Open Access
Lens  |   May 2023
MicroRNA-22-3p Regulates the Apoptosis of Lens Epithelial Cells Through Targeting KLF6 in Diabetic Cataracts
Author Affiliations & Notes
  • Xin Yin
    Medical College, Graduate School of Medicine, Qingdao University, Qingdao, China
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Ling Chen
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Jiachao Shen
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Zhaojing Bi
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Chen Chen
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Xinmei Zhao
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Shujun Liu
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Yuanbin Li
    Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China
  • Correspondence: Yuanbin Li and Shujun Liu, Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, 20 Yuhuangding East Road, Yantai 264000, China. e-mails: yuanbinli@yeah.net and liushujunqd@163.com 
  • Footnotes
    *  XY and LC contributed equally to this work.
Translational Vision Science & Technology May 2023, Vol.12, 9. doi:https://doi.org/10.1167/tvst.12.5.9
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      Xin Yin, Ling Chen, Jiachao Shen, Zhaojing Bi, Chen Chen, Xinmei Zhao, Shujun Liu, Yuanbin Li; MicroRNA-22-3p Regulates the Apoptosis of Lens Epithelial Cells Through Targeting KLF6 in Diabetic Cataracts. Trans. Vis. Sci. Tech. 2023;12(5):9. https://doi.org/10.1167/tvst.12.5.9.

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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.

Introduction
Cataract is a main cause of blindness, especially in developing countries.1 Over the past decades, understanding of the pathogenesis and risk factors of cataract has increased.1,2 In addition to irreversible risk elements, such as aging, income, sex, race, and myopia,2,3 the relation between cataract and systemic disease has attracted more attention of many researchers currently, especially diabetes mellitus (DM).1 Diabetic cataract (DC) is characterized by higher incidence, earlier development, and more complications,4,5 where high glucose levels can promote its development. The progression of DC has been reported to be involved in complicated pathological mechanisms, such as oxidative stress, autoimmunity, epithelial-mesenchymal transition (EMT), and lens epithelial cells (LECs) apoptosis.610 The generation of polyols from glucose by aldose reductase (AR) was the initial mechanism in DC formation. It is likely that the LECs apoptosis, oxidative stress, and autoimmune theory are the complex mechanism of DC formation.9 Thus, exploring the mechanism of LECs apoptosis under high hyperglycemia is significant to understand the pathology of DC. 
MicroRNAs (miRNAs) fall into the category of small, single-stranded, non-protein coding RNAs of about 18 to 23 nucleotides in length, which interact with the 3′-untranslated regions (3′-UTR) of downstream messenger RNAs (mRNAs) and affect the expression of mRNA at the post-translational level, such as mRNA degradation or translational repression.11,12 A growing body of evidence suggested that miRNAs could have been detected in various ocular tissues and had an important effect on the apoptosis, proliferation, and stress response of multiple ocular cells.1113 Studies on cataract also confirmed that miRNAs and their downstream binding genes were involved in regulating LECs’ function and significantly affect disease progression.11,1416 In recent years, accumulating evidence has showed that abnormal expression of miRNAs, such as miR-34a, miR-15a, miR-16-1, and miR-125b, were related to abnormal apoptosis of LECs during the pathogenesis of cataract.11,1719 Thus, miRNA’s regulation of LECs apoptosis through the binding target gene may be essential and the fundamental mechanisms during the development of the cataract formation in DM. 
MiR-22-3p, originally discovered as a tumor suppressor,20 has recently been linked to DM and various ophthalmic diseases,21 such as fibrotic cataract22 and retinal pigment epithelial injury.23 It was found that miR-22 was significantly decreased in the H9c2 embryonic cardiac myoblast cell line induced by high glucose, whereas upregulation of miR-22 could reduce the oxidation and apoptosis of diabetic cardiomyopathy by targeting Sirt1.24 Wang et al.22 identified that miR-22-3p is a critical regulator of lens fibrosis, whose research indicated that miR-22-3p was significantly downregulated in posterior capsule opacification cataract and the direct target of miR-22-3p, HDAC6, was activated under such conditions and contributed to lens fibrosis. More importantly, Liu et al.14 found that high glucose could induce a decreasing trend in the expression of miR-22 in lens epithelial cells, indicating that miR-22 may be a protective factor in DC. However, the specific effect and mechanism of miR-22-3p on the progression of DC and the downstream target gene of miR-22-3p remains unclear in DC. In this study, we demonstrated that miR-22-3p could inhibit the apoptotic progress in LECs by targeting Krüppel Like Factor 6 (KLF6) directly under high glucose conditions, which provide novel views into the pathogenic mechanism of DC. 
