微小RNA-203通过锌指神经转录因子2的靶向作用对胃癌细胞SGC7901侵袭和凋亡的影响

摘要

目的 观察微小RNA(miRNA,miR)-203通过锌指神经转录因子2(SNAI2)对胃癌细胞SGC7901侵袭和凋亡的影响.方法 胃癌细胞SGC7901使用含10％胎牛血清的杜尔伯科改良伊格尔(DMEM)培养基,在37℃、5％ C02培养箱培养.采用脂质体转染法将miR-203 mimics转入胃癌细胞SGC7901,转染48 h后,提取SGC7901-miRNAcon、SGC7901-miR-203 mimic两组细胞总RNA,实时定量聚合酶链反应(Real-time PCR)检测转染效果,Transwell实验和流式细胞术检测细胞侵袭和凋亡.Real-time PCR和Western blot检测转染miR-203后在胃癌细胞中SNAI2的表达水平.小干扰RNA(siRNA)干扰胃癌细胞SGC7901中SNAI2的水平,Western blot验证敲减效果,并检测敲减SNAI2后细胞侵袭和凋亡.转染miR-203 mimics后,脂质体转染SNAI2表达质粒,检测细胞侵袭和凋亡.结果 (1)胃癌细胞SGC7901转染miR-203 mimics后,miR-203的表达水平(6.68±0.53)明显高于miRNA con组(0.84±0.12),两组间差异有统计学意义(P＜0.05).(2)流式细胞术结果显示,与miRNA con组的凋亡率(9.25±1.19)％比较,转染miR-203 mimics组胃癌细胞SGC7901的凋亡率为(32.73±5.94)％,较miRNA con组明显增加,两组间差异有统计学意义(P＜0.05).(3)与miRNA con组比较,转染miR-203 mimics 48 h后,胃癌细胞株SGC7901、MGC-803、BGC-823中SNAI2的表达都下调,分别为16.52±3.28、13.37 ±3.06和14.93 ±3.49,两组间差异有统计学意义(P＜0.05).(4)敲减SNAI2之后,细胞的侵袭能力明显下降,siRNA con组、siRNA-1组和siRNA-2组的侵袭细胞数量分别为(71.04 ±6.83)×106/L,(20.47 ±2.37)×106/L和(23.29±2.58)×106/L,与对照组比较,两组间差异有统计学意义(P＜0.05).(5)流式细胞术的结果表明,转染SNAI2后,miR-203对胃癌细胞SGC7901的促凋亡作用被反转,siRNA con组、siRNA-1组和siRNA-2组的SGC7901细胞凋亡率分别为(10.74±1.26)％、(30.73±5.58)％和(16.29±2.14)％,siRNA-2组的细胞凋亡程度明显降低.结论 miR-203能够通过靶向调控SNAI2而降低胃癌细胞SGC7901的侵袭能力并促进其凋亡.%Objective To investigate the effect of microRNA (miRNA,miR)-375 on cell invasion and apoptosis in the human gastric cancer cell line SGC7901.Methods SGC7901 cells were cultured in the Dulbecco's modified Eagle's medium (DMEM) at 37 ℃ in 5％ CO2.The expression levels of miRNA-203 in SGC7901 cells were detected by real-time quantitative polymerase chain reaction (Real-time PCR) after transfection of the miRNA-203 mimic.Transwell cell invasion assay and flow cytometry were used to detect the invasion and apoptosis of SGC7901 cells respectively.The expression levels of zinc finger transcription factor 2 (SNAI2) in SGC7901 cells were assayed after transfection of the miRNA-203 mimic by Real-time PCR and Western blotting.The expression of SNAI2 was determined by Western blotting after knockdown of SNAI2 by siRNA,and the invasion and apoptosis of SGC7901 cells after knockdown of SNAI2 were detected.Finally,the SNAI2 expression after transfection of miR-203 mimic was detected,and the invasion and apoptosis of SGC7901 cells were examined.Results (1) The expression level of miR-203 (6.68 ± 0.53) was significantly higher than that in con miR-203 group (0.84 ± 0.12) after transfection of SGC7901 cells with mimics (P ＜ 0.05).(2) Flow cytometry showed that as compared with miRNA con group [(9.25 ± 1.19)％] the apoptosis rate of SGC-7901 cells in miR-203 mimics transfection group [(32.73 ± 5.94％)] was significantly increased (P ＜ 0.05).(3) As compared with miRNA con group,the expression of SNAI2 in SGC7901,MGC-803,and BGC-823 cells at 48 h after transfection of miR-203 mimics was significantly down-regulated [(16.52 ± 3.28),(13.37 ± 3.06) and (14.93 ± 3.49) respectively] (P ＜ 0.05).(4) Knock-down of SNAI2 significantly reduced the invasion ability.The number of invasive cells in siRNA con,siRNA-1 and siRNA-2 groups was (71.04 ± 6.83) x 106/L,(20.47 ± 2.37) x 106/L and (23.29 ± 2.58) x 106/L respectively,which was significantly different from that in the control group (P ＜ 0.05).(5) Flow cytometry results showed that after the transfection of SNAI2,the promoting effect of miR-203 on apoptosis of SGC7901 cells was reversed.The apoptosis rate of SGC7901 cells in con siRNA group,siRNA-1 group and siRNA-2 group was (10.74 ± 1.26) ％,(30.73 ± 5.58) ％ and (16.29 ± 2.14) ％ respectively.Conclusion MiR-203 can inhibit invasion and promote apoptosis of SGC7901 cells by targeting SNAI2.