抑癌基因MIIP对人鼻咽癌CNE-1细胞增殖与凋亡影响研究

目的迁移侵袭抑制蛋白(invasive migration inhibitor protein,MIIP)是近来发现的抑制肿瘤侵袭转移的基因,研究显示其在多种恶性肿瘤组织中表达降低。本研究探讨沉默MIIP表达对鼻咽癌CNE-1细胞增殖、细胞周期与凋亡及表皮生长因子受体(epidermal growth factor receptor,EGFR)/p38通路的影响。方法体外培养人鼻咽癌CNE-1细胞,利用MIIPsiRNA转染CNE-1细胞为MIIPsiRNA组,采用国际通用的与所有基因均无同源性序列的non-target siRNA转染为阴性对照组,以未经处理的CNE-1细胞为空白对照组,48h后收集各组细胞,利用实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)法检测MIIP mRNA表达情况,利用蛋白质印迹法检测MIIP蛋白表达情况;通过细胞计数盒8(cell counting kit 8,CCK8)法检测细胞增殖情况,利用流式细胞仪检测细胞周期分布和细胞凋亡,采用蛋白质印迹法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白Cyclin D1、Bax、Bcl-2和EGFR/p38通路相关蛋白表达情况。结果空白组、阴性对照组和MIIPsiRNA组MIIP mRNA水平分别为1.04±0.17、1.07±0.16和0.20±0.04,MIIP蛋白分别为0.94±0.13、0.92±0.14和0.13±0.03,与空白对照组和阴性对照组比较,MIIPsiRNA组MIIP mRNA、MIIP蛋白表达降低,均P<0.001;空白组、阴性对照组和MIIPsiRNA组G0/G1期细胞比例分别为(58.88±4.25)%、(58.79±6.15)%和(30.67±4.26)%,S期细胞比例分别为(33.00±4.07)%、(35.76±4.12)%和(46.35±5.20)%,G2/M期细胞比例分别为(8.12±2.35)%、(5.45±1.95)%和(22.98±3.76)%;与空白对照组和阴性对照组比较,G2/M期细胞比例增多,差异有统计学意义,P<0.001;与阴性对照组比较,MIIPsiRNA组G0/G1期细胞比例有所减少,S期变化差异无统计学意义,G2/M期细胞比例增多,P<0.001;空白组、阴性对照组和MIIPsiRNA组凋亡率分别为(20.4±4.1)%、(20.8±4.1)%和(9.5±1.9)%。与空白对照组和阴性对照组比较,MIIPsiRNA组细胞凋亡率降低,P<0.05;空白组、阴性对照组和MIIPsiRNA组PCNA蛋白分别为0.14±0.02、0.15±0.02和0.43±0.07,Cyclin D1蛋白分别为0.37±0.06、0.35±0.05和0.62±0.10,Bax蛋白分别为0.36±0.06、0.34±0.05和0.27±0.04,Bcl-2蛋白分别为0.29±0.04、0.28±0.04和0.45±0.07。与空白对照组和阴性对照组比较,MIIPsiRNA组PCNA、Cyclin D1和Bcl-2蛋白表达升高,均P<0.001;Bax蛋白表达降低,均P<0.05。结论沉默MIIP基因表达可能通过促进EGFR/p38信号通路激活,促进CNE-1细胞增殖,抑制凋亡。

Effects of tumor suppressor gene MIIPon the proliferation and apoptosis of CNE-1 cells of human nasopharyngeal carcinoma

OBJECTIVE Invasive migration inhibitor protein(MIIP)is a recently discovered gene that inhibits the invasion and metastasis of nasopharyngeal carcinoma.Studies have shown that MIIPexpression decreases in many malignant tumors.This study investigated the effects of silencing MIIPexpression on proliferation,cell cycle,apoptosis and epidermal growth factor receptor(EGFR)/p38 pathway of nasopharyngeal carcinoma CNE-1 cells.METHODS Human nasopharyngeal carcinoma CNE-1 cells were cultured in vitro,CNE-1 cells were transfected with MIIPsiRNA,internationally common non-target siRNA with non-homologous sequences of all genes was transfected into negative control group,and untreated CNE-1 cells were used as blank control group.After 48 hours,the expression of MIIP was detected by qRT-PCR,and the expression of MIIP was detected by Western blot(WB).CKK-8 method was used to detect cell proliferation,flow cytometry was used to detect cell cycle distribution and apoptosis,WB was used to detect the expressions of proliferating cell nuclear antigen(PCNA),cyclin D1,Bax,Bcl-2 and EGFR/p38 pathway-related proteins.RESULTS The levels of MIIP mRNA and MIIP protein in blank group,negative control group and MIIPsiRNA group were 1.04±0.17,1.07±0.16 and 0.20±0.04.The levels of MIIP protein in MIIPsiRNA group were 0.94±0.13,0.92±0.14 and0.13±0.03 respectively.Compared with blank control group and negative control group,the expressions of MIIPgene and MIIPprotein in MIIPsiRNA group were significantly decreased(t=3.098,7.609,11.782,14.871,12.921,13.515,P<0.05).The percentage of G0/G1 phase cells in sex control group and MIIPsiRNA group were(58.88±4.25),(58.79±6.15)%,(30.67±4.26)%,the proportion of S phase cells were(33.00±4.07)%,(35.76±4.12)%,(46.35±5.20)%,and the proportion of G2/M phase cells were(8.12±2.35)%,(5.45±1.95)%,(22.98±3.76)%.Compared with the blank control group and the negative control group,the proportion of G2/M phase cells was(8.12±2.25)%,there was a significant increase(t=8.209,10.138,P<0.001);compared with negative control group,MIIP siRNA group had a decrease in G0/G1 phase cell proportion,no significant change in S phase,and a significant increase in G2/M phase cell proportion(t=10.138,P<0.001);the apoptosis rate of blank group,negative control group and MIIP siRNA group was 20.4±4.1,20.8±4.1,9.5±1.9.Compared with the blank control group and the negative control group,the apoptotic rate of MIIPsiRNA group was significantly lower(t=5.908,6.125,P<0.05);the PCNA protein of blank group,negative control group and MIIPsiRNA group were 0.14±0.02,0.15±0.02,0.43±0.07,Cyclin D1 protein was 0.37±0.06,0.35±0.05,0.62±0.10,and Bax protein was 0.36±0.06,0.34±0.05,0.27±0.04,respectively.The white values were 0.29±0.04,0.28±0.04 and 0.45±0.07,respectively.Compared with blank control group and negative control group,the expression of PCNA,cyclin D1 and Bcl-2 protein in MIIPsiRNA group was increased significantly(t=9.757,5.251,4.861,9.421,5.915,5.165,P<0.05),while the expression of Bax protein decreased significantly(t=3.057,2.678,P<0.05).CONCLUSION Silencing MIIPgene expression may promote the proliferation of CNE-1 cells and inhibit apoptosis by promoting the activation of EGFR/p38 signaling pathway.