Long Non-coding RNA CCAT1 Is Involved In Transforming Growth Factor TGF-β2-mediated Lens Epithelial-mesenchymal Transformation

Objective: 1.Transforming growth factor-β2(TGF-β2)was applied to induce the interstitial transformation of human lens epithelial cells(HLECs)to construct the interstitial cell model.Then,the expression level of Long non-coding RNA colon cancerassociated transcript-1(lncRNA CCAT1)in lens epithelial cells was detected.2.The expression of CCAT1 was knocked down to detect the changes in the expression of interstitial markers at the molecular level.3.To investigate the role of CCAT1 in the interstitial process of HLECs induced by TGF-β2.Methods: In this study,TGF-β2 was used to induce the mesenchymal development of HLECs immortal cell line SRA01/04,and the mesenchymal cell model was established.SRA01/04 cells were cultured in low-glucose DMEM medium containing 10ng/ml TGF-β2 for 48 h as the experimental group,and SRA01/04 cells were cultured in normal low-glucose DMEM medium as the blank control group.The morphological changes of cells were observed by inverted microscope,and the changes in the relative expression levels of lncRNA CCAT1 and epithelial mesenchymal transformation markers were detected by real-time fluorescence quantitative PCR and Western blot.The expression of CCAT1 was silenced by small interfering RNA(si RNA),and the relative expression levels of lncRNA CCAT1 and epithelial mesenchymal transformation markers were detected by real-time fluorescence quantitative PCR and Western blot.To investigate the role of lncRNA CCAT1 in the interstitial process of HLECs induced by TGF-β2 and its effect on the formation of PCO.Results: 1.TGF-β2 induced human lens SRA01/04 cells,and successfully constructed mesenchymal cell model.Under inverted microscope,the morphology of cells in the experimental group changed from polygon to long spindle.Real-time quantitative PCR and Western blot analysis showed that the expression of the epithelial marker protein E-cadherin was significantly decreased in the experimental group compared with the blank control group(P< 0.05),the interstitial cell marker vimentin(p< 0.05),α-smooth muscle actin(α-SMA)(P< 0.05),and the difference was statistically significant.Fluorescence microscopy showed that compared with the blank control group,the expression of α-SMA protein and vimentin in the lens epithelial cells of the experimental group increased,the fluorescence intensity increased,and the cell morphology showed interstitial transformation.The effect of TGF-β2 at different concentrations on lncRNA CCAT1 expression was detected by real-time fluorescence quantitative PCR,and the expression of CCAT1 was concentration dependent.With the increase of TGF-β2 concentration,the expression of CCAT1 increased,and the increase was most obvious at 10ng/ml.Therefore,10ng/ml was selected as the treatment concentration for subsequent experiments.2.The si CCAT1 with high efficiency silencing expression of CCAT1 was successfully screened out.Realtime quantitative PCR results showed that siccat1-1,siccat1-2 and siccat1-3 could all silence the expression of CCAT1,but the interference effect of siccat1-2 was the most obvious.Finally,we chose siccat1-2 for subsequent experiments.3.The expression changes of epithelial marker protein E-cadcadin,interstitial marker protein vimentin and α-smooth muscle actin under different CCAT1 expression levels were detected by real-time fluorescence quantitative PCR and Western blot.The results showed that TGF-β2-treated SINC(labeled TNC),vimentin,and α-smooth muscle actin expression increased compared with SINC(labeled NC)(P < 0.05),the expression of E-cadherin decreased(P<0.05),the difference was statistically significant.Compared with the TNC group,the expression of vimentin and α-smooth muscle actin decreased in the TGF-β2-treated si CCAT1 knockdown group(labeled TLNC group)(P< 0.05),the expression of E-cadherin increased(P<0.05),the difference was statistically significant.Conclusion: LncRNA CCAT1 is involved in the interstitial differentiation of h LECs induced by TGF-β2.Silencing the expression of lncRNA CCAT1 can reverse the interstitial process of HLECs induced by TGF-β2.