HIF1α通过启动ALKBH5调控整合素蛋白αν表达的机制研究

Mechanism of HIF1αregulating integrin proteinανexpression by activating ALKBH5

目的探索缺氧诱导因子1α(hypoxia inducible factor 1α,HIF1α)启动N6-甲基腺苷(N6-methyladenosine,m6A)去甲基化酶ALKBH5改善子宫内膜容受性分子标志物整合素蛋白αν(integrinαν,ITGAV)表达的内在机制。方法利用人子宫内膜癌Ishikawa细胞,低氧以及干扰HIF1α条件下实时荧光定量PCR(real time quantity PCR,RT-PCR)或Western blotting检测ALKBH5的表达;过表达或干扰ALKBH5表达后,RT-PCR或Western blotting检测对ITGAV基因和蛋白表达的影响;荧光素酶报告基因系统分析miR-136-5p靶向ITGAV mRNA表达;RNA pull-down分析及m6A甲基化检测ALKBH5与miR-136-5p竞争性结合ITGAV mRNA上甲基化位点序列。结果低氧条件下HIF1α时间依赖性促进ALKBH5蛋白在Ishikawa细胞中的表达。过表达ALKBH5后,ITGAV的表达水平升高(P<0.001),Ishikawa细胞增殖能力增强(P<0.001);ALKBH5敲除后,ITGAV的表达水平降低(P<0.001),Ishikawa细胞的增殖能力下降(P=0.019)。miR-136-5p靶向调节ITGAV mRNA后,ITGAV mRNA(P=0.007)和蛋白表达水平(P=0.015)下调。miR-136-5p靶向调节ITGAV的序列与ALKBH5所识别的甲基化位点所在序列重叠,两者在调控ITGAV mRNA上存在竞争性。结论HIF1α通过调控ALKBH5,竞争性地结合miR-136-5p所靶向的ITGAV mRNA上特定序列,精细化调节ITGAV的表达,改善子宫内膜容受性。

Objective To explore the intrinsic mechanism of hypoxia inducible factor 1α(HIF1α)initiating N6-methyladenosine(m6A)demethylase ALKBH5 expression to improve the expression of integrin αν(ITGAV),a molecular marker of endometrial receptivity.Methods Human endometrial cells Ishikawa were used in this study.The expression of ALKBH5 was detected by real time quantity PCR(RT-PCR)and Western blotting under hypoxia and HIF1α interference conditions.After overexpression or interference ALKBH5,RT-PCR and Western blotting were used to detect ITGAV expression;Luciferase reporter gene system was used to detect miR-136-5p targeting ITGAV mRNA expression;RNA pull-down analysis and m6A methylation were used to detect the competitive binding of ALKBH5 and miR-136-5p to the methylation site sequence on ITGAV mRNA.Results HIF1α promoted ALKBH5 protein expression in a time-dependent manner under hypoxic conditions in Ishikawa cells.Overexpression of ALKBH5 enhanced the expression of ITGAV and cell proliferation(P<0.001,P<0.001),while knockout of ALKBH5 inhibited the expression of ITGAV and cell proliferation(P=0.001,P=0.019).ITGAV mRNA(P=0.007)and protein expression level(P=0.015)were significantly down-regulated by miR-136-5p.And the location of mRNA sequence by miR-136-5p targeted overlapped with the methylation site recognized by ALKBH5,furthermore competed in regulating ITGAV mRNA.Conclusion HIF1α regulates the expression of ITGAV and improves endometrial receptivity by regulating the expression of ALKBH5 and competitively binding to the specific sequence on ITGAV mRNA targeted by miR-136-5p.