%]明显高于miR-203组细胞克隆形成率[(39.55±4.91)%],差异有统计学意义(t=5.901,P<0.05)。对照组细胞G0/G1期比例[(40.27±5.71)%]明显低于miR-203组细胞G0/G1期比例[(54.90±5.98)%],差异有统计学意义(t=3.821,P<0.05)。对照组细胞S期比例[(30.16±4.20)%]明显高于miR-203组细胞S期比例[(21.85±4.09)%],差异有统计学意义(t=3.019,P<0.05)。对照组细胞G2/M期比例[(28.86±3.57)%]明显高于miR-203组细胞G2/M期细胞比例[(24.66±4.01)%],差异有统计学意义(t=2.621,P<0.05)。对照组细胞迁移数量[(146.38±16.10)个]明显高于miR-203组细胞迁移数量[(85.32±10.57)个],差异有统计学意义(t=6.926,P<0.05)。RGS17是miR-203的靶基因。对照组细胞RGS17表达水平(3.09±0.31)明显高于miR-203组细胞RGS17表达水平(1.74±0.20),差异有统计学意义(t=5.194,P<0.05)。结论 miR-203通过靶向RGS17蛋白调控食管癌细胞增殖、凋亡、周期和迁移。

Objective To investigate the effects of microRNA(miR)-203 targeting RGS17 protein on the proliferation,cell cycle and migration of esophageal cancer cells.Methods The expression levels of miR-203 in normal esophageal epithelial cells HEEC,esophageal cancer cells TE-8,EC9706 and EC-11 were analyzed by fluorescence quantitative polymerase chain reaction(PCR).The logarithmic long-term esophageal cancer cell EC9706 was divided into control group and miR-203 group.The stable cell lines were screened by lentivirus infection with overexpression of miR-203 and control group,respectively.cell counting kit-8(CCK-8)and clone formation assay were used to analyze the cell proliferation ability of the two groups.Apoptosis and cell cycle were analyzed by flow cytometry.Cell migration ability was analyzed by Transwell in two groups.The target gene of miR-203 was analyzed by bioinformatics and double luciferase reporter gene.The expression levels of target genes and related proteins were analyzed by Western blotting.The measurement data were compared by t-test.Results The expression level of miR-203 in normal esophageal epithelial heec cells(1.19±0.17)was significantly higher than that in esophageal cancer TE-8,EC9706 and EC-11 cells(0.56±0.11,0.39±0.09,0.47±0.10,t=3.719,4.498,3.909,P<0.05).The expression level of miR-203 in control group(1.07±0.12)was significantly lower than that in miR-203 group(3.41±0.19,t=5.576,P<0.05).The light absorbance value of cells in the control group(1.95±0.19)was significantly higher than that of miR-203 cells(1.36±0.16,t=3.274,P<0.05).The cell clone formation rate of the control group[(69.94±6.51)%]was significantly higher than that of the miR-203 group[(39.55±4.91)%,t=5.901,P<0.05].The proportion of G0/G1 cells in the control group[(40.27±5.71)%]was significantly lower than that in the miR-203 group[(54.90±5.98)%],and the difference was statistically significant(t=3.821,P<0.05).The proportion of S phase cells in the control group[(30.16±4.20)%]was significantly higher than that in the miR-203 group[(21.85±4.09)%,t=3.019,P<0.05].The proportion of G2/M cells in the control group[(28.86±3.57)%]was significantly higher than that in the miR-203 group[(24.66±4.01)%,t=2.621,P<0.05].The number of cell migration in the control group(146.38±16.10)was significantly higher than that in the miR-203 Group(85.32±10.57),and the difference was statistically significant(t=6.926,P<0.05).RGS17 is the target gene of miR-203.The expression level of RGS17 in control group(3.09±0.31)was significantly higher than that in miR-203 group(1.74±0.20,t=5.194,P<0.05).Conclusion MiR-203 regulates the proliferation,apoptosis,cycle and migration of esophageal cancer cells by targeting RGS17 protein.