Materials and Methods
Acquisition of Clinical Tissue Samples
Fresh human lens anterior capsular tissues from 20 cases with age-related cataract (ARC) and 20 cases with DC were collected respectively from the Department of Ophthalmology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University (Yantai, Shandong, China). This study was approved by the Ethic Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University (No. 2022-206). Patients with ARC was diagnosed by professional ophthalmologists and placed as controls. Patients with DC were diagnosed by professional endocrinologists and ophthalmologists. Patients with a history of systemic metabolic diseases, ocular surgery, or combined with other ocular diseases other than diabetic retinopathy (DR) were excluded from this study. Informed consent was obtained from patients and their families prior to the collection of all clinical tissue samples. After the patients were hospitalized, we collected the general clinical information. The value of fasting blood glucose and glycosylated hemoglobin type A1c (HbA1c) were provided by the Department of Laboratory Medicine. 
Cell Culture and Treatment
The human lens epithelial B3 (HLE-B3) cell lines were kindly donated by Eye Hospital of Wenzhou Medical University. HLE-B3 cells were cultured in Roswell Park Memorial Institute1640 (RPMI1640; Biological Industries, Israel) added with 10% fetal bovine serum (Biological Industries, Israel), penicillin (100 U/mL; Biological Industries, Israel), and streptomycin (100 µg/mL) (Sigma, USA) at 37°C cell culture chamber containing 5% CO2. HLE-B3 cells were cultured under different concentrations of glucose (50, 80, and 100 mmol/L) to simulate the high glucose microenvironment in human eyes, and 5.5 mmol/L glucose served as a control for the other groups. All cells with different glucose concentrations were cultured at 37°C for 48 hours. 
Transfection of MicroRNA
When HLE-B3 cells grew to 80% to 90% density, they were treated with trypsin (Biological Industries, Israel) digestion. Then, these cells were plated at an approximate density of 1 × 106 cells per well in 6-well plates. Then, 24 hours after that, miR-22-3p mimic, mimic negative control (mimic NC), inhibitor, and inhibitor negative control (inhibitor NC; Guangzhou RiboBio Co., China) were transfected into cells, respectively, with Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA) following the manufacturers’ recommendations. According to the instructions, cells were transfected at a final concentration of 50 nM for miR-22-3p mimic, miR-22-3p mimic NC and 100 nM for miR-22-3p inhibitor, and miR-22-3p inhibitor NC. The group only added transfection agent was named as the control. All cells were replaced with fresh complete culture medium 6 hours after transfection. After 1 day, the transfected cells were cultured in high glucose (HG; 50 mmol/L) medium for an additional 48 hours. 
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction
MicroRNA from the anterior capsular tissues of the patient's lens was isolated with miRNA First Strand cDNA Synthesis (Tailing Reaction; Sangon Biotech, China). Total RNA of cells or tissues were purified using 5 × All-In-One RT MasterMix with AccuRT (ABM, Canada) based on the reagent manufacturer's guidebook, and reverse transcribed using ChamQ Universal SYBR Qpcr Master Mix (Vazyme, China). The amplification was performed on Applied Biosystems 7500 Series Real-Time PCR system (Thermo Fisher Scientific, USA). U6 and GAPDH served as the housekeeping genes for miRNA and mRNA detection separately. Three biological replicates were used for evaluating the gene expression, which relative to the reference gene expression via the 2−ΔΔCT method. Table 1 lists the primers we used in the present study. 
Table 1.
 
Primer Sequences of MiRNA and MRNA Utilized for QRT-PCR
Table 1.
