MicroRNA-22-3p Regulates The Apoptosis Of Lens Epithelial Cells Through Targeting KLF6 In Diabetic Cataracts

Background: Diabetic cataract(DC)is a common ocular complication in people with diabetes mellitus(DM).The incidence of DC is 2-5 times that of normal people,and it is one of the causes of blindness in diabetic patients.Lens opacification in DC patients has the characteristics of early onset,rapid progression and more complications.The treatment of cataract can be effectively accomplished through phacoemulsification.The incidence of diabetic cataract has been on the rise,due to the growing population of diabetics,and this has caused a considerable financial and social strain.The prevention and treatment of DC have attracted more and more attention,but its pathogenesis is still unclear.Apoptosis is one of the forms of programmed cell death.The excessive apoptosis of human lens epithelial cells,caused by a prolonged high glucose level,is a contributing factor to lens turbidity.Apoptosis is a common cytopathic mechanism for DC,which alters the balance required for lens homeostasis and leads to cataract formation.Micro RNAs(miRNAs)are a large class of endogenous,small non-coding RNAs with a length of 18-25 nucleotides.By base pairing with the 3’-untranslated region of the target gene m RNA,miRNA can regulate the expression of post-transcriptional genes through inducing either m RNA degradation or translational inhibition.The evidence that miRNAs are implicated in the development of eye diseases such as glaucoma,diabetic retinopathy,keratopathy,and cataract is mounting,despite the fact that the majority of studies on miRNAs are associated with cancer.Studies have found that the expression of miRNAs in the anterior lens capsule of diabetic cataract patients is different from that of age related cataract(ARC).By exploring the cause of diabetic cataract through miRNAs,a novel approach to treating DC could be developed in the future.Purpose: To investigate the effect of micro NA-22-3p(miR-22-3p)on the apoptosis of lens epithelial cells in diabetic cataract and its mechanism of targeting Krüppel Like Factor 6(KLF6).Methods: The general data of DC patients and ARC patients who underwent phacoemulsification in the Department of Ophthalmology,Yantai Yuhuangding Hospital affiliated to Qingdao University were collected.The anterior capsular tissue of the lens was collected from the patients during cataract surgery,20 cases in each group.Using quantitative Real-time PCR(q RT-PCR)to detect the relative expression of miR-22-3p in the anterior capsule tissues of the two groups,and using Western Blot to detect the expression of apoptosis-related proteins B-cell lymphoma associated protein X(BAX),B-cell lymphoma-2(BCL-2)in the anterior capsule tissues of the two groups.The lens epithelial cells(HLE-B3)exposed to different concentrations of glucose were used to simulate the model in vitro.They were divided into control group(5.5mmol/L group)and high glucose apoptosis model group(50mmol/L group,80mmol/L group,100mmol/L group).After 48 hours of cell culture,the relative expression levels of miR-22-3p,BAX and BCL-2 in each group were detected by q RT-PCR,and Western Blot revealed the relative protein expression levels of BAX and BCL-2 in each group.Transfection groups:control group,miR-22-3p mimic group,miR-22-3p mimic NC group,miR-22-3p inhibitor group,miR-22-3p inhibitor NC group.After 48 hours of transfection,the relative expression levels of miR-22-3p,BAX and BCL-2 in each group were detected by q RT-PCR,and the relative protein expression levels of BAX and BCL-2 in each group were detected by Western Blot.Hoechst 33258 staining revealed the apoptosis of HLE-B3 cells following transfection.Dual luciferase reporter gene assay was used to verify the targeted binding relationship between miR-22-3p and KLF6.Using q RT-PCR,the relative expression of KLF6 in cells with varying glucose concentrations and after transfection was determined.Western blot was used to detect the relative expression of KLF6 protein in different glucose concentration media and after transfection.Results: 1.There were no significant differences in age and gender between DC and ARC patients at baseline(P>0.05).The fasting blood glucose and glycosylated hemoglobin,type A1c(Hb A1c)in DC group were significantly higher than those in ARC group(P<0.001).2.In diabetic cataract capsules,the expression of miR-22-3p showed a significant downward trend compared to ARC group(P<0.05).The level of apoptosis was higher in the DC group than in the ARC group(P<0.05).3.In the high glucose model of human lens epithelial cells HLE-B3,the expression of miR-22-3p decreased and apoptosis increased with the increase of glucose concentration(P < 0.05).4.Compared with miR-22-3p mimic NC group,the cell apoptosis was decreased in miR-22-3p mimic group(P < 0.05).Compared with miR-22-3p inhibitor NC group,the cell apoptosis was increased in miR-22-3p inhibitor group(P < 0.05).5.Dual luciferase reporter assay confirmed the targeted binding relationship between miR-22-3p and KLF6.In the high glucose model of HLE-B3,the expression of KLF6 gradually increased with the increase of glucose concentration(P < 0.05).The expression of KLF6 was decreased in the miR-22-3p mimic group(P<0.05)and increased in the miR-22-3p inhibitor group(P<0.05).Conclusions: This study suggested that the expression of miR-22-3p was down-regulated in the anterior capsule tissue of diabetic cataract and under high glucose environment in vitro.Overexpression of miR-22-3p could inhibit the apoptosis of diabetic cataract lens epithelial cells through targeting KLF6.