miR-15a-5p 通过靶向 VEGF 抑制大鼠腹膜透析诱导的腹膜纤维化。

miR-15a-5p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting VEGF in rats.

机构信息

Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA.

出版信息

Ren Fail. 2020 Nov;42(1):932-943. doi: 10.1080/0886022X.2020.1811123.

AIM

When peritoneal fibrosis (PF) causes ultrafiltration failure in peritoneal dialysis (PD) patients, PD has to be discontinued. Currently, there is no effective way to relieve PF. In this study, we aimed to determine whether miR-15a-5p is involved in PF and to determine the underlying mechanism.

METHODS

Six normal rats were used as the control group. A uremic rat model was constructed using 5/6 nephrectomy in a Sprague-Dawley model. The uremic rats were randomly divided into PD, lentivirus-transfected, negative control, VEGFR-inhibited and gavage control groups. Except for the control group, all uremia rats received continuous PD for 28 days. In the lentivirus-transfected group, the miR-15a-5p plasmid was injected into the peritoneal cavity to upregulate miR-15a-5p expression. Axitinib was used to block vascular endothelial growth factor receptor (VEGFR) in the peritoneum. The mRNA levels of miR-15a-5p and VEGF were detected by qRT-PCR and FISH. Protein levels of VEGF, E-cadherin, collagen IV, fibronectin and α-SMA were detected by western blot and immunohistochemistry.

RESULTS

PD leads to peritoneal thickening and fibrosis. The expression level of miR-15a-5p decreased and that of VEGF increased in the PD group than in the controls. Additionally, E-cadherin was significantly reduced while collagen IV, fibronectin and α-SMA were obviously increased in the PD group compared to controls. FISH showed that VEGF might be the target gene of miR-15a-5p. Overexpression of miR-15a-5p or inhibition of VEGFR could reverse PF.

CONCLUSION

miR-15a-5p may participate in the endothelial to mesenchymal transition of PF caused by PD through VEGF.

目的

当腹膜纤维化（PF）导致腹膜透析（PD）患者超滤失败时，PD 必须停止。目前，尚无有效方法缓解 PF。本研究旨在确定 miR-15a-5p 是否参与 PF，并确定其潜在机制。

方法

将 6 只正常大鼠作为对照组。通过 Sprague-Dawley 模型中的 5/6 肾切除术构建尿毒症大鼠模型。将尿毒症大鼠随机分为 PD、慢病毒转染、阴性对照、VEGFR 抑制和灌胃对照组。除对照组外，所有尿毒症大鼠均接受连续 PD 治疗 28 天。在慢病毒转染组中，将 miR-15a-5p 质粒注入腹腔以上调 miR-15a-5p 的表达。使用阿昔替尼阻断血管内皮生长因子受体（VEGFR）在腹膜中的表达。通过 qRT-PCR 和 FISH 检测 miR-15a-5p 和 VEGF 的 mRNA 水平。通过 Western blot 和免疫组化检测 VEGF、E-钙黏蛋白、IV 型胶原、纤连蛋白和α-SMA 的蛋白水平。

结果

PD 导致腹膜增厚和纤维化。与对照组相比，PD 组 miR-15a-5p 的表达水平降低，而 VEGF 的表达水平升高。此外，与对照组相比，PD 组 E-钙黏蛋白明显减少，而 IV 型胶原、纤连蛋白和α-SMA 明显增加。FISH 表明，VEGF 可能是 miR-15a-5p 的靶基因。过表达 miR-15a-5p 或抑制 VEGFR 可以逆转 PF。