 
Primer Sequences of MiRNA and MRNA Utilized for QRT-PCR
Western Blot
The tissue samples were homogenized in a tissue grinder. The tissue homogenates were placed on ice, lysed with protein lysis buffer (RIPA: PMSF = 50:1; Beyotime Biotechnology, China) for 30 minutes at 4°C. The lysates were centrifuged for 20 minutes at a speed of 16,000 rpm. The cell lysates were collected into new Eppendorf tubes. Protein concentration was detected by BCA Protein Assay Kit (Coolaber, China). Then, 20 ug of protein were resolved with 10% SDS-PAGE (Sparkjade, China) and electro-transferred to nitrocellulose membrane, which was subsequently blocked with 5% defatted milk for 1 hour at room temperature and coated with specific primary antibodies against β-tubulin (1:1000; Affinity, USA), BAX (1:3000; Proteintech, China), BCL-2 (1:2000; Abcam, USA), and KLF6 (1:2000; Proteintech, China) for overnight incubation at 4°C. The membrane was probed by the secondary antibody for 1 hour at 37°C. The electrochemiluminescence (ECL) kit (Affinity, USA) was dropped on the membranes and put into the chemiluminescence instrument ChemiScope 6200 Touch (Clinx, China) for automatic luminescence imaging. Image J software (version 1.53t; URL link: https://imagej.nih.gov/ij/download.html) was used for image analysis and processing. 
Cell Counting Kit-8 Assays
The Cell Counting Kit-8 (CCK8; Dojindo, Japan) experiment was used to detect cell viability. Approximately 2000 cells/well were cultured into a 96-well plate. After 48 hours of incubation, a mixture of 90 µL medium and 10 µL CCK8 was added to each well. Then, cells were incubated at 37° for 2 hours and protected from light. Finally, the cell viability was detected with the absorbance at 450 nm using a microplate reader. 
Hoechst 33258 Staining
The HLE-B3 cells were planted in HG (50 mmol/L) for 48 hours after transfected with miR-22-3p mimic, inhibitor, and corresponding NCs. After aspirating the culture medium, cells were fixed with 500 µl stationary liquid for 20 minutes at room temperature. Cells were then slowly and gently washed in pre-cooled phosphate-buffered saline (PBS) and stained with 500 µL Hoechst 33258 (Beyotime Biotechnology, China) for 20 minutes under light exclusion. The cells were washed with PBS again, and then fluorescence quencher was added. Fluorescence microscope Olympus IX51 (Olympus Corporation, Japan) were used to examine morphological changes in the cells. 
Luciferase Reporter Assay
First, TargetScan (http://www.targetscan.org/), an online website tool, was used to predict possible targets of miR-22-3p. The results were subsequently confirmed by dual luciferase reporting assays. MiR-22-3p mimic and corresponding negative control were co-transferred with KLF6-3′UTR wild plasmids or KLF6-3′UTR mutant plasmids into HLE-B3 cell lines to assess whether miR-22-3p could target to the downstream KLF6 gene. Cells were collected 48 hours after transfection and lysed to detect Luciferase activity according to the instructions of the luciferase detection kit (Vazyme, China). 
Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 8.0 software. All experimental data were presented as mean ± SD, with each experiment performed repeatedly in triplicate. Experiments with two groups were analyzed using unpaired Student's t-test when the data are normally distributed or using Wilcoxon test when the data are not normally distributed. Experiments with more than two groups were analyzed using 1-way ANOVA. The P values < 0.05 were considered statistically significant. 
Results
General Feature of Age-Related Cataract and Diabetic Cataract
The opacity of the lens in DC mostly occurs in the anterior capsule (Fig. 1A, left) and the posterior capsule (see Fig. 1A, right). The clinical information of patients with ARC and DC is shown in Table 2. No significant differences were found between ARC and DC in age (P = 0.748). However, fasting blood glucose and glycosylated HbA1c in the DC group were significantly higher than those in the ARC group (P < 0.001; Figs. 1B, 1C). 
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 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).
Table 2.
 
General Feature of the Two Groups (Mean ± SD)
Table 2.
 
General Feature of the Two Groups (Mean ± SD)
MiR-22-3p and Apoptosis-Related Markers’ Expression in Human DC Tissues
We collected ARC and DC tissues from the patients who accepted cataract surgery to detect the expression level of miR-22-3p and apoptosis-related markers. The results suggested that miR-22-3p expression in the DC group was downregulated compared to the ARC group by real-time polymerase chain reaction (qRT-PCR; Fig. 2A). The expression of the pro-apoptotic marker BAX was significantly upregulated in patients with DC, whereas BCL-2, a marker of anti-apoptosis, decreased significantly through Western blot (Figs. 2B, 2C, 2D). 
