Expression And Function Of MiR-30a In Lens Epithelial Cells Of Diabetic Cataract

BackgroundDiabetic cataract(DC)is one of the common complications of diabetes The characteristics of DC are the younger ages when the lens becomes opaque,faster progress and severity of lens opacity.Currently,the only treatment is to remove the opaque lens through surgery.Although the surgical devices and techniques have been improved,the incidence of surgery complications in diabetic patients is greater.With the large increase of diabetic patients in recent years,DC is getting more and more attention.However,the molecular mechanism during the process of DC formation remains unknown.Epithelial-mesenchymal transition(EMT)occurs when lens epithelial cells(LECs)lose the normal differentiation phenotype and become mesenchyme cells.The presence of EMT in LECs has been involved in the progress of cataract formation.Autophagy is essential for cell survival and development to remove abnormal proteins and organelles so as to realize recycle nutrients.The degradation and clearance of LECs protein aggregation and organelles through an autophagosomal-lysosomal pathway is critically important for LECs function as well as maintaining lens transparency.It plays pathophysiologic roles in many types of cataracts.MicroRNAs(miRNAs)are a group of non-coding small RNAs,approximately 22 nucleotides in length.They bind to the mRNA 3' UTR of the target gene to regulate the protein coding gene from the post-transcriptional level.The regulations of miRNAs on EMT and autophagy have been shown in multiple tissues and become a new perspective study,which make miRNAs a potential molecule for the treatment of EMT and autophagy disorder diseases.Recently,the functions of miRNAs in multiple eye diseases have been revealed,including diabetic keratopathy,primary angle closure glaucoma,retinopathy and cataract.By now,the regulations of miRNAs on EMT and autophagy in DC remain unknown.miRNA microarray is a standard method for detecting miRNAs expression profiles,which that can be used to identify the differentially expressed miRNAs between LECs attaching on normal lens and DC tissues.Combined with the bioinformatic prediction of miRNAs and function study can help find the role of miRNAs in pathogenesis of DC.ObjectiveIn this study,we identified the expression of miRNAs in LECs attaching on DC tissues.By using the high glucose(HG)models in vitro,we studied the regulatory mechanism of miR-30a on EMT and autophagy in LECs so as to find the role of miR-30a in the DC pathogenesis.Methods1.To investigate the expression of miRNAs in LECs attaching on normal lens and DC tissues,a miRNA microarray analysis was performed.Quantitative RT-PCR(qRT-PCR)was employed to confirm the expression of miRNAs(miR-30a).2.According to the bioinformatic database,the target genes of miR-30a were found.A dual luciferase reporter assay was performed to test the direct link between miR-30a and the predicted target genes.3.To test the levels of EMT and autophagy in DC LECs,immunohistochemistry and Western blotting were carried out for expressions of E-cadherin(marker of epithelial cells),vimentin and ?-SMA(late markers of EMT).Transmission electron microscopy(TEM)was used to test the morphology of autophagosome.4.To simulate the states of LECs in vivo,a lens capsule model in vitro and human LECs-B3 cells were cultured in high glucose.5.In HG models in vitro,expressions of miR-30a were regulated through its mimic and inhibitor to test its regulation on EMT and autophagy.Western blotting were used to identify the protein expression of target genes(SNAI1 and BECN1),the late markers of EMT(vimentin and a-SMA),and the markers of autophagy(LC3BI and LC3BII).The mRNA levels of the above genes were tested by qRT-PCR.The apoptotic rate of LECs was also tested by flow cytometry.ResultsPart 1.MiR-30a regulated EMT by targeting SNAI 1 in LECs1.1 The expression of miR-30a and EMT in LECs attaching on DC tissues(1)By immunohistochemistry and Western blot analyses,the protein expression of E-cadherin was found to be lower in LECs attaching on DC tissues than normal lens,while vimentin and a-SMA showed a higher expression.(2)The miRNA microarray analysis showed that 73 miRNAs were unregulated more than two folds in DC LECs,while 199 miRNAs were downregulated more than two folds.The downregulation of miR-30a was more than 180 folds,which was confirmed by qRT-PCR.1.2 The levels of EMT and miR-30a in HG model in vitro(1)In immunohistochemistry analysis,the staining intensity of vimentin and a-SMA increased in HG condition.