沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响

Effects of silencing Icmt on the proliferation,apoptosis,and cell cycle of CAL-27 and SCC-4 cells

目的 :探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprenylcysteine carboxyl methyltransferase,Icmt)对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡和细胞周期的影响及其相关机制。方法:针对人Icmt基因序列设计并构建3条小干扰RNA(small interfering RNA,siRNA),采用脂质体载体瞬时转染Icmt-siRNA抑制舌鳞癌CAL-27和SCC-4细胞Icmt表达,将实验组分为Icmt-siRNA-1组、Icmt-siRNA-2组、Icmt-siRNA-3组;同时将脂质体转染NC-siRNA作为阴性对照组,只加转染试剂作为空白对照组。采用实时荧光定量PCR(qRT-PCR)和蛋白质免疫印记法(Western blot)检测转染后各组细胞Icmt、K-Ras的mRNA和蛋白表达及K-Ras膜蛋白的表达;Western免疫印迹检测Cyclin D1、p21、Akt、p-Akt蛋白表达;细胞增殖活性检测试剂盒和流式细胞术检测细胞的增殖活性、周期变化和凋亡能力。应用GraphPad Prism 8.2.1软件对实验数据进行统计学分析。结果:qRT-PCR和Western免疫印迹检测结果显示,与对照组相比,实验组Icmt mRNA和蛋白表达显著下降(P<0.05),K-Ras mRNA和蛋白表达无显著差异(P>0.05),K-Ras膜蛋白表达显著下降(P<0.05);周期相关蛋白Cyclin D1表达显著下调,p21表达显著上调(P<0.05);Ras/PI3K/Akt/mTOR信号通路相关蛋白Akt表达无统计学差异(P>0.05),但p-Akt表达显著下降(P<0.05)。细胞增殖活性检测结果表明,与对照组相比,实验组细胞增殖能力显著下降(P<0.05);流式细胞术检测结果表明,实验组细胞凋亡水平较对照组显著增加,细胞周期被阻滞在G1/S期(P<0.05)。结论:体外沉默Icmt基因可有效抑制CAL-27和SCC-4细胞增殖且诱导凋亡,其作用可能是通过影响K-Ras膜蛋白靶向膜定位,负性调控细胞周期和下调Ras/PI3K/Akt/mTOR信号通路而实现。

PURPOSE: This study was aimed to explore the effects and regulatory mechanisms of silencing Icmt on cell proliferation, apoptosis, and cell cycle of cell line CAL-27 and SCC-4 in vitro. METHODS: Three siRNAs were designed and constructed for Icmt gene sequence, and then transfected into CAL-27 and SCC-4 cells to silence Icmt expression.The tested cells were divided as follows: RNA interference groups including Icmt-siRNA-1, Icmt-siRNA-2, and IcmtsiRNA-3, negative control group, and blank control group. The mRNA and protein expression of Icmt and K-Ras were examined by real-time PCR and Western blot, respectively. The expression of Cyclin D1, p21, Akt, and p-Akt were examined by Western blot. The proliferation abilities of CAL-27 and SCC-4 cells were determined by cell counting kit-8 assay. Cell cycle analysis and apoptosis abilities of CAL-27 and SCC-4 cells were detected by flow cytometry. Statistical analysis and presentation was performed using GraphPad Prism 8.2.1 software. RESULTS: The expression of Icmt mRNA and protein in CAL-27 and SCC-4 cells was reduced significantly after Icmt siRNAs were transfected(P<0.05). No significant difference in K-Ras mRNA and protein expression was detected(P>0.05), but the expression of K-Ras membrane protein was decreased significantly compared with the negative control group and the blank control group(P<0.05). Cyclin D1 expression was decreased, whereas p21 expression was increased significantly. The expression of Akt was invariant(P>0.05), but the expression of p-Akt was significantly decreased(P<0.05). The cell cycle was altered in G1/S, the growthproliferative activity was inhibited and apoptosis was significantly induced(P<0.05). CONCLUSIONS: Silencing Icmt can effectively reduce the proliferation and induce apoptosis of CAL-27 and SCC-4 cells by affecting K-Ras membrane targeting localization, and then negatively regulating cell cycle and down-regulating K-Ras/PI3 K/Akt/mTOR signaling pathway.