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 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).
Expression of MiR-22-3p and Apoptosis In Vitro
To further confirm this result, HLE-B3 cells were cultured with HG to determine the expression of miR-22-3p and apoptosis levels in vitro. The HLE-B3 cells were cultured with 50 mmol/L, 80 mmol/L, and 100 mmol/L D-glucose to mimic the DC pathological microenvironment, with 5.5 mmol/L D-glucose as the control condition (normal glucose [NG]). From the results of qRT-PCR, it can be found that the expression of miR-22-3p was decreased significantly with the increase of glucose concentration (Fig. 3A). The expression of BAX increased at both transcription and protein levels under the HG condition (Figs. 3B, 3D), whereas the BCL-2 expression decreased (Figs. 3C, 3E). The protein ratio of BAX/BCL-2 showed the cells in HG are sensitive to apoptosis (Fig. 3F). The cell viability of HLE-B3 cultured with different concentrations of glucose for 48 hours was decreased with the increase of glucose concentrations (Fig. 3G). Therefore, these results showed that miR-22-3p expression level was negatively correlated with the concentration of glucose, whereas the apoptosis level was upregulated under the HG condition. 
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 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).
Downregulation of MiR-22-3p Resulted in LECs Apoptosis In Vitro
For the purpose of investigating the potential mechanism of miR-22-3p on apoptosis of LECs, miR-22-3p mimic, inhibitor, and their respective negative controls were transfected into HLE-B3 cells followed by treatment of HG. First, transfection efficiency was confirmed by qRT-PCR (Fig. 4A). Then, the gene and protein levels of BAX and BCL-2 were detected. As illustrated in Figures 4B, 4C, and 4D, the expression of BAX was downregulated both at mRNA and protein level in miR-22-3p mimic group under HG (50 mmol/L glucose), whereas the BCL-2 was upregulated. The BAX was upregulated after miR-22-3p inhibitor transfection both at mRNA and protein level, whereas the BCL-2 was downregulated, compared with inhibitor NCs under HG (50 mmol/L glucose). In addition, the Hoechst 33258 staining suggested that the apoptotic cells in the miR-22-3p mimic group decreased under the same microscope field, whereas the apoptosis level in the miR-22-3p inhibitor group increased (Fig. 4E). The viability of cells analyzed by the CCK-8 experiment, can be seen that the activity of cells in the miR-22-3p mimic group increased. On the contrary, the activity of cells in the miR-22-3p inhibitor group decreased (Fig. 4F). Accordingly, these results suggested that miR-22-3p inhibition contributed to LECs apoptosis in DC. 
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 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).
MiR-22-3p Exerts Anti-Apoptosis Effect by Targeting KLF6
It has been found that miR-22-3p showed a decreasing trend in DC, thus the regulatory mechanism of miR-22-3p in DC was further investigated. KLF6 could be served as a potential target of miR-22-3p through the analysis of the online website (TargetScan, http://www.targetscan.org/; Fig. 5A). Dual luciferase reporter assays were applied to determine whether miR-22-3p target the 3′-UTR of KLF6 mRNA. The results showed that relative level of luciferase activity was significantly inhibited when KLF6 3′UTR-wt and miR-22-3p mimic were co-transfected into cells, compared with NC mimic or KLF6 3′UTR-mu group, which indicated KLF6 as a representative direct binding target of miR-22-3p (Fig. 5B). In addition, we further investigated the regulatory effect of miR-22-3p on KLF6 in vitro. It was found that the KLF6 mRNA as well as protein expression increased significantly under the high concentrations of glucose (Figs. 5C, 5D). The expression of KLF6 was largely inhibited after that miR-22-3p mimic were transferred into LECs compared with the respective NCs. On the contrary, the downstream gene KLF6 was increased after miR-22-3p inhibitor transfection (Figs. 5E, 5F). These findings demonstrated that miR-22-3p could inhibit lens apoptotic progression by targeting KLF6 directly to affect the DC development. 
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).
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).
