A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy
A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy
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DOI：
10.1371/journal.pone.0031021
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发表时间：
2012-02-02
期刊：
影响因子：
3.7
通讯作者：
Deltas, Constantinos
中科院分区：
文献类型：
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作者：
Papagregoriou, Gregory;Erguler, Kamil;Deltas, Constantinos
肝素结合表皮生长因子 (HBEGF) 在足细胞中表达，并被证明在肾小球生理学中发挥作用。使用 miRWalk 算法预测 HBEGF 3'UTR 上的 MicroRNA 结合位点，并对 103 名诊断为轻度或重度肾小球病的患者进行 DNA 测序。在 HBEGF 的 3'UTR 处鉴定出一个单核苷酸多态性 miRSNP C1936T (rs13385)，对应于 hsa-miR-1207-5p 种子区域的第二个碱基。当 AB8/13 未分化足细胞用 hsa-miR-1207-5p 的 miRNA 模拟物转染时，HBEGF 蛋白水平降低了约 50%。将包含 miRSNP 等位基因-1936C 的 DNA 片段克隆到 pMIR-Report 荧光素酶载体中，并与 hsa-miR-1207-5p 的 miRNA 模拟物共转染到 AB8/13 足细胞中。与蛋白质印迹数据一致，这导致荧光素酶表达减少，证明 hsa-miR-1207-5p 直接调节 HBEGF 表达的能力。相反，在存在 mirRSNP 1936T 等位基因的情况下，该调节被废除。总的来说，这些结果表明该 miRSNP 的变体 1936T 可防止 hsa-miR-1207-5p 下调足细胞中的 HBEGF。我们假设该变体具有作为遗传修饰剂的功能作用。为此，我们发现，在 78 名被诊断患有 CFHR5 肾病（也称为 C3 肾病）的患者队列中，miRSNP 1936T 等位基因的遗传显着增加，在长期随访中表明该组患者进展为慢性肾功能衰竭。在薄基底膜肾病患者队列中没有检测到类似的关联。这是第一份将 miRRSNP 作为基因修饰剂与单基因肾病联系起来的报告。
Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3'UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.