miR-17-5p调控LIMK1/cofilin1通路对子宫内膜异位症发生发展的影响

The effect of miR-17-5p regulation of LIMK1/cofilin1 pathway on the occurrence and development of endometriosis diseases

目的:探究微小核糖核酸-17-5p(miR-17-5p)调控LIM激酶1(LIMK1)/丝切蛋白1(cofilin1)通路对子宫内膜异位症发生发展的影响。方法:选取行子宫切除术的30例子宫内膜异位症患者作为子宫内膜异位症在位内膜组,另选取30例非雌激素依赖性疾病患者作为正常子宫内膜组。对两组患者的在位内膜细胞中LIMK1、cofilin1表达量采用实时荧光定量PCR法检测,对所获取的在位内膜细胞进行转染,Transwell法检测细胞的侵袭能力;CCK-8法检测细胞的增殖能力;ELISA法检测子宫内膜细胞中细胞因子ICAM-1、VEGF、MMP-9表达。结果:在位内膜组患者的miR-17-5p表达水平低于正常子宫内膜组(P<0.05);在位内膜组的LIMK1蛋白及其mRNA、cofilin1蛋白及其mRNA相对表达水平均高于正常子宫内膜组(P<0.05);在位内膜组未转染组(EC组)及转染阴性对照(EC-C组)情况下细胞侵袭能力显著高于正常子宫内膜组(NC组),但转染情况下(EC-miR-17-5p-RNAi组)细胞侵袭能力显著低于正常子宫内膜组(NC-miR-17-5p-cDNA组),两组患者细胞转染阴性对照(EC-C组、NC-C组)与未转染状态(EC组、NC组)差异无统计学意义(P>0.05);转染情况在位内膜组患者(EC-miR-17-5p-RNAi组)细胞侵袭力低于未转染状态(EC组),但正常子宫内膜组(NC-miR-17-5p-cDNA组)高于未转染组(NC组,P>0.05)。EC组细胞增殖能力在24 h、48 h、72 h、96 h均高于NC组细胞(P<0.05);EC-miR-17-5p-RNAi组各时间段细胞增殖能力与EC组相比较均升高(P<0.05);EC组与EC-C组在各时间段的细胞增殖能力比较差异无统计学意义(P>0.05);NC-miR-17-5p-cDNA组在各时间段的增殖能力均低于NC组(P<0.05);NC-C组各时间段的细胞增殖能力与NC组比较差异无统计学意义(P>0.05)。基础状态下,EC组ICAM-1、MMP-9和VEGF水平高于NC组,EC-miR-17-5p-RNAi组与EC组比较明显升高(P<0.05);EC组与EC-C组各指标比较差异无统计学意义(P>0.05);NC组ICAM-1、MMP-9和VEGF水平明显高于NC-miR-17-5p-cDNA组(P<0.05),但与NC-C组比较差异无统计学意义(P>0.05)。结论:miR-17-5p在内异症细胞中表达水平下调,通过负向调控LIMK1/cofilin1通路抑制细胞增殖、侵袭,在内异症发生及进展过程中发挥基因调节功能。

Objective To investigate the effect of miR-17-5p regulation of LIMK1/cofilin1 pathway on the occurrence and development of endometriosis diseases.Method 30 patients with endometriosis who underwent hysterectomy were selected as the eutopic endometrium group of endometriosis,and 30 patients with non estrogen dependent diseases were selected as the normal endometrium group.The expressions of LIMKL and cofilinl in the eutopic endometrial cells of the two groups were detected by real-time fluorescent quantitative PCR.The obtained eutopic endometrial cells were transfected,and the invasiveness of the cells was detected by Transwell method;CCK-8 method was used to detect the proliferation ability of cells;The expression of cytokines ICAM-1,VEGF and MMP-9 in endometrial cells was detected by ELISA.Results The expression level of miR-17-5p in eutopic endometrium group was lower than that in normal endometrium group(P<0.05).The relative expression levels of LIMK1 protein and its mRNA,cofilin1 protein and its mRNA in eutopic endometrium group of endometriosis were higher than those in normal endometrium group(P<0.05).The invasiveness of eutopic endometrium group without transfection(EC group)and negative transfection control(EC-C group)was significantly higher than that of normal endometrium group(NC group),but the invasiveness of eutopic endometrium group(EC-miR-17-5p-RNAi group)was significantly lower than that of normal endometrium group(NC-miR-17-5p-cDNA group),There was no significant difference between the cell transfection negative control(EC-C group,NC-C group)and the non transfection status(EC group,NC group)between the two groups(P>0.05);Transfection status:The invasive ability of endometriosis patients in eutopic endometrium group(EC-miR-17-5p-RNAi group)was lower than that in untransfected endometrium group(EC group),but that in normal endometrium group(NC-miR-17-5p-cDNA group)was higher than that in untransfected endometrium group(NC group)(P>0.05).The cell proliferation ability of EC group was higher than that of NC group at 24,48,72,96 h(P<0.05);The cell proliferation ability of EC-miR-17-5p-RNAi group was higher than that of EC group(P<0.05);There was no significant difference in cell proliferation between EC group and EC-C group at each time(P>0.05);The proliferative capacity of NC-miR-17-5p-cDNA group was lower than that of NC group at all times(P<0.05);There was no significant difference in cell proliferation ability between NC-C group and NC group at different time periods(P>0.05).At baseline,the levels of ICAM-1,MMP-9 and VEGF in EC group were higher than those in NC group,and those in EC-miR-17-5p-RNAi group were significantly higher than those in EC group(P<0.05);There was no significant difference between EC group and EC-C group(P>0.05);The levels of ICAM-1,MMP-9 and VEGF in NC group were significantly higher than those in NC-miR-17-5p-cDNA group(P<0.05),but there was no significant difference compared with NC-C group(P>0.05).Conclusion The expression level of miR-17-5p is down regulated in endometriosis cells,which inhibits cell proliferation and invasion by negatively regulating LIMK1/cofilin1 pathway,and plays a role in gene regulation during the occurrence and progression of endometriosis.