Discussion
Many epidemiology studies have been reported that DM is a chronic systemic disease, whose incidence keeps increasing over time.25 Cataract is one of the main reasons for blindness globally, especially in people with diabetes who are two to five times higher in incidence and much earlier in development.4 The aberrant expression of miRNAs during the cataract development has been reported by many studies, and the role of miRNAs in the formation of DC has attracted more and more attention.1416,26 In previous studies, microRNAs, such as miRNA-199a-5p and miRNA-30a, have been reported to affect the development and progression of DC by targeting downstream genes.14,16 The development of cataract in patients with diabetes could be partly due to the aging process. In the present study, to control the influence of age bias, we choose the lens capsule of patients with age-related cataract, but not healthy people, as the control samples. We identified that the expression of miR-22-3p was downregulated in the anterior capsules of patients with DC, which was confirmed in vitro as well. We further confirmed that miRNA-22-3p regulated the process of apoptosis of LECs by targeting KLF6, hoping to provide a basis for the understanding of pathogenesis and development of DC therapies in clinical practice. 
MiR-22-3p is an evolutionarily highly conserved microRNAs in vertebrates,27 which was initially considered as a tumor suppressor gene and played a crucial role in the development and progression of lung cancer, breast cancer, and so on.28,29 A growing number of studies have demonstrated that miR-22-3p was closely related to the hyperproliferative diseases, such as cancer and fibrosis. Recent studies have showed that miR-22-3p could inhibit the potential ability of cell proliferation and induce cell apoptosis in several types of tumors.28,30 In fibrotic cataract, miR-22-3p acted as an antifibrotic factor to inhibit the progression of lens fibrosis by targeting downstream gene HDAC6 and promoting α-tubulin acetylation.22 It was also closely associated with diabetes and diabetes-related diseases. In diabetes-induced salivary gland dysfunction and HG treated human submandibular glands (SMGs) C6 cells, the miR-22-3p showed a decreasing trend and the paracellular permeability decreased in SMG-C6. The overexpression of miR-22-3p could reduce the inhibition of paracellular permeability induced by HG.31 It was found that miR-22-3p was downregulated in in the islet tissues of mice with gestational diabetes mellitus (GDM). Upregulation of miR-22-3p could improve hepatic insulin resistance in mice with GDM by regulating cytokine signaling 3 (Socs3).32 In diabetic cardiomyopathy, lncMALAT1 participates in the process of cardiomyocyte apoptosis through the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/miR-22/ATP binding cassette subfamily a member 1 (ABCA1) signaling pathway, and the aberrant upregulation of miR-22 can alleviate cardiomyocytes injury, which was in keeping in line with the fact that miR-22-3p reduces the apoptotic damage of LECs in our experiment.33 Although miR-22 has been shown to be involved in multiple metabolic pathways of glucose,34 how miR-22-3p affect DC progression remains a mystery, and the specific mechanism of miR-22-3p in the progression of DC still was an attractive topic. 
Apoptosis was a common cellular pathological mechanism for initiation of DC, which altered the balance required for LECs homoeostasis and led to cataract formation.9 The presence of a high-glucose microenvironment may serve to further strengthen or reinforce these effects by inducing endoplasmic reticulum (ER) stress and causing oxidative stress damage to lens fibers.9 Moreover, recent studies also found that microRNA-211 upregulation promotes the proliferation and inhibits apoptosis of LECs in DC mice by targeting downstream gene SIRT1.15 Likewise, the study proved that miR-22-3p played an inhibitory role in the apoptosis process of LECs by targeting KLF6 in patients with DC. 
