内质网应激通过miRNA-155作用PI3K-Akt-mTOR信号通路促进狼疮性肾炎进展的研究

Endoplasmic Reticulum Stress Promotes the Progression of Lupus Nephritis by MiRNA-155 Acting on PI3K-Akt-mTOR Signaling Pathway

目的内质网应激(ERS)是狼疮性肾炎(LN)进展期间的致病因素之一。虽然microRNAs(miRNAs)已经被证明与狼疮性肾炎的发生发展有密切关系,但内质网应激与miRNA在肾系膜细胞中的作用仍不清楚。本研究探讨miRNA-155在肾系膜细胞内质网应激中的作用及其在LN中作用机制。方法通过在40只小鼠(10只正常和30只LN小鼠)模型中进行肾组织活检来检验内质网应激的活性;分别在CTL和LN组间,ERS阳性/阴性亚组间,通过qRT-PCR实验检测miRNA-155的表达水平,通过Western Blot实验检测肾炎组织中PI3K-Akt-mTOR信号通路相关蛋白(P85、Akt、p-Akt、m-TOR、p-mTOR、S6、p-S6)以及内质网应激诱导型分子伴侣GRP78的表达情况;在体外肾系膜细胞中诱导内质网应激后,构建miRNA-155对照组和干扰组(沉默miRNA-155,或者添加PI3K-Akt-mTOR信号通路抑制剂),分别通过qRT-PCR实验检测细胞中miRNA-155的表达水平,通过MTT实验检测系膜细胞增殖能力,通过qRT-PCR、Western Blot实验检测PI3K-Akt-mTOR信号通路相关蛋白在mRNA水平和蛋白质水平的情况。结果(1)在大多数狼疮性肾炎模型中均表现出内质网应激反应为阳性。(2)在内质网应激活性为阳性的狼疮均表现出miRNA-155高表达。(3)在肾系膜细胞中,诱导内质网应激后发现miRNA-155表达增高,且细胞增殖能力增强;在诱导内质网应激后,沉默miRNA后,细胞增殖能力下降。(4)在ERS(+)肾系膜细胞中,发现miRNA-155表达增高的同时,Western Blot实验显示PI3K-Akt-mTOR信号通路相关蛋白水平均增高;在肾系膜细胞中,诱导内质网应激后,沉默miRNA-155后,Western Blot实验显示PI3K-Akt-mTOR信号通路相关蛋白水平均下降;在ERS(+)肾系膜细胞中,发现miRNA-155表达增高的同时,添加PI3K通路抑制剂后,细胞增殖能力下降。结论内质网应激可使狼疮性肾炎患者中miRNA-155表达增加,进而作用于PI3K-Akt-mTOR信号通路促进狼疮性肾炎进展。

Objective Endoplasmic reticulum stress(ERS)is one of the causative factors during the progression of lupus nephritis(LN).Although microRNAs(miRNAs)have been shown to be closely related to the development of lupus nephritis,the role of ERS and miRNAs in human renal mesangial cells(HMC)remains unclear.In light of this,we conducted this study to investigate the role of miRNA-155 in ERS of HMC and its underlying mechanism of LN.Methods The activity of endoplasmic reticulum stress was tested by detecting renal biopsy in 40 mice(10 normal and 30 ln mice),and these subjects were divided into two groups with ERS positive/negative.The expression of miRNA-155 was detected by qRT-PCR in two groups of LN mice with ERS negative/positive,and expression of PI3K-Akt-mTOR signaling pathway-associated protein(P85,Akt,p-Akt,m-TOR,p-mTOR,S6,p-S6)and endoplasmic reticulum stress inducible molecular chaperone GRP78 in normal and LN mice was detected by western blot assay.After induction of ERS in mesangial cells in vitro group,silencing miRNA-155 or adding PI3K-Akt-mTOR signaling pathway inhibitor was conducted,then detecting the expression level of miRNA-155 in cells by qRT-PCR,and detecting the proliferation of mesangial cells by MTT assay were performed.Western blot assay was used to detect the protein levels of PI3K-Akt-mTOR signaling pathway-related proteins.Results(1)ERS was positive in most subjects with lupus nephritis.(2)Subjects with LN of ERS positive showed a high expression of miRNA-155.(3)The expression of miRNA-155 was increased after induction of ERS positive in HMC,and the cell proliferation ability was enhanced.After induction of ERS positive in HMC,the cell proliferation ability decreased by silencing miRNA.(4)In LN mice of ERS positive,western blot experiment showed that the level of protein associated with the PI3K-Akt-mTOR signaling pathway was increased.After silencing miRNA-155 and adding the inhibitor of PI3K pathway in HMC of ERS positive,western blot showed that the level of protein related to PI3K-Akt-mTOR signaling pathway decreased,while MTT array showed that the cell proliferation ability was decreased.Conclusion Endoplasmic reticulum stress can increase the expression of miRNA-155 in subjects with lupus nephritis,which may act on PI3K-Akt-mTOR signaling pathway to promote the progression of lupus nephritis.