Regulation By The LncRNA-MALAT1/miRNA-26a-5p/TET1 Signalling Axis Of The Inflammatory Response,Proliferation,Migration,and Epithelial-mesenchymal Transition Of Human Lens Epithelial Cells

Background: HMC(Highly myopic cataract)is a complex cataract with high blindness.Due to the special manifestations of high myopia,HMC is considered to be a relatively independent cataract phenotype.Inflammatory microenvironment in the eyes has been reported to be closely associated with the occurrence and progression of HMC,however,the underlying mechanisms is still unclear.LncRNA-MALAT1(long non-coding RNA metastasis associated lung adenocarcinoma transcript 1)could be involved in inducing cellular inflammatory response by up-regulating the expression of inflammatory mediators.Objective: 1.To detect the expression levels of lnc RNA-MALAT1 and inflammatory factors in the anterior capsular tissue of the lens of HMC patients;2.To establish the inflammatory injury model of lens epithelial cells in vitro and simulate the inflammatory environment in the eyes of patients with HMC.3.To investigate the effect and mechanism of lnc RNA-MALAT1/mi R-26a-5p/TET1 signaling pathway on inflammatory reaction,proliferation,migration,apoptosis and EMT of human len epithelial cells(HLECs).Materials and methods: 1.In vivo,ARC patients and HMC patients with axial length over 26 mm were selected,and the anterior capsule tissues of ARC and HMC patients were obtained during cataract surgery.A part of the anterior capsule tissues were used for the whole-transcriptome sequencing.2.The other part of anterior capsule tissues were used for tissue milling,and then RT-q PCR and Western blotting were conducted to analyze the expression of MALAT1,inflammatory factors,mi R-26a-5p and TET1 at m RNA or protein levels.3.In vitro,TNF-α(20ng/m L)was used to induce inflammatory response in HLECs to simulate the inflammatory microenvironment in HMC patient eyes.4.Shengxin website predicted and screened downstream target genes with binding sites to MALAT1.5.MALAT1 gene expression was knocked down by Si-MALAT1,and specific inhibitors and mimics were used to inhibitor overexpress mi R-26a-5p,respectively.6.q RT-PCR,Western blotting were used to detect the expression levels of MALAT1,mi R-26a-5p,TET1,inflammatory cytokines(IL6,MMP2)and EMT markers(E-cadherin,N-cadherin,Vimentin,Slug,α-SMA).7.Changes in cell proliferation and migration ability before and after treatment were detected by CCK8,Transwell and wound healing assay.8.Flow cytometry was used to detect the changes of apoptosis before and after treatment.Results: 1.After the whole-transcriptome sequencing of anterior capsular tissues,MALAT1 with significantly up-regulated differential expression was selected for follow-up experiments.2.The results of RT-q PCR showed that the expression levels of MALAT1,TET1 and inflammatory factors TNF-α,IL6 and MMP2 in the anterior capsular tissues of HMC were significantly higher than those of ARC,while the expression levels of mi R-26a-5p in anterior capsular tissues of HMC were significantly lower than those of ARC.3.TNF-α(20ng/m L)could cause inflammatory injury in normal HLECs,and significantly up-regulate the expression of MALAT1 and EMT-related genes in HLECs.4.Bioinformatic softwares predicted and screened out that mi R-26a-5p had gene binding sites with MALAT1 and TET1.5.Si-MALAT1 significantly down-regulated the expression of MALAT1,and specific inhibitors and mimics effectively inhibitor overexpressed the expression level of mi R-26a-5p.6.The expression of TET1,inflammatory factors and EMT markers in HLECs decreased after MALAT1 knockdown,while the expression of mi R-26a-5p was up-regulated.Meanwhile,the expression level of MALAT1 was up-regulated or down-regulated after mi R-26a-5p was inhibited or overexpressed.7.TNF-αenhanced the proliferation and migration ability of HLECs,while the proliferation and migration ability of HLECs were decreased after MALAT1 knockout.8.TNF-αaccelerated the apoptosis process of HLECs,and the knockout of MALAT1 further increased the apoptosis rate.Conclusion: 1.The expression levels of lnc RNA-MALAT1 and inflammatory factors in the anterior capsular tissues of HMC patients were increased,and there was an inflammatory microenvironment in the eyes of HMC patients.2.Inflammatory response,cell proliferation,migration and EMT of HLECs were induced by TNF-α,and an inflammatory injury model of lens epithelial cells was successfully established in vitro.3.MALAT1 may act as ce RNA to regulate TNF-α-induced HLECs inflammatory response,proliferation,migration,and EMT through "sponge" mi R-26a-5p and targeting TET1,and may be a potential therapeutic target for prevention of HMC.