The specificity protein/Krüppel-like factor (SP/KLF) transcription factor family played several important roles in a variety of biological cellular functions. KLF6, as an important member of this family, had a special zinc finger structure and played a special role in cell proliferation and apoptosis.35 It was a nuclear transcriptional regulatory factor initially recognized in placental cells and subsequently found to be ubiquitously expressed in various tissues.36 KLF6 has been demonstrated to exert an antineoplastic effect through various mechanism,37 such as induction of apoptosis,38 cell-cycle arrest,39,40 and inhibition of angiogenesis.41 In addition to cancers, the roles of KLF6 in ophthalmic diseases have attracted increasing attention in recent years. Nakamura et al. initially reported that KLF6 was detected in the mouse cornea and lens and played a core effect in lens development.42 Subsequently, it was found that KLF6 aberrant upregulation led to LECs apoptosis under ultraviolet radiation-B by activating transcription factor 4 (ATF4) - activating transcription factor 3 (ATF3) - DNA Damage inducible transcript 3 (CHOP) axis,43 and miR-181 could promote the ability to proliferate and migrate of retinal endothelial cells by targeting KLF6 in DR.44 Moreover, recent research has revealed that miR-22-3p also could moderate fatty infiltration involved in muscle atrophy by regulating target KLF6 gene, and the miR-22-3p/KLF6/matrix metallopeptidase 14 (MMP-14) axis could be likely to act as a potential therapeutic target for muscle degenerative diseases.45 Therefore, our research is the first time to find that miRNA-22-3p could regulate apoptosis in DCs through targeting KLF6, which complements the possible pathogenic mechanism of miR-22-3p/KLF6 axis in DC disease development. 
In conclusion, our work indicated that miR-22-3p was down expression in DC, and regulated cell apoptosis by targeting the KLF6 gene. MiR-22-3p/KLF6 axis could be served as a novel underlying pathway for the diagnosis and therapy of DC. Nevertheless, further experimental work in vivo and clinical researches are needed to validate our findings, which is a limitation of this study. 
Acknowledgments
Supported by the Yantai Technology Development Plan (grant no. 2017WS109), Fund Project of Yantai Science and Technology Development Plan (grant no. 2021YD025). 
Authors’ Contributions: Y.B.L. and S.J.L. were responsible for the topic selection, research design and framework of the paper. X.Y. and L.C. participated in the design of the trial, study implementation, and data collection. J.C.S. and Z.J.B. assisted in completing part of the qRT-PCR and Western blot. X.M.Z. is responsible for the purchase of test materials and test technical guidance. X.Y. and L.C. wrote the manuscript, which was reviewed by C.C. All authors read and approved the final manuscript. 
Disclosure: X. Yin, None; L. Chen, None; J. Shen, None; Z. Bi, None; C. Chen, None; X. Zhao, None; S. Liu, None; Y. Li, None 
References
Ang MJ, Afshari NA. Cataract and systemic disease: a review. Clin Exp Ophthalmol. 2021; 49(2): 118–127. [CrossRef] [PubMed]
Lim JC, Caballero Arredondo M, Braakhuis AJ, et al. Vitamin C and the lens: new insights into delaying the onset of cataract. Nutrients. 2020; 12(10): 3142. [CrossRef] [PubMed]
Lee CM, Afshari NA. The global state of cataract blindness. Curr Opin Ophthalmol. 2017; 28(1): 98–103. [CrossRef] [PubMed]
Greenberg MJ, Bamba S. Diabetic cataracts. Dis Mon. 2021; 67(5): 101134. [CrossRef] [PubMed]
Kumar PA, Reddy PY, Srinivas PN, et al. Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity. J Nutr Biochem. 2009; 20(7): 553–562. [CrossRef] [PubMed]
