Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract
Exosomal miR-29b found in aqueous humour mediates calcium signaling in diabetic patients with cataract
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房水中发现的外泌体 miR-29b 介导糖尿病白内障患者的钙信号传导
DOI：
10.18240/ijo.2021.10.03
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发表时间：
2021-10-18
影响因子：
1.4
通讯作者：
Luo, Yi
中科院分区：
文献类型：
--
作者：
Gao, Chao;Liu, Xin;Luo, Yi
目的：探讨胞外体miR-29b和钙离子在调节人晶状体上皮细胞(HLECs)功能中的作用。方法：采用超速离心法从人房水中分离胞外体，并用纳米粒子示踪和透射电子显微镜观察。对糖尿病合并白内障(DMC)组和老年性白内障(ARC)组进行外体miRNA测序，以确定差异表达的miRNAs。用TargetScan预测某些miRNA的潜在靶点。实时定量聚合酶链式反应检测CACNA1C基因的表达，Western blotting检测CACNA1C蛋白的表达。用钙离子测定试剂盒检测人脑脊液中钙离子浓度及细胞培养上清。结果：从人AH中分离到外切体，具有典型的杯状表型，其颗粒大小分布与细胞外微泡一致。外体miRNA测序显示，与ARC组相比，DMC组miR-29b表达显著下调。人AH在DMC中的Ca~(2+)浓度高于ARC。转导miR-29b抑制剂的细胞培养上清液中钙离子浓度高于转导miR-29b模拟物的细胞。MIR-29b通过上调CACNA1C的表达降低HLECs的活性。结论：从人AH分离的外切体中含有丰富的miRNAs。一个显著表达的miRNA miR-29b可以通过上调CACNA1C来影响钙离子浓度并调节HLEC过程。
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 miR29b 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.