ALKBH5调控GEFT的m6A修饰对胆管癌转移及EMT的实验研究

Experimental Study of GEFT m6A Modification Regulated by ALKBH5 on Cholangiocarcinoma Metastasis and EMT

目的探究alkB同源蛋白5(ALKBH5)调控鸟嘌呤核苷酸交换因子T(guanine nucleotide exchange factors T,GEFT)的m6A修饰对胆管癌转移和上皮间充质转化(epithelial-mesenchymal transition,EMT)的影响。方法将HuCCT1细胞按照转染类别分为Control组,NC-sh组、ALKBH5-sh组、NC-LV组、ALKBH5-LV组、ALKBH5-LV+NC-sh组和ALKBH5-LV+GEFT-sh组。Control组细胞不进行转染,使用Lipofectamine 2000对其他组细胞分别转染相应的慢病毒。采用MTT法检测细胞增殖水平,流式细胞仪检测细胞凋亡水平,Transwell实验检测细胞迁移和侵袭水平。采用RT-qPCR分析HuCCT1细胞中的ALKBH5,GEFT,Bax,Bcl-2,MMP2,MMP9,E-cadherin,N-cadherin和Vimentin的mRNA水平。采用Western blot分析HuCCT1细胞中的ALKBH5和GEFT蛋白水平。采用MeRIP-qPCR分析HuCCT1细胞中GEFT m6A甲基化水平。结果与Control组和NC-sh组比较,ALKBH5-sh组ALKBH5和GEFT的mRNA和蛋白水平降低,相对细胞活力降低,细胞凋亡率和Bax mRNA水平升高,Bcl-2 mRNA水平降低,迁移和侵袭细胞数量降低,MMP2和MMP9 mRNA水平降低,E-cadherin mRNA水平升高,N-cadherin和Vimentin水平降低,GEFT m6A甲基化水平升高,差异具有统计学意义(F=43.347~1995.868,均P<0.001)。与Control组和NC-LV组比较,ALKBH5-LV组ALKBH5和GEFT的mRNA和蛋白水平升高,相对细胞活力升高,细胞凋亡率和Bax mRNA水平降低,Bcl-2 mRNA水平升高,迁移和侵袭细胞数量升高,MMP2和MMP9 mRNA水平升高,E-cadherin mRNA水平降低,N-cadherin和Vimentin水平升高,GEFT m6A甲基化水平降低,差异具有统计学意义(F=42.421~720.275,均P<0.001)。与ALKBH5-LV+NC-sh组比较,ALKBH5-LV+GEFT-sh组GEFT的mRNA和蛋白水平降低,相对细胞活力降低,细胞凋亡率和Bax mRNA水平升高,Bcl-2 mRNA水平降低,迁移和侵袭细胞数量降低,MMP2和MMP9 mRNA水平降低,E-cadherinmRNA水平升高,N-cadherin和Vimentin水平降低,差异具有统计学意义(t=7.175~77.872,均P<0.001)。结论ALKBH5和GEFT均促进胆管癌细胞的生长和转移,ALKBH5可能通过调控GEFT m6A甲基化修饰来影响胆管癌的转移及EMT。

Objective To explore the effect of m6A modification of guanine nucleotide exchange factor T(GEFT)regulated by alkB homolog 5(ALKBH5)on metastasis and epithelial-mesenchymal transformation(EMT)of cholangiocarcinoma.Methods HuCCT1 cells were divided into control group,NC-sh group,ALKBH5-sh group,NC-LV group,ALKBH5-LV group,ALKBH5-LV+NC-sh group and ALKBH5-LV+GEFT-sh group according to the type of transfection.The cells in control group were not transfected,while the cells in other groups were transfected with lentivirus with Lipofectamine 2000.Cell proliferation was detected by MTT method,apoptosis was detected by flow cytometry,and cell migration and invasion were detected by Transwell test.The mRNA levels of ALKBH5,GEFT,Bax,Bcl-2,MMP2,MMP9,E-cadherin,N-cadherin and Vimentin in cells were detected by RT-qPCR.The protein levels of ALKBH5 and GEFT in cells were detected by Western blot.The methylation level of GEFT m6A in cells was detected by MeRIP-qPCR.Results Compared with control group and NC-sh group,ALKBH5 and GEFT mRNA and protein levels decreased,relative cell viability decreased,apoptosis rate and Bax mRNA level increased,Bcl-2 mRNA level decreased,the number of migratory and invasive cells decreased,MMP2 and MMP9 mRNA levels decreased,E-cadherin mRNA level increased,N-cadherin and vimentin levels decreased,GEFT m6A methylation level increased in ALKBH5-sh group,the differences were statistically significant(F=43.347~1995.868,all P<0.001).Compared with control group and NC-LV group,ALKBH5 and GEFT mRNA and protein levels increased,relative cell viability increased,apoptosis rate and Bax mRNA level decreased,Bcl-2 mRNA level increased,the number of migratory and invasive cells increased,MMP2 and MMP9 mRNA levels increased,E-cadherin mRNA level decreased,N-cadherin and vimentin levels increased,GEFT m6A methylation level decreased in ALKBH5-sh group,the differences were statistically significant(F=42.421~720.275,all P<0.001).Compared with ALKBH5-LV+NC-sh group,GEFT mRNA and protein levels decreased,relative cell viability decreased,apoptosis rate and Bax mRNA level increased,Bcl-2 mRNA level decreased,the number of migratory and invasive cells decreased,MMP2 and MMP9 mRNA levels decreased,E-cadherin mRNA levels increased,N-cadherin and Vimentin levels decreased in ALKBH5-LV+GEFT-sh group,and the differences were statistically significant(t=7.175~77.872,all P<0.001).Conclusion Both ALKBH5 and GEFT promoted the growth and metastasis of cholangiocarcinoma cells.ALKBH5 may affect the metastasis and EMT of cholangiocarcinoma by regulating the methylation of GEFT m6A.