Drinkwater JJ, Davis WA, Davis TME. A systematic review of risk factors for cataract in type 2 diabetes. Diabetes Metab Res Rev. 2019; 35(1): e3073. [CrossRef] [PubMed]
Chung SS, Ho EC, Lam KS, et al. Contribution of polyol pathway to diabetes-induced oxidative stress. J Am Soc Nephrol. 2003; 14(8 Suppl 3): S233–S236. [PubMed]
Wu TT, Chen YY, Chang HY, et al. AKR1B1-induced epithelial-mesenchymal transition mediated by RAGE-oxidative stress in diabetic cataract lens. Antioxidants (Basel). 2020; 9(4): 273. [CrossRef] [PubMed]
Kiziltoprak H, Tekin K, Inanc M, et al. Cataract in diabetes mellitus. World J Diabetes. 2019; 10(3): 140–153. [CrossRef] [PubMed]
Wang Y, Zhang G, Kang L, et al. Expression profiling of DNA methylation and transcriptional repression associated genes in lens epithelium cells of age-related cataract. Cell Mol Neurobiol. 2017; 37(3): 537–543. [CrossRef] [PubMed]
Yu X, Zheng H, Chan MT, et al. MicroRNAs: new players in cataract. Am J Transl Res. 2017; 9(9): 3896–3903. [PubMed]
Lu TX, Rothenberg ME. MicroRNA. J Allergy Clin Immunol. 2018; 141(4): 1202–1207. [CrossRef] [PubMed]
Van Meter E N, Onyango JA, Teske KA. A review of currently identified small molecule modulators of microRNA function. Eur J Med Chem. 2020; 188: 112008. [CrossRef] [PubMed]
Liu X, Gong Q, Yang L, et al. microRNA-199a-5p regulates epithelial-to-mesenchymal transition in diabetic cataract by targeting SP1 gene. Mol Med. 2020; 26(1): 122. [CrossRef] [PubMed]
Zeng K, Feng QG, Lin BT, et al. Effects of microRNA-211 on proliferation and apoptosis of lens epithelial cells by targeting SIRT1 gene in diabetic cataract mice. Biosci Rep. 2017; 37(4): BSR20170695. [CrossRef] [PubMed]
Zhang L, Wang Y, Li W, et al. MicroRNA-30a regulation of epithelial-mesenchymal transition in diabetic cataracts through targeting SNAI1. Sci Rep. 2017; 7(1): 1117. [CrossRef] [PubMed]
Li Y, Liu S, Zhang F, et al. Expression of the microRNAs hsa-miR-15a and hsa-miR-16-1 in lens epithelial cells of patients with age-related cataract. Int J Clin Exp Med. 2015; 8(2): 2405–2410. [PubMed]
Qin Y, Zhao J, Min X, et al. MicroRNA-125b inhibits lens epithelial cell apoptosis by targeting p53 in age-related cataract. Biochim Biophys Acta. 2014; 1842(12 Pt A): 2439–2447. [PubMed]
Chien KH, Chen SJ, Liu JH, et al. Correlation between microRNA-34a levels and lens opacity severity in age-related cataracts. Eye (Lond). 2013; 27(7): 883–888. [CrossRef] [PubMed]
Xu D, Takeshita F, Hino Y, et al. miR-22 represses cancer progression by inducing cellular senescence. J Cell Biol. 2011; 193(2): 409–424. [CrossRef] [PubMed]
Guo J, Yang P, Li YF, et al. MicroRNA: crucial modulator in purinergic signalling involved diseases. Purinergic Signal. 2022; 19: 329–341. [CrossRef] [PubMed]
Wang X, Wang L, Sun Y, et al. MiR-22-3p inhibits fibrotic cataract through inactivation of HDAC6 and increase of α-tubulin acetylation. Cell Prolif. 2020; 53(11): e12911. [CrossRef] [PubMed]
Hu Z, Lv X, Chen L, et al. Protective effects of microRNA-22-3p against retinal pigment epithelial inflammatory damage by targeting NLRP3 inflammasome. J Cell Physiol. 2019; 234(10): 18849–18857. [CrossRef] [PubMed]
Tang Q, Len Q, Liu Z, et al. Overexpression of miR-22 attenuates oxidative stress injury in diabetic cardiomyopathy via Sirt 1. Cardiovasc Ther. 2018; 36(2): 12318. [CrossRef]
Majmudar FD . A review: cataract, a common ocular complication in diabetes, 2016. Available at: https://www.semanticscholar.org/paper/A-review%3A-Cataract%2C-a-common-ocular-complication-in-Majmudar/dd1d7dd7992ae67546b22744f1feaf5d63aa2b95.
