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MicroRNA (miRNA) is a kind of small non-coding RNA, which can specifically bind to the 3' untranslated region of the target RNA, inducing the degradation or inhibiting the translation of the target mRNA, and ultimately affecting the important biological processes such as cell proliferation, differentiation, and apoptosis.Cataract, a leading blinding eye disease in the world, is a kind of disease that causes blindness because of lens opacification, including age-related cataracts, diabetic cataracts, congenital cataracts and posterior capsule opacification.In recent years, it has been found that many kinds of miRNA are expressed in lens and participate in the development of cataract, having significant influences on the proliferation, migration, epithelial-mesenchymal transition and apoptosis of lens epithelial cells, and take part in the occurrence and development of cataracts.The advances of different miRNAs in cataract were reviewed in this article so as to provide new ideas and methods for the prevention and treatment of cataract.
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Objective:To investigate the effect of microRNA-15a (miR-15a) on the anti-oxidative stress ability of human lens epithelial cells (LECs) induced by high glucose and its possible mechanism.Methods:The anterior lens capsule specimens from patients with diabetic cataract (DC) and healthy donors were collected.The expressions of miR-15a and silent information regulator 1 (SIRT1) in the specimens were detected by real-time quantitative PCR (RT-qPCR) and Western blot, respectively.The human lens epithelial cell line HLEB-3 cells were cultured with 0, 10, 20, or 50 mmol/L glucose for 24 hours.The expression of miR-15a in the cells was detected by RT-qPCR.The expressions of SIRT1, forkhead transcription factor 3a (FOXO3a), and p53 proteins in the cells were determined by Western blot.The cell apoptosis was assayed by flow cytometry.The endogenous reactive oxygen species (ROS) content in the cells was measured by 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) activities in the cells were identified.HLEB-3 cells were transfected with miR-15a control or miR-15a inhibitor, then incubated with 50 mmol/L glucose for 24 hours.Cell apoptosis of the transfected cells was detected by flow cytometry.The endogenous ROS expression in the transfected cells was determined by DCFH-DA.The T-AOC, SOD, and GSH-Px activities as well as MDA concentration were measured.The relationship between miR-15a and SIRT1 was verified by dual-luciferase reporter assay.The SIRT1, FOXO3a, and p53 protein expressions in the transfected cells were detected by Western blot.This study was approved by an Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (No.ZDEFY201803160023). Written informed consent was obtained from each subject.Results:The relative expression of miR-15a in normal lens anterior capsule tissue was 0.21±0.02, which was lower than 0.96±0.10 in lens anterior capsule tissue of DC patients, and the difference was statistically significant ( t=12.231, P<0.001). The relative expression of SIRT1 in the anterior lens capsule tissue was 0.89±0.09, which was higher than 0.31±0.05 in the anterior lens capsule tissue of DC patients, showing a statistically significant difference ( t=8.964, P<0.001). With the increase of glucose concentration, the relative expression of miR-15a, FOXO3a, and p53 in cells increased, and the relative expression of SIRT1 decreased; the apoptosis rate of cells increased; the ROS content and MDA concentration increased; the activities of T-AOC, SOD and GSH-Px decreased, and the differences were statistically significant (all at P<0.05). The apoptosis rate, ROS content, and MDA concentrations were higher in miR-15a control group than miR-15a inhibitor group, and the activities of T-AOC, SOD, and GSH-Px were lower in miR-15a control group than miR-15a inhibitor group, with statistically significant differences (all at P<0.05). The luciferase activity of the SIRT1-3'-untranslated region (UTR)-wild type (WT) reporter gene in miR-15a control group was significantly lower than that in miR-15a inhibitor group, and the difference was statistically significant ( t=5.978, P=0.004). No significant difference was found in the luciferase activity of the SIRT1-3'-UTR-mutant type (MUT) reporter gene ( t=0.432, P=0.688). The relative expressions of FOXO3a and p53 proteins were significantly higher in miR-15a control group than miR-15a inhibitor group, and the relative expression of SIRT1 protein was significantly lower in miR-15a control group than miR-15a inhibitor group, showing statistically significant differences (all at P<0.05). Conclusions:miR-15a can inhibit the anti-oxidative stress damage ability of LECs induced by high glucose, which may be achieved by inhibiting the expression of SIRT1 to up-regulate the activities of FOXO3a and p53, and aggravating apoptosis.
