DNMT3a 通过负向调控 PTEN 来激活 PI3K/AKT 通路，从而加重肾脏纤维化。

DNMT3a negatively regulates PTEN to activate the PI3K/AKT pathway to aggravate renal fibrosis.

机构信息

Department of Nephrology, Wuhan No.1 Hospital, Wuhan, 430022, China.

Department of Nephrology, Wuhan No.1 Hospital, Wuhan, 430022, China..

出版信息

Cell Signal. 2022 Aug;96:110352. doi: 10.1016/j.cellsig.2022.110352. Epub 2022 Jun 5.

DOI:10.1016/j.cellsig.2022.110352

PMID:35523401

Abstract

BACKGROUND

Renal fibrosis has become one of the major diseases threatening global public health and harming human life and health. PTEN methylation plays an important role in fibrotic diseases of many organs. However, the relationship between PTEN methylation and renal fibrosis is still elusive.

METHODS

In the present study, we established a unilateral ureteral obstruction (UUO) mouse model in vivo and a transforming growth factor β1 (TGF-β1)-stimulated renal tubular epithelial cell (HK-2) model in vitro. The degree of renal interstitial fibrosis was detected by haematoxylin-eosin (HE) staining and Masson's trichrome staining. Western blot (WB), qRT-PCR, immunohistochemistry (IHC) and methylation-specific PCR (MSP) analyses were used to determine the mechanism by which PTEN methylation regulates renal fibrosis. The α-SMA fibrosis marker was detected by immunofluorescence (IF). Additionally, the relationship of PTEN and DNMT3a in UUO was determined by ChIP-qRT-PCR.

RESULTS

Our results showed that the promoter region of PTEN was methylated in UUO. Compared to the sham group, the expression of PTEN was significantly reduced in the UUO group. However, the demethylation reagent significantly inhibited epithelial-mesenchymal transition (EMT), which showed increased expression of E-cadherin and decreased expression of α-SMA and fibronectin. Moreover, treatment of HK-2 cells with 5-aza-dc reversed the activation of the TGF-β1-induced PI3K/AKT signalling pathway, which inhibited renal fibrosis. WB analysis demonstrated that TGF-β1 inhibited the PTEN protein expression level and DNMT3a knockdown reversed the inhibitory effect of TGF-β1 on PTEN expression. Furthermore, ChIP-qRT-PCR showed that DNMT3a interacted with PTEN. Finally, we found that DNMT3a negatively regulated PTEN to activate the PI3K/AKT signalling pathway and aggravate renal fibrosis in vitro and in vivo.

CONCLUSION

In summary, these results indicated that renal fibrosis is related to the downregulation of PTEN. Additionally, DNMT3a negatively regulates PTEN to activate the PI3K/AKT signalling pathway and induce EMT in renal tubular epithelial cells, thereby aggravating renal fibrosis.

摘要

背景

肾纤维化已成为威胁全球公众健康、危害人类生命健康的主要疾病之一。PTEN 甲基化在许多器官的纤维化疾病中发挥着重要作用。然而，PTEN 甲基化与肾纤维化之间的关系仍不清楚。

方法

本研究在体内建立单侧输尿管梗阻（UUO）小鼠模型和体外转化生长因子β1（TGF-β1）刺激的肾小管上皮细胞（HK-2）模型，通过苏木精-伊红（HE）染色和 Masson 三色染色检测肾间质纤维化程度。采用 Western blot（WB）、qRT-PCR、免疫组化（IHC）和甲基化特异性 PCR（MSP）分析来确定 PTEN 甲基化调节肾纤维化的机制。通过免疫荧光（IF）检测α-SMA 纤维化标志物。此外，通过 ChIP-qRT-PCR 确定 UUO 中 PTEN 与 DNMT3a 的关系。

结果

我们的结果表明，PTEN 的启动子区域在 UUO 中发生甲基化。与假手术组相比，UUO 组 PTEN 的表达明显降低。然而，去甲基化试剂显著抑制上皮-间充质转化（EMT），表现为 E-钙黏蛋白表达增加，α-SMA 和纤维连接蛋白表达减少。此外，用 5-aza-dc 处理 HK-2 细胞逆转了 TGF-β1 诱导的 PI3K/AKT 信号通路的激活，从而抑制了肾纤维化。WB 分析表明，TGF-β1 抑制了 PTEN 蛋白表达水平，DNMT3a 敲低逆转了 TGF-β1 对 PTEN 表达的抑制作用。此外，ChIP-qRT-PCR 表明 DNMT3a 与 PTEN 相互作用。最后，我们发现 DNMT3a 负调控 PTEN 以激活 PI3K/AKT 信号通路，并在体外和体内加重肾纤维化。