(2)The results of qRT-PCR showed that the expression of miR-30a was decreased in HG conditions,suggesting that our HG model in vitro adequately simulated the states of LECs in vivo.1.3 SNAI1 was the target gene of miR-30a(1)According to the bioinformatic database(TargetScanHuman,http://www.targetscan.org/),miR-30a could target SNAI1 3'-UTR(706-713).(2)In a dual luciferase reporter assay,when pmiR-RB-REPORT-SNAI1-3'UTR and miR-30a mimic were cotransfected,the relative luciferase activity decreased significantly.Moreover,mutation of the complementary sites in the SNAI1-3'UTR abolished the suppressive effect of miR-30a and the relative luciferase activity increased.The results revealed that BECN1 is the target gene of miR-30a.(3)In the HG model in vitro,miR-30a targeted SNAI1 and regulated its expression.In qRT-PCR,SNAI1 mRNA levels were significantly downregulated after transfection with the miR-30a mimic(mimic NC as control,P<0.05)and upregulated after tansfection with the inhibitor(inhibitor NC as control,P<0.05).In Western blotting,SNAI1 protein levels were significantly downregulated after transfection with the miR-30a mimic(mimic NC as control,P<0.05)and upregulated after transfection with the inhibitor(inhibitor NC as control,P<0.05).1.4 The inverted regulation of miR-30a on EMT through SNAI1(1)In Western blotting,the expressions of both vimentin and a-SMA proteins were induced by HG and could be downregulated by miR-30a mimic(mimic NC as control,P<0.05).(2)MiR-30a could downregulate the level of EMT induced by TGF-?2.In the TGF-?2 group,the protein expressions of vimentin and ?-SMA were high expressed,revealing a high level of EMT induced by TGF-?2 in HG condition.However,the expression of vimentin and ?-SMA protein decresed in the present of TGF-?2 and miR-30a mimic.Part 2.MiR-30a regulated autophagy through its target gene BECN1 in LECs1.1 Downregulation of miR-30a in human DC tissuesThe miRNA microarray analysis showed that the top 5 upregulated miRNAs were miR-431-5p,miR-3169,miR-371a-3p,miR-1537 and miR-593-3p;the top 5 downregulated miRNAs were miR-30a-5p,miR-193a-3p,miR-204-5p,miR-184 and miR-29b-3p.The result of qRT-PCR validated the downregulated expression of miR-30a in LECs attaching on DC tissues,which was corresponding to the miRNA microarray analysis.1.2 The change of autophagy in DC tissuesThe single autophagosome in LECs attaching on DC tissues was larger than that in normal LECs and included several mitochondria to be degraded.1.3 BECN1 was the target gene of miR-30a(1)It indicated that miR-30a could target BECN1 3'UTR(240-247)using a bioinformatic database(TargetScanHuman,http://www.targetscan.org/).(2)In the result of a dual luciferase reporter assay,miR-30a could target BECN1 directly.When pmiR-RB-REPORT-BECN1 3'UTR and miR-30a agomir are cotransfected,the luciferase activity is significantly decreased.While the relative luciferase activity was observed to increase significantly when the mutant of BECN13'UTR abolished the suppressive effect of miR-30a on BECN1.(3)Western blotting showed that the protein expression of BECN1 was decreased by miR-30a agomir(agomir NC as control,P<0.05)in LECs in the presence of HG and increased by miR-30a antagomir(antagomir NC as control,P<0.05).1.4 MiR-30a repressed HG-induced autophagy in LECs(1)HG induced autophagy by BECN1 dependent pathway.As LC3B was measured as an autophagosome-associated marker,the high ratio of LC3B?/LC3BI represented the increase of autophagosome formation;otherwise,the low ratio of LC3B?/LC3BI represented the decrease of autophagosome formation.In the HG environment,the ratio of LC3BII/LC3BI increased significantly in LECs,which could be repressed by BECN1 siRNA.(2)HG-induced autophagy was regulated by miR-30a.The radio of LC3B?/LC3BI was decresed by miR-30a agomir(agomir NC as control,P<0.05).The expression of LC3B mRNA could be decreased by miR-30a(agomir NC as control,P<0.05).1.5 MiR-30a decreased apoptotic rate of LECs.The apoptotic rate of LECs increased in HG condition;meanwhile,which could be decreased by miR-30a agomir(agomir NC as control,P<0.05).Conclusions1.In LECs attaching on DC tissues,the expressions of miRNAs were different with those in normal lens.MiR-30a was downregulated more than 180 folds.2.In HG model in vitro,miR-30a can regulate the level of EMT by directly targeting SNAI1 in LECs.3.MiR,30a can inhibit autophagy in LECs by targeting BECN1 in HG conditon.4.MiR-30a can reduce the rate of apoptosis induced by HG and protect LECs in HG conditon.