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Objective:To investigate the role of microRNA (miR)-155 in hydrogen peroxide (H 2O 2)-induced oxidative stress injury in lens epithelial cells (LECs) and its mechanism regulating silent information regulator factor related enzymes 1 (SIRT1). Methods:The HLE-B3 at the logarithmic growth phase was taken and cultured for 24 hours under different concentrations of H 2O 2 (0, 50, 100, 200, 400, 800 μmol/L), and the cell viability was detected by MTT assay to determine the optimal concentration of H 2O 2 for establishing an oxidative stress injury model.HLE-B3 cells were divided into 6 groups, untreated blank control group, model control group cultured with 100 μmol/L H 2O 2, miR-155 mimics group transfected with miR-155 mimics, miR-155 mimics negative control group transfected with miR-155 mimics negative control, miR-155 inhibitor group transfected with miR-155 inhibitor, and miR-155 inhibitor negative control group transfected with miR-155 inhibitor negative control.Transfected cells were cultured with 100 μmol/L H 2O 2.Cells in various groups were cultured for 24 hours, and cell morphology was observed under an inverted microscope.The relative expression of miR-155 and SIRT1 mRNA in cells was assayed by fluorescent quantitative PCR.Cell apoptosis rates were detected by flow cytometry.Reactive oxygen species (ROS) content was identified by 2', 7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe method.Superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration were measured by ELISA method.The targeting of SIRT1 by miR-155 was tested by dual luciferase reporter gene system.Expressions of SIRT1, B-cell lymphoma/leukemia-2 gene (bcl-2), bcl-2 associated X protein (bax), cleaved-cysteine aspartase 3 (cleaved-Caspase-3) proteins were determined by Western blot. Results:With the increase of H 2O 2 concentration, the cell viability gradually decreased, and the differences in cell viability among different concentrations were statistically significant (all at P<0.05), and 100 μmol/L was selected as the experimental concentration.Cells in blank control group grew well adherently.The number of cells in model control group decreased, and the morphology of some surviving cells changed, and their boundaries were blurred.There were fewer cells in miR-155 mimics group than model control group, and the cell morphology changed.There were more cells in miR-155 inhibitor group than model control group, and the cells grew well.Compared with model control group, the relative expression level of miR-155, the apoptosis rate, ROS content, MDA concentration, as well as the relative expression levels of bax and cleaved-Caspase-3 proteins were increased, and the relative expression level of SIRT1 mRNA, the SOD activity, the relative expression of SIRT1 and bcl-2 proteins, as well as bcl-2/bax were decreased in miR-155 mimics group, and the differences were statistically significant (all at P<0.05). Compared with model control group, the relative expression of miR-155, the apoptosis rate, ROS content, MDA concentration, as well as the relative expression levels of bax and cleaved-Caspase-3 proteins were decreased, and the relative expression level of SIRT1 mRNA, SOD activity, the relative expression levels of SIRT1 and bcl-2 protein, as well as bcl-2/bax were significantly increased in miR-155 inhibitor group, and the differences were statistically significant (all at P<0.05). The relative luciferase activity of wild-type SIRT1 in cells transfected with miR-155 mimics was 0.41±0.07, which was significantly weaker than 1.00±0.11 in cells transfected with miR-155 mimics negative control, and the relative luciferase activity of wild-type SIRT1 in cells transfected with miR-155 inhibitor was 1.98±0.17, which was significantly higher than 1.00±0.12 in cells transfected with miR-155 inhibitor negative control, showing statistically significant differences ( t=7.838, 8.157; both at P<0.05). No obvious effect on the relative luciferase activity of mutant SIRT1 was found in transfected cells. Conclusions:miR-155 is involved in H 2O 2-induced oxidative damage of LECs, and its overexpression can target the expression of SIRT1 and play a role in cell injury.
