MicroRNA-124 facilitates lens epithelial cell apoptosis by inhibiting SPRY2 and MMP-2

Abstract

Age-related cataract (ARC) is the primary cause of blindness worldwide. Abnormal expression of microRNAs (miRNAs/miRs) has been reported to be associated with multiple diseases, including ARC. However, the potential role of miR-124 in ARC remains unclear. The present study used the human lens epithelial cell line, SRA01/04, to investigate the potential role of miR-124 in ARC. Reverse transcription-quantitative PCR analysis was performed to detect the expression levels of miR-124, protein sprouty homolog 2 (SPRY2) and matrix metalloproteinase-2 (MMP-2) in ARC tissues, while western blotting was performed to detect the protein levels of SPRY2 and MMP-2. Cell viability and apoptosis of SRA01/04 cells were assessed via Cell Counting Kit-8 and TUNEL assays, respectively. The interaction between miR-124 and SPRY2 or MMP-2 was confirmed via the dual-luciferase reporter and RNA immunoprecipitation assays. The results of the present study demonstrated that miR-124 expression was significantly upregulated in ARC tissues, and knockdown of miR-124 increased SRA01/04 cell viability and suppressed apoptosis. In addition, SPRY2 and MMP-2 expression was decreased in ARC tissues, and were demonstrated to directly bind to miR-124. Overexpression of SPRY2 or MMP-2 increased SRA01/04 cell viability and repressed apoptosis, the effects of which were reversed following overexpression of miR-124. Taken together, these results suggested that miR-124 facilitates lens epithelial cell apoptosis by modulating SPRY2 or MMP-2 expression, providing a novel treatment approach for ARC.

Keywords: microRNA-124; protein sprouty homolog 2; matrix metalloproteinase-2; lens epithelial cells; age-related cataract.

Figure 1.

miR-124 expression is increased in ARC tissues and miR-124 inhibitor inhibits SRA01/04 cells apoptosis. (A) RT-qPCR assay was performed to determine miR-124 expression in anterior lens capsules of ARC tissues compared with the normal tissues, n=28. (B) RT-qPCR assay was employed to measure miR-124 expression in SRA01/04 cells transfected with NC inhibitor or miR-124 inhibitor. (C) Cell Counting Kit-8 assay was used to evaluate cell viability in SRA01/04 cells transfected with miR-124 inhibitor or NC inhibitor. (D) TUNEL assay (magnification, ×200; scale bar, 50 µm) was performed to analyze cell apoptosis rate of SRA01/04 cells transfected with miR-124 inhibitor or NC inhibitor. *P<0.05 vs. control group. miR, microRNA; ARC, age-related cataract; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.

Figure 2.

SPRY2 is a target of miR-124. (A) The binding sequence between miR-124 and SPRY2 was predicted using the StarBase website. (B) Dual-luciferase reporter assay showed the luciferase activity of SPRY2-wt or SPRY2-mut in SRA01/04 cells transfected with NC mimics or miR-124 mimics. (C) Dual-luciferase reporter assay showed the luciferase activity of SPRY2-wt or SPRY2-mut in SRA01/04 cells transfected with NC inhibitor or miR-124 inhibitor. (D) RIP assay showed the abundance of SPRY2 enriched by Ago2 or IgG in SRA01/04 cells transfected with NC mimics or miR-124 mimics. (E) RT-qPCR was performed to determine miR-124 expression in SRA01/04 cells transfected with miR-124 mimics or NC mimics. (F) RT-qPCR assay showed the expression of SPRY2 in SRA01/04 cells transfected with NC mimics, miR-124 mimics, NC inhibitor or miR-124 inhibitor. *P<0.05 vs. control group. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; SPRY2, protein sprouty homolog 2; wt, wild-type; mut, mutant; RIP, RNA immunoprecipitation; Ago2, argonaute 2; IgG, immunoglobin G.

