hTERT上调FOXM1乙酰化影响胃癌细胞侵袭能力

hTERT promotes FOXM1 acetylation and subsequent invasion of gastric cancer cells

目的探讨人端粒酶逆转录酶(human telomerase reverse enzyme,hTERT)促进胃癌细胞侵袭的分子机制。方法构建稳定过表达hTERT的细胞株SGC-7901-hTERT,采用Western blot检测叉头盒蛋白(forkhead box M1,FOXM1)的表达,q RT-PCR和Western blot检测FOXM1下游侵袭相关基因基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(MMP-9)的m RNA和蛋白表达;构建FOXM1的sh RNA干扰质粒,在SGC-7901-hTERT细胞中转染该质粒,Transwell法检测细胞侵袭能力,q RT-PCR和Western blot检测MMP-2、MMP-9的m RNA和蛋白表达;构建FOXM1启动子活性报告质粒,在SGC-7901-hTERT细胞中用双荧光素酶报告实验检测FOXM1启动子活性,q RT-PCR检测FOXM1 m RNA表达;构建稳定过表达FOXM1的细胞株SGC-7901-HA-FOXM1,并在此细胞中转染hTERT过表达质粒,免疫共沉淀(co-immunoprecipitation,Co-IP)检测FOXM1乙酰化水平变化,免疫荧光实验检测hTERT与FOXM1的细胞定位。结果与对照组比较,过表达hTERT促进FOXM1及其下游分子MMP-2、MMP-9的蛋白表达,同时也促进肿瘤细胞侵袭(P<0.01),但不影响FOXM1的启动子活性及m RNA表达;与过表达hTERT组比较,干扰FOXM1可抑制MMP-2、MMP-9的m RNA和蛋白表达,同时也抑制肿瘤细胞侵袭(P<0.01);hTERT与FOXM1存在细胞共定位,且过表达hTERT可上调FOXM1乙酰化水平。结论 hTERT通过上调FOXM1乙酰化促进胃癌细胞侵袭。

Objective To investigate the molecular mechanism of human telomerase reverse transcriptase （hTERT） in promoting the invasion of gastric cancer cells. Methods A cell line with stable expression of hTERT, SGC7901-hTERT was constructed based on human gastric cancer SGC-7901 cells. Western blotting was used to detect the expression of forkhead box M1 （FOXM1）, and it combined with qRT-PCR to measure the expression of FOXM1 downstream molecules, matrix metalloproteinase-2 （MMP-2） and matrix metalloproteinase-9 （MMP-9） at protein and mRNA levels. The shRNA interference plasmid of FOXM1 was constructed and transfected into the SGC-7901-hTERT cells. Transwell chamber test was used to detect the invasive ability of the cells. qRT-PCR and Western blotting were used again for the expression of MMP-2 and MMP-9. The activity of FOXM1 promoter was detected by double luciferase reporter assay in SGC-7901hTERT cells and corresponding control cells, and then the mRNA expression of FOXM1 was detected by qRT-PCR. After the construction of SGC-7901 FOXM1-HA cells with FOXM1-HA lentivirus, the cells were transfected with hTERT overexpression plasmid. The acetylation of FOXM1 was detected by coimmunoprecipitation （Co-IP）. The immunofluorescence assay was used to detect the co-localization of hTERT and FOXM1. Results Compared with the control group, hTERT overexpression promoted the expression of FOXM1 protein, which facilitated the expression of FOXM1 target genes, MMP-2 and MMP9, and also promoted tumor cell invasion （P〈0.01）. However, the expression of FOXM1 mRNA and its promoter activity was not affected. Compared with the overexpressing hTERT group, interference of FOXM1 partially decreased the effect of hTERT-promoted FOXM1 target gene expression and reduced the hTERT-mediated invasion of SGC-7901 cells （P〈0.01）. FOXM1 and hTERT were co-localized in the SGC-7901 cells, and overexpression of hTERT up-regulated the level of acetylation of FOXM1. Conclusion hTERT affects the invasion of gastric cancer cells by up-regulation of FOXM1 acetylation.