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Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.
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Posterior capsular opacification (PCO) is a common complication after extracapsular cataract extraction,which is drawing more attentions because of secondary vision loss,and the study on PCO pathogenesis mechanism is a key for the targeting prevention and treatment of PCO.The study of PCO pathogenesis mechanism showed that autophagy and apoptosis are associated with PCO,and it was also determined that the activation of related signal-transduction pathway plays an important role in PCO formation,for example,the release of inflammatory factors and cytokines following cataract extraction activate the signal transduction and genetic transcription of lens epithelial cells (LECs) and further promote the proliferation,migration and epithelial-mesenchymal transition (EMT) of residual LECs,which is a pathological basis of PCO.It is a challenge for us to investigate the effective treating method of PCO basis on its pathogenesis.Up to now,the studies of drugs targeting PCO and genetic therapy which based on the advances in epigenetics have made great progress.Ophthalmic researchers should pay close attention to the latest trends of basic research,track the methodology and exploit the emerging spotlight,explore the novel means of treatments of PCO,and expand the promising future of PCO prevention and treatment.
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Background Ultraviolet B (UVB) is one of the main causes of cataract formation,and its mechanism is associated with the apoptosis of lens epithelial cells (LECs).MiroRNA-133b (miR-133b) can regulate oxidative stress-induced LECs apoptosis.However,whether miR-133b is associated with UVB-induced cataract is not elucidated.Objective This study was to observe the inhibitory effects of miR-133b on UVB-induced cataract and its regulating mechanism.Methods Twenty 8-week-old C57BL/6 mice were randomized into cataract model group and normal control group.The mouse eyes in the cataract model group exposed to 302 nm UVB for 5 minutes once per day for consecutive 1 week,with the irradiation intensity of 300 W/cm2,and the mice in the normal control group did not receive any intervention.Five mice in each group were sacrificed and 10 eyeball sections were prepared.Human LECs (SRA01/04) were exposed to UVB for 25 minutes and served as UVB-induced group,the cells in the normal control group did not receive any intervention.The UVB-induced cells were inoculated to 96-well plate and divided into 4 groups,and 50 nmol/L miR-133b mimic,miR-133b mimic control agent,miR-133b inhibitor and miR-133b inhibitor control agent were transfected into the cells with lipofectamine2000.The expression of miR-133b mRNA and a target gene BCL2L2,which was identified by online miRNA database (www.mirab.org) in the cells were detected by real-time quantitative PCR to evaluate the transfected efficacy,and the apoptosis of the cells in mouse lens tissue and different transfected groups were assayed by TUNEL.The use and care of the mice followed ARVO Statement.Results The arrangement of LECs was regular and no apoptotic cell was seen in the normal control group,and the apoptotic cells showed the red fluorescence in the cataract model group.The apoptotic rate of human LECs was (43.90±9.30) ％ in the UVB-induced group,and that in the normal control group was (1.08±0.49)％,showing a significant difference between the two groups (t =-7.963,P =0.015).The relative expression levels of miR-133b mRNA in the model mouse lens and UVB-induced human LECs were evidently lower and relative expression levels of BCL2L2 mRNA were higher than those in normal mice and normal LECs (miR-133b mRNA:t =-2.958,P =0.042;t =-6.195,P =0.003;BCL2L2 mRNA:t =3.761,P =0.020;t =12.437,P =0.000).The relative expression level of miR-133b mRNA was significantly increased and the relative expression level of BCL2L2 mRNA was reduced in the miR-133b mimic group in comparation with the miR-133b mimic control group (t=10.883,-5 927.617;both at P＜ 0.01);compared with the miR-133b inhibitor control group,the relative expression level of miR-133b mRNA was significantly decreased and that of BCL2L2 mRNA was evidently increased in the miR-133b inhibitor group (t =-1 606.622,17.556;both at P ＜ 0.01).The apoptotic rate of human LECs was (43.62 ± 9.19) ％ and (17.55 ± 4.24) ％ in the miR-133b mimic control group and miR-133b mimic group,with a significant difference between them (t =-4.462,P =0.011),and the apoptotic rate in the miR-133b inhibitor group was (78.23 ± 12.42) ％,which was significantly higher than (48.01 ±9.68) ％ in the miR-133b inhibitor control group (t =3.324,P =0.029).Conclusions miR-133b can prevent UVB-induced cataract probably by negatively targeting the BCL2L2 expression to regulate the apoptosis of LECs.
