hTERT上调FOXM1乙酰化影响胃癌细胞侵袭能力

发布时间：2018-04-15 09:22

本文选题：端粒酶逆转录酶 + 叉头盒蛋白　；参考：《第三军医大学学报》2017年23期

【摘要】：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(P0.01),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(P0.01);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。
[Abstract]:Objective to investigate the molecular mechanism of human telomerase reverse transcriptase telomerase reverse enzyme hTERT in promoting the invasion of gastric cancer cells.Methods A cell line SGC-7901-hTERT with stable expression of hTERT was constructed. The expression of forkhead box M1-FOXM1 was detected by Western blot. The expression of m RNA and protein of matrix metalloproteinase-2 metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were detected by Western blot and Western blot.The sh RNA interference plasmid of FOXM1 was constructed and transfected into SGC-7901-hTERT cells to detect the invasion ability of SGC-7901-hTERT cells. The m RNA and protein expression of MMP-2mMMP-9 were detected by Western blot and RT-PCR, and the FOXM1 promoter activity reporter plasmid was constructed.The expression of FOXM1 m RNA was detected by double luciferase report assay in SGC-7901-hTERT cell line SGC-7901-HA-FOXM1, and the hTERT overexpression plasmid was transfected into SGC-7901-HA-FOXM1 cell line.Co-immunoprecipitation Co-IPP was used to detect the acetylation level of FOXM1 and the cellular localization of hTERT and FOXM1 was detected by immunofluorescence assay.Results compared with the control group, overexpression of hTERT promoted the expression of FOXM1 and its downstream MMP-2mMMP-9 protein, and also promoted the invasion of tumor cells by P0.01T, but did not affect the promoter activity and m RNA expression of FOXM1, compared with the overexpression of hTERT group.Interfering with FOXM1 could inhibit the expression of m RNA and protein of MMP-9 and inhibit the invasion of tumor cells by P0.01hTERT and FOXM1, and overexpression of hTERT could up-regulate the acetylation level of FOXM1.Conclusion hTERT promotes the invasion of gastric cancer cells by up-regulating FOXM1 acetylation.
【作者单位】：第三军医大学新桥医院消化内科;
【基金】：重庆市基础科学与前沿技术研究项目(CSTC2016jcyjA0570)~~
【分类号】：R735.2

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