Abstract：
Chronic kidney disease (CKD) is a serious threat to human health worldwide, and its incidence is increasing annually. A growing amount of information is emerging about the role of micoRNAs (miRNAs) in the regulation of renal fibrosis, which has aroused interest in the development of drugs that block pathogenic miRNAs or restore protective miRNAs levels. To clarify the role of miRNAs in CKD, we selected patients with significant renal fibrotic disease (diabetic nephropathy (DN) and focal segmental glomerulosclerosis (FSGS)) as the disease group, and patients with little or no renal fibrotic disease (minimal change disease (MCD) and renal carcinoma adjacent to normal kidney) as controls. Significantly differentially expressed miRNAs were obtained by human kidney tissue sequencing, subsequently verified in mice models of DN and FSGS, and subsequently inhibited or overexpressed in human renal tubular epithelial cells (HK-2) stimulated by high glucose (HG) and TGF-β1 in vitro. Therefore, the mechanism of its action in renal fibrosis was further elaborated. Finally, the downstream target genes of the corresponding miRNAs were verified by bioinformatics analysis, qRT-PCR, western blot and double luciferase report analysis. Two novel miRNAs, hsa-miR-1470-3p (miR-1470) and hsa-miR-4483-3p (miR-4483), were detected by renal tissue sequencing in the disease group with significant renal fibrosis (DN and FSGS) and the control group with little or no renal fibrosis (MCD and normal renal tissue adjacent to renal carcinoma). Subsequent human renal tissue qRT-PCR verified that the expression of miR-1470 was significantly increased, while the expression of miR-4483 was markedly decreased in the disease group (p < 0.05). Moreover, in vivo DN and FSGS mice models, the expression levels of miR-1470 and miR-4483 were consistent with the results of human kidney tissue. In vitro, miR-4483 was suppressed, whereas miR-1470 was induced by treatment with TGF-β1 or HG. Inhibition of miR-1470 or overexpression of miR-4483 promoted HG or TGF-β1-induced fibrosis in HK-2 cells. Further study revealed that MMP-13 and TIMP1 were the target genes ofmiR-1470 and miR-4483, respectively. Our study identifies newly dysregulated miRNA profiles related to fibrosis kidneys. miR-1470 and miR-4483 are demonstrated to participate in kidney fibrosis by regulation of MMP-13, TIMP1 respectively. Our results may represent a promising research direction for renal disorders and help identify new biomarkers and therapeutic targets for CKD.
摘要：
慢性肾脏病(CKD)是全球范围内严重威胁人类健康的疾病,发病率每年都在增加。关于micoRNAs（miRNAs）在调节肾纤维化中的作用的信息越来越多，这引起了人们对开发阻断致病性miRNA或恢复保护性miRNA水平的药物的兴趣。为了阐明miRNA在CKD中的作用，我们选择有显著肾纤维化疾病（糖尿病肾病（DN）和局灶节段肾小球硬化（FSGS））的患者作为疾病组，和很少或没有肾脏纤维化疾病（微小病变（MCD）和与正常肾脏相邻的肾癌）的患者作为对照。通过人肾组织测序获得显著差异表达的miRNA，随后在DN和FSGS的小鼠模型中验证，随后在体外高葡萄糖（HG）和TGF-β1刺激的人肾小管上皮细胞（HK-2）中抑制或过表达。因此,进一步阐述了其在肾纤维化中的作用机制。最后,通过生物信息学分析验证相应miRNAs的下游靶基因,qRT-PCR,蛋白质印迹和双荧光素酶报告分析。两个新的miRNA,hsa-miR-1470-3p（miR-1470）和hsa-miR-4483-3p（miR-4483），通过肾组织测序检测到有明显肾纤维化的疾病组（DN和FSGS）和很少或没有肾纤维化的对照组（MCD和肾癌附近的正常肾组织）。随后的人肾组织qRT-PCR证实miR-1470的表达显著增加,而miR-4483的表达在疾病组中明显降低(p<0.05)。此外，体内DN和FSGS小鼠模型，miR-1470和miR-4483的表达水平与人肾组织的结果一致。体外，miR-4483被抑制，而miR-1470是通过TGF-β1或HG治疗诱导的。miR-1470的抑制或miR-4483的过表达促进HK-2细胞中HG或TGF-β1诱导的纤维化。进一步研究发现MMP-13和TIMP1分别是miR-1470和miR-4483的靶基因。我们的研究鉴定了与纤维化肾脏相关的新的失调的miRNA谱。miR-1470和miR-4483分别通过调节MMP-13和TIMP1参与肾纤维化。我们的研究结果可能代表了肾脏疾病的一个有希望的研究方向，并有助于确定CKD的新生物标志物和治疗靶点。