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Background Excessive production,deposition and contraction of extracellular matrix (ECM) of human lens endothelial cells (LECs) is one of main causes to posterior capsular opacification (PCO).Researches indicated that Wnt3a protein was involved in production of ECM and fibrosis in epithelial cells,but its effect on LECs is still unclear.Objective This study aimed to elucidate the roles of Wnt3a in production of ECM and gel contraction of LECs.Methods Lipofectamine-mediated transient transfection technique was used to introduce cDNA of Wnt3a gene and pcDNA3-HA vector into the LEC cell line SRA01/04 to act as Wnt3a transfection group and control group respectively.After 48 h of transfection,Western blot was used to detect the expression of Wnt3a,main components of ECM Col-Ⅰ,Col-Ⅳ and integrin β1.Immunofluorescence was used to detect the expression and distrubition of α-SMA and F-actin;collagen contraction was observed by mingling SRA01/04 cells with Col-Ⅰ.Results After 48 hours of transfection using lipofectamine 2000,the expressions of wnt3a protein in SRA01/04 cells was 0.703 ±0.105 in the wnt3a transfected group,and that in the control group was 0.290 ± 0.066,showing a significant difference between the two groups (t =5.782,P＜0.01).Western blot assay showed that the expression levels of Col-Ⅰ and Integrin β1 was 0.697±0.021 and 0.875±0.055 in the Wnt3a transfected group,and which was significantly higher than 0.370±0.020 and 0.580±0.030 in the control group (t =19.600,8.156,both P＜0.01).The expression level of Col Ⅳ in the Wnt3a transfected group was higher than that of the control group (0.430±0.020 vs 0.383 ±0.031),but the difference was not significant (t =2.514,P＞0.05).Immunofluorescence assay revealed that F-actin and α-SMA were weakly expressed in the cell membrane primarily in the control group,while they were strongly expressed in the cell membrane,cytoplasma in the Wnt3a transfected group.Tewnty-four hours after addtion of Col-Ⅰ,the gel contraction area ratio appeared to be more obvious in comparison with the 8 hours (64.1％ ±2.3％ vs 98.9％ ± 1.0％),and gel contraction area ratio was lower in the Wnt3a transfected group than that in the control group (64.1％ ± 2.3％ vs 93.9％ ± 3.1％).Conclusions The overexpression of Wnt3a activates the production of ECM,and the remodeling of celluar skeleton and cellular contraction.
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Posterior capsular opacification (PCO),also known as after-cataract,is the most frequent complication and the primary cause for visual decrease after extracapsular cataract surgery.At present,there is no effective way to treat PCO,so more attentions are focused on preventive reseaching of PCO and treatment methods.Although a variety of studies have increased our understanding of the pathogenesis of PCO,the cellular mechanisms responsible for PCO are still unclear.Cultured capsular bag model in vitro could effectively simulate lens capsular membrane and cells survival environment after cataract extraction and IOL implantation.However,lens capsular bag cultivation with different methods have their own characreristics.The material source,preparation methods of capsular bag model,characreristics of materials which maintain capsular bag contours and its application in PCO were reviewed.
