Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract

Abstract

AIM: To investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells (HLECs). METHODS: Exosomes were isolated from human aqueous humour (AH) by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes with cataracts (DMC) group and age-related cataracts (ARC) group. TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time polymerase chain reaction and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ in human AH and the culture supernatant of cells were detected by the calcium assay kit. Cell counting kit-8 was used to determine cell viability. RESULTS: Exosomes were isolated from human AH, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in DMC group compared with ARC. Ca2+ concentration of human AH in DMC was higher than that in ARC. The culture supernatant of cells transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of HLECs by upregulating CACNA1C expression. CONCLUSION: Exosomes isolated from human AH contains abundant miRNAs. A significantly expressed miRNA, miR-29b, can affect the concentration of Ca2+ and regulate HLEC processes by upregulating CACNA1C.

Status of higher TGF-β1 and TGF-β2 levels in the aqueous humour of patients with diabetes and cataracts

“…In our previous study, we successfully isolated abundant exosomes from AH samples in the DMC and ARC groups and found that exosomal miRNAs could target the mRNAs of certain genes to affect the viability and apoptosis of human lens epithelial cells. For example, high expression of miR-551b could downregulate CRYAA expression, thus decreasing the transparency of the lens [ 26 ], and low expression of miR-29b could upregulate CACNA1C expression, resulting in an increased concentration of Ca 2+ in the AH of DMC eyes [ 27 ]. The miR-26a-5p/ITGAV/TGF-β/Smad3 axis was also reported to be involved in cell viability, migration and epithelial–mesenchymal transition in diabetic cataracts [ 22 ].…”

Extracellular vesicles in the retina - putative roles in physiology and disease

“…EM revealed EVs of an appropriate size but the presence of EV tetraspanins was not examined. Differences in the miRNA content of the different samples of AH-derived EVs were reported (C. Gao et al, 2021 ), but as noted above, the EV samples were not pre-treated with appropriate enzymes (RNAse, trypsin, proteinase K, see Valadi et al, 2007 ) to eliminate extravesicular contaminants, limiting what can be concluded with regards to biomarker discovery. In another study, Mass Spectrometry analysis of the EV cargo of AH-derived EVs from patients with AMD revealed elevated levels of apolipoprotein A1, C3 convertase, opticin and clusterin proteins, compared to healthy controls ( Tsai et al, 2022 ); the study did not perform RNA anlysis.…”

“…Specific miRNAs have been found in AH derived EVs from uveal melanoma patients (Luz Pessuti et al, 2022), while Gao et al, 2021 provide convincing evidence of the presence of EVs in AH fluids from patients with diabetes and cataracts (DMC), compared with patients with age-related cataracts (ARC) (C. Gao et al, 2021). EM revealed EVs of an appropriate size but the presence of EV tetraspanins was not examined.…”

“…EM revealed EVs of an appropriate size but the presence of EV tetraspanins was not examined. Differences in the miRNA content of the different samples of AH-derived EVs were reported (C. Gao et al, 2021), but as noted above, the EV samples were not pre-treated with appropriate enzymes (RNAse, trypsin, proteinase K, see Valadi et al, 2007) analysis of the EV cargo of AH-derived EVs from patients with AMD revealed elevated levels of apolipoprotein A1, C3 convertase, opticin and clusterin proteins, compared to healthy controls (Tsai et al, 2022); the study did not perform RNA anlysis. Although the technical details of the LC-MS are available in this study, further evidence that the protein sample originates from EVs would strengthen their conclusions.…”

The regulatory role of microRNAs in common eye diseases: A brief review

“…It was also noticed that mimics of miR-551b reduced the viability of HLECs drastically, and its inhibitors lessened the apoptosis of these cells ( Gao et al, 2021a ). Additionally, Gao et al (2021b) found that in exosomes isolated from AH, miR-29b was substantially downregulated in a DC cohort than an ARC cohort, thus triggering the expression of its potential target Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C). High CACNA1C level is associated with increased concentration of Ca 2+ inducing apoptosis of HLECs in patients with DC ( Gao et al, 2021b ).…”