miR-515-5p靶向ITLN2对子宫内膜基质细胞增殖、迁移和侵袭的影响

Effect of miR-515-5p targeting ITLN2 on proliferation,migration and invasion of endometrial stromal cells

目的探讨微小RNA-515-5p(miR-515-5p)是否可靶向内凝集素2(lectin 2,ITLN2)影响子宫内膜异位症(endometriosis,EMT)子宫内膜基质细胞(endometrial stromal cells,ESCs)的增殖、迁移和侵袭。方法收集2020年12月至2021年12月期间在济宁医学院附属医院生殖医学科就诊治疗的24例EMT患者的在位内膜和异位内膜组织及18例行腹腔镜手术的非EMT患者的正常内膜组织。RT-qPCR检测内膜组织miR-515-5p、ITLN2 mRNA相对表达量;Western blotting法检测内膜组织中ITLN2蛋白表达量。原代分离、培养EMT患者的ESCs,利用Lipofectamine 2000试剂转染ESCs,并将其分为空白组、miR-515-5p抑制剂组、miR-515-5p抑制剂阴性对照(negative control,NC)组,CCK-8法检测细胞增殖情况;划痕愈合和Transwell实验分别评估细胞迁移、侵袭能力;双荧光素酶报告基因检测miR-515-5p和ITLN2的靶向关系;RT-qPCR技术检测各组细胞miR-515-5p、ITLN2 mRNA相对表达量;Western blotting技术检测ITLN2、白细胞介素(interleukin,IL)-1β、IL-6及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白相对表达量。结果EMT患者在位和异位子宫内膜组织miR-515-5p相对表达量显著高于正常子宫内膜组织(均P<0.001),ITLN2 mRNA相对表达量显著低于正常子宫内膜组织,异位子宫内膜组织ITLN2 mRNA相对表达量显著低于在位子宫内膜组织(均P<0.001),EMT患者异位子宫内膜组织ITLN2蛋白质水平显著低于EMT在位和正常子宫内膜组织(均P<0.001),EMT患者在位子宫内膜组织ITLN2蛋白质水平显著低于正常子宫内膜组织(P<0.001)。miR-515-5p抑制剂组细胞存活率[(54.71±2.78)%]、划痕愈合率[(38.31±3.78)%]及侵袭数[(286.67±25.15)个]、miR-515-5p相对表达量、IL-1β、IL-6、TNF-α蛋白相对表达量均显著低于空白组[(100.00±0.00)%,P<0.001;(84.08±5.14)%,P<0.001;(515.67±22.19)个,P<0.001;P<0.001;P=0.012;P=0.010;P<0.001],ITLN2 mRNA和蛋白相对表达量均显著高于空白组(P<0.001;P<0.001)。经生物信息学分析和双荧光素酶报告基因检测,ITLN2为miR-515-5p的潜在靶基因。结论降低miR-515-5p水平可抑制EMT患者ESCs增殖、迁移和侵袭,这可能与靶向提高ITLN2表达进而降低炎症因子水平有关。

Objective To investigate whether microRNA-515-5p(miR-515-5p)can target the endogenous lectin 2(ITLN2)to affect the proliferation,migration and invasion of endometrial stromal cells(ESCs)in endometriosis(EMT).Methods Eutopic and ectopic endometrium tissues of 24 patients with EMT who attended Department of Reproductive Medicine,the Affiliated Hospital of Jining Medical College for treatment from December 2020 to December 2021,and normal endometrium tissues of 18 non-EMT patients who underwent laparoscopic surgery were selected.RT-qPCR was used to detect the relative expression of miR-515-5p and ITLN2 mRNA,and Western blotting was used to detect ITLN2 protein expression in endometrium tissues.ESCs from EMT patients were isolated and cultured in primary culture.ESCs were transfected with Lipofectamine 2000 reagent,and were divided into blank group,miR-515-5p inhibitor group,miR-515-5p inhibitor negative control(NC)group.Cell proliferation was detected by CCK-8 method.The ability of cell migration and invasion was evaluated by scratch healing and Transwell test.The target relationship between miR-515-5p and ITLN2 was detected by double luciferase reporter gene.RT-qPCR was used to detect the relative expression of miR-515-5p and ITLN2 mRNA in each group.Western blotting was used to detect the relative expression of ITLN2,interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)protein.Results The relative expression of miR-515-5p in EMT eutopic and ectopic endometrium was significantly higher than that in normal endometrium(both P<0.001).The ITLN2 mRNA level in EMT eutopic and ectopic endometrium was significantly lower than that in normal endometrium,and ITLN2 mRNA level in ectopic endometrium was significantly lower than that in eutopic endometrium(all P<0.001).The level of ITLN2 protein in EMT ectopic endometrium was significantly lower than that in EMT eutopic endometrium and normal endometrium(both P<0.001),and the protein level of ITLN2 in EMT eutopic endometrium was significantly lower than that in normal endometrium(P<0.001).In miR-515-5p inhibitor group,the cell survival rate[(54.71±2.78)%],the scratch healing rate[(38.31±3.78)%],invasion number(286.67±25.15),the relative expressions of miR-515-5p,IL-1β,IL-6 and TNF-αwere significantly lower than those of the blank group[(100.00±0.00)%,P<0.001;(84.08±5.14)%,P<0.001;515.67±22.19,P<0.001;P<0.001;P=0.012;P=0.010;P<0.001],the relative expression levels of ITLN2 mRNA and protein were significantly higher than those in the blank group(P<0.001;P<0.001).Through bioinformatics analysis and double luciferase reporter gene detection,ITLN2 was a potential target gene of miR-515-5p.Conclusion Lowering the level of miR-515-5p can inhibit the proliferation,migration and invasion of ESCs in EMT patients,which may be related to the targeted increase of ITLN2 expression,thereby reducing inflammatory factors level.