MicroRNA-22-3p Regulates the Apoptosis of Lens Epithelial Cells Through Targeting KLF6 in Diabetic Cataracts

Abstract

Purpose: The purpose of this study was to identify novel abnormally expressed microRNAs (miRNAs) and their downstream target in diabetic cataract (DC).

Methods: General feature, fasting blood glucose, glycosylated hemoglobin, and type A1c (HbA1c) expression level of patients were collected. DC capsular tissues were obtained from patients and the lens cells (HLE-B3) exposed to different concentrations of glucose were used to simulate the model in vitro. Both mimic and inhibitor of miR-22-3p were transferred into HLE-B3 to up- and downregulate miR-22-3p expression, respectively. The cellular apoptosis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The downstream target gene of miR-22-3p was identified by dual luciferase reporter.

Results: In DC capsules and HLE-B3 under hyperglycemia, miR-22-3p showed a significant downward trend. The expression of BAX was upregulated and the BCL-2 was downregulated following high glucose. The expression of BAX was significantly down- or upregulated in HLE-B3 cells following transfection of mimic or inhibitor of miR-22-3p, respectively. Conversely, BCL-2 was significantly increased or decreased. Dual luciferase reporter assay showed that miR-22-3p directly targeted Krüppel Like Factor 6 (KLF6) to regulate cell apoptosis. In addition, the expression of KLF6 were significantly up- or downregulated following transfection of inhibitor or mimic of miR-22-3p.

Conclusions: This study suggested that miR-22-3p could inhibit lens apoptosis by targeting KLF6 directly under high glucose condition. The miR-22-3p/KLF6 signal axis may provide novel insights into the pathogenesis of DC.

Translational relevance: Differential expression of miR-22-3p may account for the pathogenesis of DC and lead to a new therapeutic strategy for DC.

Figure 1.

General feature of age-related cataract and diabetic cataract. (A) Representative image of the lens capsular opacification in patients with DC. The opacity of the lens in DC occurred in the anterior capsule (left) and posterior capsule (right). (B, C) The fasting blood glucose and HbA1c levels in the ARC group and the DC group. DC, diabetic cataract; ARC, age-related cataract; n = 20, ***P < 0.001).

Figure 2.

Expression of miR-22-3p and markers for apoptosis in human diabetic cataract LECs. (A) The qRT-PCR results of miRNA in capsule tissues from patients with DC and patients with ARC (n = 20, **P < 0.01). (B, C, D) The protein expression of apoptosis makers detected by Western blot (n = 3, *P < 0.05).

Figure 3.

Expression level of miR-22-3p and apoptosis in vitro. The relative expression of miR-22-3p in HLE-B3 under different glucose concentration (A); BAX mRNA and protein relative expression levels under different glucose concentration (B, D); BCL-2 mRNA and protein relative expression levels under different glucose concentration (C, E); The protein ratio of BAX/BCL-2 (F). The cell viability of HLE-B3 under HG detected by the CCK-8 assay(G) (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

MiR-22-3p repressed LECs apoptosis in vitro (A). Validation of transfection efficiency of miR-22-3p. (B, C, D) The mRNA and protein expression of BAX following miR-22-3p mimic or inhibitor transfection; (C, D) The mRNA and protein expression of the BCL-2 following miR-22-3p mimic or inhibitor transfection. (E) Hoechst 33258 staining was performed to show the apoptosis of cells transferred with miR-22-3p mimic or inhibitor. (F) The activity of cells transferred with miR-22-3p mimic or miR-22-3p inhibitor (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

MiR-22-3p worked by targeting KLF6 in vitro (A). Online websites prediction results of the miR-22-3p target sequence position. (B) Luciferase reporter assay showed that the luciferase activity of KLF6 3′-UTR-wt significantly decreased with miR-22-3p transfection, comparing to that of the NC mimic or KLF6 3′- UTR-wt group. (C, D) The mRNA and protein expression of KLF6 following the increase of sugar concentration. (E, F) KLF6 mRNA and protein levels following the miR-22-3p mimic or inhibitor transfection (n = 3, *P < 0.05, **P < 0.01).