Figure 3.

miR-124 increases SRA01/04 cell apoptosis by targeting SPRY2. (A) RT-qPCR assay was employed to assess SPRY2 expression in ARC. (B) RT-qPCR and western blotting assays showed SPRY2 expression in SRA01/04 cells transfected with shSPRY2 or shNC. (C) CCK-8 assay showed the viability of SRA01/04 cells transfected with shSPRY2 or shNC. (D) TUNEL assay (magnification, ×200; scale bar, 50 µm) showed the apoptosis of SRA01/04 cells transfected with shSPRY2 or shNC. (E) RT-qPCR and western blotting assays showed SPRY2 expression in SRA01/04 cells transfected with pcDNA3.1/SPRY2 or pcDNA3.1. (F) CCK-8 assay showed the proliferation of SRA01/04 cells transfected with pcDNA3.1, pcDNA3.1/SPRY2, pcDNA3.1/SPRY2 + miR-124 mimics. (G) TUNEL assay (magnification, ×200; scale bar, 50 µm) showed the apoptosis of SRA01/04 cells transfected with pcDNA3.1, pcDNA3.1/SPRY2, pcDNA3.1/SPRY2 + miR-124 mimics. *P<0.05 vs. control group. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; SPRY2, protein sprouty homolog 2; sh-, short hairpin RNA; CCK-8, Cell Counting Kit-8; ARC, age-related cataract.

Figure 4.

MMP-2 is targeted by miR-124. (A) The binding sequence between MMP-2 and miR-124 was predicted using StarBase software. (B) Dual-luciferase reporter assay showed the luciferase activity of MMP-2-wt or MMP-2-mut in SRA01/04 cells transfected with NC mimics or miR-124 mimics. (C) Dual-luciferase reporter assay showed the luciferase activity of MMP-2-wt or MMP-2-mut in SRA01/04 cells transfected with NC inhibitor or miR-124 inhibitor. (D) RIP assay showed the enrichment of MMP-2 by Ago2 or IgG in SRA01/04 cells transfected with NC mimics or miR-124 mimics. (E) Reverse transcription-quantitative PCR assay revealed MMP-2 expression in SRA01/04 cells transfected with NC mimics or miR-124 mimics, NC inhibitor or miR-124 inhibitor. *P<0.05. miR, microRNA; MMP-2, matrix metalloproteinase-2; wt, wild-type; mut, mutant; RIP, RNA immunoprecipitation; Ago2, argonaute 2; IgG, immunoglobin G; NC, negative control.

Figure 5.

miR-124 depends on MMP-2 to regulate SRA01/04 cell apoptosis. (A) RT-qPCR was used to analyze MMP-2 expression in ARC tissues. (B) RT-qPCR and western blotting assays were performed to determine MMP-2 expression in SRA01/04 cells transfected with shMMP-2 or shNC. (C) CCK-8 assay showed the proliferation of SRA01/04 cells transfected with shMMP-2 or shNC. (D) TUNEL assay (magnification, ×200; scale bar, 50 µm) showed the cell apoptosis of SRA01/04 cells transfected with shMMP-2 or shNC. (E) RT-qPCR and western blotting assays were conducted to measure MMP-2 expression in SRA01/04 cells transfected with pcDNA3.1/MMP-2 or pcDNA3.1. (F) CCK-8 assay showed the proliferation of SRA01/04 cells transfected with pcDNA3.1, pcDNA3.1/MMP-2, pcDNA3.1/MMP-2 + miR-124 mimics. (G) TUNEL assay (magnification, ×200; scale bar, 50 µm) showed the apoptosis of SRA01/04 cells transfected with pcDNA3.1, pcDNA3.1/MMP-2, pcDNA3.1/MMP-2 + miR-124 mimics. *P<0.05 vs. control group. ARC, age-related cataract; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; MMP-2, matrix metalloproteinase-2; sh-, short hairpin RNA; CCK-8, Cell Counting Kit-8.