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Background Human LECs can express telomerase activity,which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis,but whether estrogen exerts its role on human LECs is still unclear.Objective This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs.Method Human LECs line was cultured and passaged in vitro,and 1×10-6 mol/L β-E2 was added in the medium for 0,6,12,24 and 48 hours,and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1％ was added in the solvent control group,and 1 × 10-8,1 × 10-7 or 1 × 10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups,respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8,and the apoptosis rate of the cells was examined by Hoechst33258 staining.Results The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015,0.712±0.013 and 0.914±0.031 in the 1 ×10-8,1 ×10-7 and 1 ×10-6 mol/L β-E2 group,which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group (all at P＜0.05).The telomerase activity values (absorbance) were 0.711 ±0.015,0.941±0.010 and 1.249±0.047 in the 1×10-8,1×10-7and 1×10-6 mol/L β-E2 group,which were higher than 0.535±0.013 in the blank control group and 0.543 ±0.013 in the solvent control group (all at P =0.000).The proliferantive values of the cells (absorbance) were significantly raised in the 1 × 10-8 mol/L β-E2 group compared with l × 10-7 and 1 × 10-6 mol/L β-E2 group (both at P =0.000),and no significnant difference was found in the proliferetive values between the blank control group and the solvent control group (P =0.718,0.856).The apoptosis rates of the cells in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L β-E2 group were lower than those in the the blank control group and the solvent control group (all at P=0.000),and there was no significant difference between the blank control group and the solvent control group (P =0.777).No obvious correlation was found between the HLECs preliferative values and hTERT mRNA expression levels or telomerase activity values (r=-0.299,P=0.278;r=-0.157,P=0.576).However,significantly negative correlations were seen between apoptosis rates and hTERT mRNA expression levels or telomerase activity values (r =-0.975,P=0.000;r=-0.981,P=0.000).Conclusions β-E2can increase the activity of telomerase in human LECs,and high dose of β-E2can inhibit apoptosis,but it dose not promote proliferation.
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Background Researchers showed that posterior capsule opacification (PCO) may be associated with the materials of intraocular lenses (IOLs).However,there have long been controversies about the influence of IOLs materials on PCO pathogenesis.Objective This study was to compare the capsule biocompatibility of different materials of IOLs by observing the biological behavior of human lens epithelial cells (LECs) on the surface of IOLs,including adhesion,proliferation,epithelial mesenchymal transition (EMT) and the secretion of transforming growth factor-β2(TGF-β2),interleukin-6 (IL-6),matrix metalloproteinase-2 (MMP-2) and MMP-9 in vitro.Methods Human lens epithelial cell line (HLE-B3) was cultured on the surface of hydrophobic acrylic (Acrysof SA60AT) IOLs,silicone (Crystalens HD) IOLs and polymethyl methacrylate (PMMA) IOLs for 6 hours and 24 hours,respectively.The number and morphology of HLE-B3 cells on the surface of IOLs were observed under the optical microscope.The proliferation states of the cells on the IOL surface were detected with cell counting kit-8 (CCK-8).The expression of α-smooth muscle actin (α-SMA),a mesenchymal cell marker,in the cells was detected by immunofluoreseence technology,and the EMT rates of HLE-B3 cells were calculated.The contents of TGF-β2,IL-6,MMP-2 and MMP-9 in the medium of IOL surface were measured by ELISA assay.The examination results were compared among different IOLs.Results Six hours after cultured,attached cells showed polygon and round in shape on the surface of Acrysof IOLs and Crystalens IOLs,while those on the PMMA IOLs showed the fusiform.Twenty-four hours after cultured,the cells extended obviously.On the surface of Acrysof IOLs and Crystalens IOLs,the adherent cells showed less cobble-stone like and more spindle shape;while those on the PMMA IOLs showed the typical fiber and some cells clustered the pearl rolls.The number of the cells on the Acrysof IOLs,Crystalens IOLs and PMMA IOLs was 0.238 4 ± 0.007 1,0.178 1 ±-0.006 6 and 0.158 9 ± 0.006 9 respectively,showing a significant difference among the three types of IOLs (F =475.947,P =0.000),and the number of the cells was much more on the Acrysof IOLs than that on the Crystalens IOLs or PMMA IOLs (both at P＜0.001).The EMT rats of the cells on the Acrysof IOLs,Crystalens IOLs and PMMA IOLs were (9.99±3.80) ％,(17.33±5.71) ％ and (84.16±10.48) ％,with a significant difference among them (F =127.411,P =0.000),and the EMT rates of the cells on the PMMA IOLs were significantly higher than those on the Crystalens IOLs and A.crysof IOLs(both at P＜0.001).There were significant differences in the contents of TGF-β2,IL-6,MMP-2 and MMP-9 in the medium on the Acrysof IOLs,Crystalens IOLs and PMMA IOLs (F =0.846,0.947,0.255,0.922,all at P ＞ 0.05).Conclusions The hydrophobic acrylic IOLs have the best capsule biocompatibility followed by the silicone IOLs and then the PMMA IOLs.
