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