| objective: 1.Construction of transforming growth factor(TGF)-?2-induced epithelial-mesenchymal transdifferentiation(EMT)of human lens epithelial cells(HLECs)cell model and investigation of the expression of long non-coding RNA(lnc RNA)HOTAIR of HLECs.2.To explore the role of HOTAIR in the proliferation and migration of HLECs and to observe the role of HOTAIR in TGF-?2-induced proliferation,migration and EMT of HLECs.3.Detecting the expression of the main signal molecules of TGF-?/Smad pathway in TGF-?2-induced EMT in HLECs and confirming whether HOTAIR is involved in EMT of HLECs through TGF-?/Smad pathway.Methods: 1.To construct TGF-?2-induced EMT of HLECs(SRA01/04),and to investigate the expression of HOTAIR in SRA01/04 cells.SRA01/04 cells were cultured and stimulated with 10 ng/ml TGF-?2 for 24 h.Observing changes in cell morphology using a microscope.Western blotting and quantitative RT-PCR(qRT-PCR)were then performed to analyze the expression levels of EMT markers including epithelial markers E-cadherin?zonula occluden-1(ZO-1)and mesenchymal markers vimentin?alpha smooth muscle actin(?-SMA).HLECs were incubated with TGF-?2(10 ng/m L)for indicated times(0h,12 h,24h,48 h,72h)and then the expression of HOTAIR was detected by qRT-PCR.2.Down-regulate the expression of HOTAIR and explore the role of HOTAIR in the proliferation and migration of human lens epithelial cells.Transfection of si HOTAIR-1,si HOTAIR-2,si HOTAIR-3 and control si NC in SRA01/04 cells using RNA small interference technique,and detection of transfection efficiency.Then CCK-8 assay,Ed U staining,transwell migration assay were used to analyze the viability,proliferation and migration of SRA01/04 cells.3.To observe the role of HOTAIR in TGF-?2-induced HLECs proliferation,migration and EMT,and to detect the expression of EMT-related transcription factors.The cells were divided into four groups: si NC group: cells were transfected with si NC and routinely cultured for 24 hours;si HOTAIR: cells were transfected with si HOTAIR and routinely cultured for 24 hours;si NC+TGF-?2 Group: cells were transfected with si NC and followed by treatment with 10 ng/ml TGF-?2 for 24 hours;si HOTAIR+TGF-?2 group: cells were transfected with si HOTAIR and followed by treatment with 10 ng/ml TGF-?2 for 24 hours.The morphology of the cells was observed by microscopy.The expressions of EMT-related proteins(E-cadherin,ZO-1,vimentin,?-SMA)were detected by immunofluorescence and western blot.Western blot was used to detect the expression of transcription factors ZEB1,Snail and Slug.The viability of SRA01/04 cells was analyzed by CCK-8 assay,the proliferation of SRA01/04 cells was analyzed by Ed U staining.The migration of SRA01/04 cells was evaluated by wound-healing assay and transwell migration assay.4.The expression of the main signaling molecules of TGF-?/Smad pathway in HLECs stimulated by TGF-?2.After HOTAIR in SRA01/04 cells was down-regulated,the expression of TGF-?/Smad pathway proteins was detected.SRA01/04 cells were treated with TGF-?2(10 ng/ml).The expression of p-Smad2,p-Smad3 and total Smad2,Smad3 were examined by western blot.SRA01/04 cells transfected with si HOAIR or si NC were treated with TGF-?2(10 ng/ml).The expression of p-Smad2,p-Smad3 and total Smad2,Smad3 was examined by western blot.Results: 1.Constructing a cell model of TGF-?2-induced EMT of HLECs(SRA01/04)successfully and investigating the up-regulation of the expression of HOTAIR in SRA01/04 cells.Inverted microscope observed that compared with the control group(not treated TGF-?2),after incubated with TGF-?2 for 24 h,the experimental group cells underwent an EMT transition,as confirmed by its morphological change from elliptical-shaped cells to a spindle-shaped cells.qRT-PCR and Western blotting were then performed to detect the TGF-?2-induced EMT of HLECs and found that compared with the control group,the experimental group cells underwent an EMT transition,as confirmed by decreased expression of epithelial markers E-cadherin and ZO-1,as well as enhanced expression of mesenchymal markers,including vimentin and ?