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Background Epithelial-mesenchymal transition (EMT) is one of the pathogenesis mechanisms of posterior capule opcification (PCO).Studying EMT is of important significance for the prevention and treatment of PCO.However,EMT model is lack.Objective This study was to establish an in vitro EMT model for the study of human PCO.Methods In vitro mimic cataract enucleation was performed on 40 donor eyes,including anterior capsulorhexis,nucleus hydroexpression,and aspiration of lens fibers.The capsular bag of lens was dissected free during the surgery and pinned flat on a plastic culture dish with DMEM/F12 supplemented containing 10％ fetal bovine serum for 4 weeks.The proliferation of LECs on the capsular bag was observed by phase-contrast and dark-field microscope in 0,3,7,14 and 28 days after culture.The capsular bag tissues were collected in cultured 0,3,7 and 28 days for the preparation of sections and hematoxylin-eosin stain,and the growth and morphology of LECs were examined with optical microscope.The expression and location of α-SMA,E-cadherin and Vimentin were assessed by immunochemistry.The expression levels of α-SMA,E-cadherin and Vimentin mRNA and proteins were detected by using real-time fluorescnce quantitative PCR and Western blot in different time points.Results No LECs were seen on the uncultured capsular bag.LECs appeared in cultured day 3 on the periphery capsular bag and grew toward the center and covered the posterior capsule 7 days after cultured,with a cobblestone-like appearance.Wrinkles occurred and extended gradually along with the enhancement of bag tension.Immunochestry showed that the expression intensity of E-cadherin in the capsular bag gradually weakened,and that of α-SMA or Vimentin was gradually enhanced during the culture duration.The relative expression levels of E-cadherhin mRNA at 0,3,7,14 and 28 days after culture were 3.35±0.13,1.47±0.20,1.13±0.14,1.00±0.85 and 0.23±0.03,and relative expression levels of Vimentin mRNA were 1.00 ± 0.73,1.05 ± 0.14,2.24 ± 0.43,2.84 ± 0.34 and 8.57 ± 0.40,and those of α-SMA mRNA were 1.00 ± 0.06,2.68 ± 0.28,4.24 ± 0.05,2.05 ± 0.90 and 15.30 ± 0.19,showing significant differences among different time points (E-cadherhin mRNA:F =23.430,P =0.000;Vimentin mRNA F =8.915,P =0.002;α-SMA mRNA:F =103.500,P =0.000),with the lowest expression levels in the E-cadherhin mRNA and the highest expression levels in the Vimentin mRNA and α-SMA mRNA at 28 days during the culture period (all at P＜0.01).The gray values of E-cadherin protein expression were 1.443 ± 0.017,1.023 ± 0.003 and 0.568 ± 0.018,and those of Vimentin protein were 0.565±0.012,1.156±0.007 and 1.241±0.009,and those of α-SMA protein were 0.195±0.045,0.693±0.036 and 1.501±0.005 at 0,3 and 28 days,with significnant differences among various time points (E-cadherin:F =2 787.000,P =0.000;Vimentin:F =4 488.000,P =0.000;α-SMA:F =1 173.000,P =0.000).The expression levels were significantly declined in E-cadherhin protein and elevated in Vimentin and α-SMA proteins at 3 and 28 days after culture in comparison with before culture.Conclusions A novel in vitro EMT model of LECs is established in this study.This model can mimic a natural EMT procedure after extracapsular cataract enucleation and therefore is a useful model for the further research of the mechanism and prevention and treatment of PCO.