Zhang Lihua, Zhu Honglin, Li Yisha, Dai Xiaodan, Zhou Bin, Li Quanzhen, Zuo Xiaoxia, Luo Hui
a Department of Rheumatology , Xiangya Hospital, Central South University , Changsha , Hunan , People's Republic of China.
b Department of Immunology and Internal Medicine , University of Texas Southwestern Medical Center , Dallas , TX , USA.
Mod Rheumatol. 2017 Nov;27(6):1010-1018. doi: 10.1080/14397595.2016.1270387. Epub 2017 Jan 13.
It's reported that multiple genes in the IFN-γ/STAT1 pathway were hypomethylated and associated with the pathogenesis of lupus nephritis (LN). Our previous study using microarray analysis suggested that interferon induced 35-kDa protein (IFI35) was hypomethylated and increased in LN. However, the role of IFI35 in LN and related mechanism remains to be elucidate.
The expressions of IFNγR, STAT1, IFI35 and MBD2 in the human kidneys tissues was detected by real-time PCR and Western blot. The protein levels of IFI35 in the human kidney tissues were detected by immunohistochemistry. The methylation status of IFNγR, STAT1 and IFI35 were detected by methylation specific PCR. Cell proliferation assay was evaluated using cell counting kit 8; pcDNA-IFI35 (pcDNA-MBD2) or IFI35 RNAi (MBD2 RNAi) was used to upregulated or downregulated the expression of the IFI35 and MBD2.
The expressions of IFNγR, STAT1 and IFI35 in the LN kidneys were significantly higher than controls. IFI35 was expressed in mesangial cells, and positively correlated with the proliferation of mesangial cells. IFNγR, STAT1and IFI35 was hypomethylated and MBD2 was increased in LN kidneys. In vitro data confirmed those findings: after stimulating with the serum from LN patients, the proliferation of human renal mesangial cells (HRMCs) was increased. The expressions of the three members of IFNγ signal pathway were hypomethylated and upregulated. However, this effect was reversed by MBD2 knockdown. IFI35 promoted the proliferation of HRMCs and was regulated by MBD2.
Our results demonstrated that IFI35 enhances the proliferation of mesangial cells and was regulated by MBD2 in LN.
据报道，IFN-γ/STAT1信号通路中的多个基因发生低甲基化，并与狼疮性肾炎（LN）的发病机制相关。我们之前使用微阵列分析的研究表明，干扰素诱导的35 kDa蛋白（IFI35）在LN中发生低甲基化且表达增加。然而，IFI35在LN中的作用及其相关机制仍有待阐明。
采用实时PCR和蛋白质免疫印迹法检测人肾组织中IFNγR、STAT1、IFI35和MBD2的表达。采用免疫组织化学法检测人肾组织中IFI35的蛋白水平。采用甲基化特异性PCR检测IFNγR、STAT1和IFI35的甲基化状态。使用细胞计数试剂盒8评估细胞增殖情况；使用pcDNA-IFI35（pcDNA-MBD2）或IFI35 RNA干扰（MBD2 RNA干扰）上调或下调IFI35和MBD2的表达。
LN肾组织中IFNγR、STAT1和IFI35的表达明显高于对照组。IFI35在系膜细胞中表达，并与系膜细胞的增殖呈正相关。LN肾组织中IFNγR、STAT1和IFI35发生低甲基化，MBD2表达增加。体外实验数据证实了这些发现：用LN患者的血清刺激后，人肾小球系膜细胞（HRMCs）的增殖增加。IFNγ信号通路的三个成员的表达发生低甲基化并上调。然而，MBD2基因敲低可逆转这种效应。IFI35促进HRMCs的增殖，并受MBD2的调控。
我们的结果表明，IFI35增强系膜细胞的增殖，并在LN中受MBD2的调控。