Zeng K, Wang Y, Yang N, et al. Resveratrol inhibits diabetic-induced Müller cells apoptosis through microRNA-29b/specificity protein 1 pathway. Mol Neurobiol. 2017; 54(6): 4000–4014. [CrossRef] [PubMed]
Ibarra I, Erlich Y, Muthuswamy SK, et al. A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells. Genes Dev. 2007; 21(24): 3238–3243. [CrossRef] [PubMed]
Wang X, Yao Z, Fang L. miR-22-3p/PGC1β suppresses breast cancer cell tumorigenesis via PPARγ. PPAR Res. 2021; 2021: 6661828. [CrossRef] [PubMed]
Zhang K, Li XY, Wang ZM, et al. MiR-22 inhibits lung cancer cell EMT and invasion through targeting Snail. Eur Rev Med Pharmacol Sci. 2017; 21(16): 3598–3604. [PubMed]
Yang X, Su W, Li Y, et al. MiR-22-3p suppresses cell growth via MET/STAT3 signaling in lung cancer. Am J Transl Res. 2021; 13(3): 1221–1232. [PubMed]
Huang Y, Liu HM, Mao QY, et al. High glucose reduces the paracellular permeability of the submandibular gland epithelium via the MiR-22-3p/Sp1/claudin pathway. Cells. 2021; 10(11): 3230. [CrossRef] [PubMed]
Zhang H, Wang Q, Yang K, et al. Effects of miR-22-3p targeted regulation of Socs3 on the hepatic insulin resistance in mice with gestational diabetes mellitus. Am J Transl Res. 2020; 12(11): 7287–7296. [PubMed]
Wang C, Liu G, Yang H, et al. MALAT1-mediated recruitment of the histone methyltransferase EZH2 to the microRNA-22 promoter leads to cardiomyocyte apoptosis in diabetic cardiomyopathy. Sci Total Environ. 2021; 766: 142191. [CrossRef] [PubMed]
Senese R, Cioffi F, Petito G, et al. miR-22-3p is involved in gluconeogenic pathway modulated by 3,5-diiodo-L-thyronine (T2). Sci Rep. 2019; 9(1): 16645. [CrossRef] [PubMed]
Hu K, Zheng QK, Ma RJ, et al. Krüppel-like factor 6 splice variant 1: an oncogenic transcription factor involved in the progression of multiple malignant tumors. Front Cell Dev Biol. 2021; 9: 661731. [CrossRef] [PubMed]
Koritschoner NP, Bocco JL, Panzetta-Dutari GM, et al. A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene. J Biol Chem. 1997; 272(14): 9573–9580. [CrossRef] [PubMed]
Difeo A, Martignetti JA, Narla G. The role of KLF6 and its splice variants in cancer therapy. Drug Resist Updat. 2009; 12(1-2): 1–7. [CrossRef] [PubMed]
Ito G, Uchiyama M, Kondo M, et al. Krüppel-like factor 6 is frequently down-regulated and induces apoptosis in non-small cell lung cancer cells. Cancer Res. 2004; 64(11): 3838–3843. [CrossRef] [PubMed]
Narla G, Heath KE, Reeves HL, et al. KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science. 2001; 294(5551): 2563–2566. [CrossRef] [PubMed]
Slavin DA, Koritschoner NP, Prieto CC, et al. A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function. Oncogene. 2004; 23(50): 8196–8205. [CrossRef] [PubMed]
Difeo A, Narla G, Hirshfeld J, et al. Roles of KLF6 and KLF6-SV1 in ovarian cancer progression and intraperitoneal dissemination. Clin Cancer Res. 2006; 12(12): 3730–3739. [CrossRef] [PubMed]
Nakamura H, Chiambaretta F, Sugar J, et al. Developmentally regulated expression of KLF6 in the mouse cornea and lens. Invest Ophthalmol Vis Sci. 2004; 45(12): 4327–4332. [CrossRef] [PubMed]
Tian F, Zhao J, Bu S, et al. KLF6 induces apoptosis in human lens epithelial cells through the ATF4-ATF3-CHOP axis. Drug Des Devel Ther. 2020; 14: 1041–1055. [CrossRef] [PubMed]
Liang X, Xu W. miR-181a-5p regulates the proliferation and apoptosis of glomerular mesangial cells by targeting KLF6. Exp Ther Med. 2020; 20(2): 1121–1128. [CrossRef] [PubMed]
Lin Y, Wen-Jie Z, Chang-Qing L, et al. mir-22-3p/KLF6/MMP14 axis in fibro-adipogenic progenitors regulates fatty infiltration in muscle degeneration. Faseb J. 2020; 34(9): 12691–12701. [CrossRef] [PubMed]
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 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 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 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 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).
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).
Table 1.
 
Primer Sequences of MiRNA and MRNA Utilized for QRT-PCR
Table 1.
 
Primer Sequences of MiRNA and MRNA Utilized for QRT-PCR
Table 2.
 
General Feature of the Two Groups (Mean ± SD)
Table 2.
 
General Feature of the Two Groups (Mean ± SD)
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