mA 去甲基化酶 FTO 通过减少 lncRNA GAS5 的 mA 修饰促进肾小管上皮-间充质转化。

The mA demethylase FTO promotes renal epithelial-mesenchymal transition by reducing the mA modification of lncRNA GAS5.

机构信息

Department of Pediatrics, The Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, PR China; Laboratory of Pediatrics Nephrology, Institute of Pediatrics, The Second Xiangya Hospital, Central South University, Changsha 410011, Hunan Province, PR China.

出版信息

Cytokine. 2022 Nov;159:156000. doi: 10.1016/j.cyto.2022.156000. Epub 2022 Sep 1.

DOI:10.1016/j.cyto.2022.156000

PMID:36058192

Abstract

BACKGROUND

Renal interstitial fibrosis (RIF) is the main pathological change of a variety of chronic kidney diseases (CKD). Epigenetic modifications of fibrosis-prone genes regulate RIF progression. This study aimed to investigate long non-coding RNA (lncRNA) N6-methyladenosine (mA) modification and its role in regulating RIF progression.

METHODS

Unilateral ureteral occlusion (UUO) was employed to construct the RIF in vivo model; and TGF-β1-treated HK-2 and HKC-8 cells were used for in vitro experiments. The mRNA and protein expressions were assessed using qRT-PCR and western blot. The proliferation and migration were evaluated by EdU assay and transwell assay, respectively. In addition, levels of inflammatory cytokines were determined by ELISA assay and qRT-PCR. Moreover, lncRNA GAS5 mA level was detected using Me-RIP assay. HE and Masson staining were employed to evaluate fibrotic lesions of the kidney.

RESULTS

FTO expression was elevated in HK-2 and HKC-8 cells after TGF-β1 treatment and mouse kidney tissue following UUO, and lncRNA GAS5 was downregulated. LncRNA GAS5 overexpression or FTO silencing suppressed TGF-β1-induced the increase of EMT-related proteins (Vimentin, Snail and N-cadherin) and inflammatory cytokines (IL-6, IL-1β and TNF-α) levels in HK-2 cells. FTO suppressed lncRNA GAS5 expression by reducing the m6A modification of lncRNA GAS5. Additionally, FTO knockdown could suppress EMT process and inflammation response induced by TGF-β1 and UUO in vitro and in vivo. As expected, FTO knockdown abrogated the promotion effects of lncRNA GAS5 silencing on TGF-β1-induced EMT process and inflammation response in HK-2 and HKC-8 cells.

CONCLUSION

FTO promoted EMT process and inflammation response through reducing the mA modification of lncRNA GAS5.

摘要

背景

肾间质纤维化（RIF）是多种慢性肾脏病（CKD）的主要病理改变。纤维化倾向基因的表观遗传修饰调节 RIF 的进展。本研究旨在探讨长非编码 RNA（lncRNA）N6-甲基腺苷（mA）修饰及其在调节 RIF 进展中的作用。

方法

采用单侧输尿管结扎（UUO）构建体内 RIF 模型；用 TGF-β1 处理 HK-2 和 HKC-8 细胞进行体外实验。采用 qRT-PCR 和 Western blot 检测 mRNA 和蛋白表达。通过 EdU 检测和 Transwell 检测评估增殖和迁移。此外，通过 ELISA 检测和 qRT-PCR 检测炎症细胞因子水平。此外，通过 Me-RIP 检测检测 lncRNA GAS5 mA 水平。采用 HE 和 Masson 染色评估肾脏纤维化病变。

结果

TGF-β1 处理 HK-2 和 HKC-8 细胞及 UUO 后，FTO 表达上调，lncRNA GAS5 下调。lncRNA GAS5 过表达或 FTO 沉默抑制 TGF-β1 诱导的 HK-2 细胞 EMT 相关蛋白（波形蛋白、Snail 和 N-钙粘蛋白）和炎症细胞因子（IL-6、IL-1β 和 TNF-α）水平升高。FTO 通过降低 lncRNA GAS5 的 m6A 修饰抑制 lncRNA GAS5 的表达。此外，FTO 敲低可抑制 TGF-β1 和 UUO 在体外和体内诱导的 EMT 过程和炎症反应。不出所料，FTO 敲低消除了 lncRNA GAS5 沉默对 TGF-β1 诱导的 HK-2 和 HKC-8 细胞 EMT 过程和炎症反应的促进作用。