Department of Nephrology, The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin, China.
Department of Chinese Formulae, Heilongjiang University of Chinese Medicine, Harbin, China.
Kidney Blood Press Res. 2022;47(1):1-12. doi: 10.1159/000516791. Epub 2021 Nov 16.
The present study aimed to elucidate the potential function of microRNA 1228 (miR-1228) on the high glucose (HG)-damaged human renal proximal tubule cells (HK-2) and the underlying mechanism.
The datasets GSE47185 and GSE51674 were downloaded from the Gene Expression Omnibus database for mining differently expressed mRNAs and miRNAs, respectively. Bioinformatics online tools were applied to predict the binding sites between miR-1228 and thrombospondin 2 (THBS2), which was confirmed by dual-luciferase assay. Real-time quantitative polymerase chain reaction was used to detect the mRNA level of miR-1228/THBS2. Western blot was used to detect the protein level of THBS2 and the PI3K/AKT signaling pathway-associated markers. HK-2 cells were cultured in HG (30 mM) to mimic hyperglycemia. Cell counting kit 8 and flow cytometry assays were utilized to determine the cell proliferation and apoptosis.
The expression of THBS2 was significantly upregulated in diabetic nephropathy (DN) based on bioinformatics tools and identified as a direct target of miR-1228. miR-1228 was downregulated in DN and HG-damaged HK-2 cells. HG notably reduced HK-2 cell proliferation. This negative effect was attenuated by transfecting with an miR-1228 mimic and aggravated by transfecting with an miR-1228 inhibitor. However, under basal condition, there was no significant effect on the HK-2 cell proliferation among blank control, mimic, and inhibitor groups. Overexpression of THBS2 abolished the elevating effect of the miR-1228 mimic on the HG-damaged HK-2 cell proliferation, while restored the inhibitory effects of the miR-1228 mimic on the cell apoptosis. On the contrary, the suppressive effects on the proliferation and the enhancive effects on the apoptosis by silencing miR-1228 in HK-2 cells stimulated with HG can be weakened by recommendation of THBS2 small interference RNAs. Furthermore, we also found that HG significantly enhanced the phosphorylation levels of PI3K and AKT. In terms of overexpression and knockdown experiments, Western blot analysis further revealed that miR-1228 inhibited the activation of the PI3K/AKT signaling pathway in HG-damaged HK-2 cells by regulating THBS2.
The findings illustrated that miR-1228 improved survivability and inhibited apoptosis in HK-2 cells stimulated with HG partly by restraining the activation of the PI3K/AKT signaling pathway.
本研究旨在阐明 microRNA 1228（miR-1228）在高糖（HG）损伤的人近端肾小管细胞（HK-2）中的潜在功能及其作用机制。
从基因表达综合数据库中分别下载数据集 GSE47185 和 GSE51674，以挖掘差异表达的 mRNA 和 miRNA。应用生物信息学在线工具预测 miR-1228 与血小板反应蛋白 2（THBS2）之间的结合位点，通过双荧光素酶报告基因实验进行验证。实时定量聚合酶链反应检测 miR-1228/THBS2 的 mRNA 水平。Western blot 检测 THBS2 及 PI3K/AKT 信号通路相关标记物的蛋白水平。采用 30 mM HG 培养 HK-2 细胞模拟高血糖。细胞计数试剂盒 8 和流式细胞术检测细胞增殖和凋亡。
基于生物信息学工具，发现 THBS2 在糖尿病肾病（DN）中表达显著上调，被鉴定为 miR-1228 的直接靶标。miR-1228 在 DN 和 HG 损伤的 HK-2 细胞中表达下调。HG 显著抑制 HK-2 细胞增殖。转染 miR-1228 模拟物可减弱这种抑制作用，而转染 miR-1228 抑制剂则加重这种抑制作用。然而，在基础条件下，空白对照、模拟物和抑制剂组之间对 HK-2 细胞增殖无显著影响。过表达 THBS2 可消除 miR-1228 模拟物对 HG 损伤的 HK-2 细胞增殖的促进作用，同时恢复 miR-1228 模拟物对细胞凋亡的抑制作用。相反，在 HG 刺激下，沉默 miR-1228 对 HK-2 细胞增殖的抑制作用和对细胞凋亡的促进作用可因 THBS2 小干扰 RNA 的推荐而减弱。此外，我们还发现 HG 显著增强了 PI3K 和 AKT 的磷酸化水平。在过表达和敲低实验中，Western blot 分析进一步表明，miR-1228 通过调节 THBS2 抑制 HG 损伤的 HK-2 细胞中 PI3K/AKT 信号通路的激活。
研究结果表明，miR-1228 通过抑制 PI3K/AKT 信号通路的激活，提高 HG 刺激的 HK-2 细胞的存活率并抑制细胞凋亡。
