南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 46-51.doi: 10.12122/j.issn.1673-4254.2023.01.06

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环状RNA circRSF1结合HuR促进辐射诱导的肝星状细胞炎性表型

周佩涛，程炳霖，孙一宁，吴德华，陈宇翰   

  1. 南方医科大学南方医院放疗科，广东 广州 510515；南方医科大学第一临床医学院，广东 广州 510515
  • 出版日期:2023-01-20 发布日期:2023-02-23

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells

ZHOU Peitao, CHENG Binglin, SUN Yining, WU Dehua, CHEN Yuhan   

  1. Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China
  • Online:2023-01-20 Published:2023-02-23

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摘要： 目的 探讨环状RNA circRSF1能否通过结合HuR蛋白并阻遏其功能发挥，进而调控辐射诱导的肝星状细胞（HSC）炎性表型。方法 在人HSC细胞株LX2过表达或敲低HuR后给予8 Gy X射线照射。通过qRT-PCR检测炎性因子（IL-1β、IL-6和TNF-α）的表达，采用Western blot检测IκBα表达和NF-κB磷酸化。采用RNA pull down和RNA结合蛋白免疫沉淀(RIP)验证circRSF1与HuR的结合。改变circRSF1和HuR的作用水平后检测炎性因子和IκBα的表达以及NF-κB的磷酸化。结果 敲低HuR可明显上调受照的LX2中IL-1β、IL-6、TNF-α的表达，并减少IκBα表达和促进NF-κB磷酸化，而过表达HuR则相反（P<0.05）。过表达或敲低circRSF1对HuR的表达无影响。RNA pull down和RIP实验证实了两者存在相互结合。过表达circRSF1减少HuR与IκBα结合并下调IκBα的表达（P<0.05）。过表达circRSF1联合过表达HuR可部分逆转过表达circRSF1所致的LX2受照后炎性因子表达上调、IκBα表达下调以及NFκB磷酸化增加，而敲低circRSF1联合敲低HuR的效应则相反（P<0.05）。结论 circRSF1通过结合HuR降低IκBα表达，促进NF-κB通路激活，从而促进辐射诱导的HSC炎性表型。

关键词: 辐射；肝星状细胞；环状RNA；HuR；炎性表型

Abstract: Objective To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function. Methods Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR. Results Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P<0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P<0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P<0.05). Conclusion circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation-induced inflammatory phenotype of HSCs.

Key words: radiation; hepatic stellate cells; circular RNA; HuR; inflammatory phenotype