-SMA.The expression of HOTAIR in HLECs was detected by qRT-PCR after TGF-?2 stimulated at different time points.The results showed that the expression of HOTAIR began to increase at 12 h after TGF-?2 treatment and reached an apex at 48h(P < 0.01).2.Screening si HOTAIR to reduce the HOTAIR expression level successfully.HOTAIR knockdown in lens epithelial cells can inhibite the survival,proliferation and migration of HLECs.The results of qRT-PCR showed that compared with the control group,si HOTAIR-2 and si HOTAIR-3 could effectively down-regulate the expression of HOTAIR(P < 0.01)and the difference was statistically significant.Finally,si HOTAIR-3 was selected for subsequent experiments.After SRA01/04 cells were transfected with si HOTAIR,the results of CCK-8 assay,EDU staining,transwell migration assay showed that compared with the control group,cell viability ability,proliferation ability and migration ability were significantly decreased(P < 0.01),the difference is statistically significant.3.Down-regulating the HOTAIR expression inhibits TGF-?2-induced lens epithelial cell proliferation,migration and EMT,as well as decreases TGF-?2-induced upregulation of EMT-associated transcription factors.Inverted microscope observation showed that compared with the control group,after incubated with TGF-?2 for 24 h,the experimental group cells underwent an EMT transition,as confirmed by its morphological change from elliptical-shaped cells to a spindle-shaped cells,and after HOTAIR knockdown,the cell morphology was reversed.Cellular immunofluorescence analysis and western blot analysis showed that compared with si NC group,after treatment with TGF-?2 for 24 hours,the expression of E-cadherin and ZO-1 was decreased and the expression of Vimentin and ?-SMA was increased in si NC+TGF-?2 group(P < 0.01).Compared with si NC+TGF-?2 group,after transfected with si HOTAIR-3,the expression of E-cadherin and ZO-1 was increased and the expression of Vimentin and ?-SMA was decreased in si HOTAIR+TGF-?2 group(P < 0.01),the difference is statistically significant. Western blot analysis showed that the expression of Snail,Slug and ZEB1 in si HOTAIR group was significantly decreased compared with si NC group(P < 0.01),the difference was statistically significant.The expression of Snail,Slug and ZEB1 in si NC+TGF-?2 group was upward compared with si NC group(P < 0.01),the difference was statistically significant.The expression of Snail,Slug and ZEB1 in si HOTAIR+TGF-?2 group was significantly decreased compared with si NC+TGF-?2 group(P < 0.01),the difference was statistically significant.The results of CCK-8 assay,EDU staining,wound-healing assay and transwell migration assay showed that the survival ability,proliferation ability and migration ability of si HOTAIR group were lower than those of si NC group(P < 0.05).In comparison to si NC group,the survival ability,proliferation ability and migration of si HOTAIR+TGF-?2 group were raised(P < 0.01).The survival,proliferation and migration ability of si HOTAIR+ TGF-?2 group were significantly decreased compared with si NC+TGF-?2 group(P < 0.01),the difference was statistically significant.4.The main signaling molecules of TGF-?/Smad pathway are involved in TGF-?2-induced EMT in lens epithelial cells,and HOTAIR plays a role in EMT of HLECs through TGF-?/Smad pathway.Western blot analysis showed that compared with the control group,phosphorylated Smad2 and Smad3 protein expression in SRA01/04 cells in TGF-?2 treatment group increased(P < 0.01),the difference was statistically significant.The expression levels of p Smad2 and p Smad3 in si HOTAIR group was significantly decreased compared with si NC group(P < 0.01).After treated with TGF-?2,in comparsion to si NC group,p Smad2 and p Smad3 expression were raised in si NC+TGF-?2 group.si HOTAIR was silenced by si HOTAIR-3.The expression levels of p Smad2 and p Smad3 in si HOTAIR+TGF-?2 group was significantly decreased compared with si NC+TGF-?2 group.Conclusion: 1.10ng/ml TGF-?2 can induce HLECs undergo EMT.The expression of HOTAIR is up-regulated in HLECs.2.HOTAIR knockdown inhibits survival,proliferation and migration of human lens epithelial cells.3.HOTAIR is involved in TGF-?2-induced survival,proliferation,migration and EMT of lens epithelial cells,suggesting that HOTAIR could play a significant role in the pathogenesis of PCO and provide a new understanding for its diagnosis and treatment.However,the lack of the experiments in vivo,it is necessary to improve in the future.4.HOTAIR can promote survival,proliferation,migration and EMT of HLECs via TGF-?/Smad pathway. |
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