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Abstract：
This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson\'s staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1\'s 3\'-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.
摘要：
本研究旨在阐明miR-483-3p在高糖条件下对人肾小管上皮细胞(HK-2)的影响及其作用机制。人近端肾小管上皮细胞(HK-2)暴露于50mmol/L葡萄糖48h,建立肾小管上皮细胞损伤模型,表示为高葡萄糖组(HG组)。细胞也在含有5.5mmol/L葡萄糖的培养基中培养48小时，作为低葡萄糖组。在不同的组进行转染：HK-2+低糖（对照组），高葡萄糖（50mM）（HG组），高糖+miR-483-3p模拟物（HG+模拟物组），高糖+miR-483-3p抑制剂（HG+抑制剂组），和相应的阴性对照。实时定量聚合酶链反应（qPCR）评估miR-483-3p的mRNA表达，bax,bcl-2和caspase-3。Western印迹测定相应的蛋白质水平。使用CCK-8测定评估增殖，用荧光TUNEL法分析细胞凋亡。Westernblot和Masson's染色观察miR-483-3p转染后细胞纤维化的变化。此外，双荧光素酶试验研究了miR-483-3p和IGF-1之间的靶向关系.CCK8实验证明HG+模拟组抑制HK-2细胞增殖,而荧光TUNEL法显示该组细胞诱导凋亡。相反，HG+抑制剂组促进细胞增殖，抑制细胞凋亡。HG+模拟组上调促凋亡标志物(bax和caspase-3)的mRNA和蛋白表达,同时下调抗凋亡标志物（bcl-2）表达。相比之下,HG+抑制剂组表现出相反的作用。与对照组相比,HG+模拟物组的胶原I和FN蛋白水平显著升高(P<0.05)。相反，在HG+抑制剂组中，与HG组相比，CollagenI和FN的蛋白表达明显降低（P<0.05）。双荧光素酶报告基因实验证实miR-483-3p可以抑制IGF-1的3'-UTR区的荧光素酶活性(P<0.05)。miR-483-3p靶向调控IGF-1，促进高糖诱导的肾小管上皮细胞凋亡和纤维化。
