微小RNA-106a-5p/集落刺激因子1轴对狼疮性肾炎患者免疫球蛋白G诱导的足细胞损伤的影响

Effect of microRNA-106a-5p/colony stimulating factor 1 axis on immunoglobulin G induced podocyte injury in patients with lupus nephritis

目的探讨微小RNA-106a-5p/集落刺激因子1(miR-106a-5p/CSF1)轴对狼疮性肾炎(LN)患者免疫球蛋白G(LN-IgG)诱导的足细胞损伤的影响。方法采用实时荧光定量聚合酶链式反应(qRT-PCR)和Western blot法检测LN患者和健康志愿者血清及LN-IgG或阴性对照(NC)-IgG(提取自健康志愿者)诱导的足细胞中miR-106a-5p和CSF1的表达。采用双荧光素酶报告实验检测miR-106a-5p与CSF1的靶向关系。采用CCK-8实验、流式细胞术、酶联免疫吸附试验(ELISA)和Western blot实验检测miR-106a-5p或CSF1表达修饰后的足细胞活性、凋亡、炎症反应和自噬。结果与健康志愿者相比,LN患者体内miR-106a-5p低表达,而CSF1高表达,差异有统计学意义(P<0.05);miR-106a-5p在LN-IgG诱导的足细胞中表达降低,而CSF1表达上调,差异有统计学意义(P<0.05)。功能实验表明,过表达miR-106a-5p可逆转LN-IgG诱导的足细胞活性抑制以及凋亡促进,同时抑制LN-IgG诱导细胞中白细胞介素-6(IL-6)以及肿瘤坏死因子-α(TNF-α)含量的升高,同时诱导足细胞自噬,差异有统计学意义(P<0.05)。CSF1是miR-106a-5p的功能靶标。实验结果显示,在LN-IgG诱导的足细胞中,上调CSF1抑制miR-106a-5p过表达介导细胞活力增强、凋亡抑制、炎症反应以及自噬增强,差异有统计学意义(P<0.05)。结论miR-106a-5p通过靶向抑制CSF1减弱LN-IgG诱导的足细胞凋亡和炎症反应以及促进足细胞自噬,表明miR-106a-5p可能是延迟或缓解LN的有效治疗靶标。

Objective To investigate the effect of microRNA-106a-5p/colony stimulating factor 1(miR-106a-5p/CSF1)axis on podocyte injury induced by lupus nephritis(LN)-immunoglobulin G(LN-IgG).Methods Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expressions of miR-106a-5p and CSF1 in the serum of LN patients and healthy volunteers,as well as in podocytes stimulated by negative control(NC)-IgG(isolated from healthy volunteers)or LN-IgG.Dual-luciferase report assay was used to detect the targeting relationship between miR-106a-5p and CSF1.The effects of modified miR-106a-5p or CSF1 expression on podocyte viability,apoptosis,inflammatory response,and autophagy were studied in vivo by using CCK-8 assay,flow cytometry,enzyme-linked immunosorbent assay(ELISA),and Western blot assay respectively.Results Compared with healthy volunteers,LN patients showed significant low expression of miR-106a-5p and high expression of CSF1(P<0.05);miR-106a-5p expression was significantly reduced in LN-IgG induced podocytes,while CSF1 expression was significantly upregulated(P<0.05).Functional experiments showed that overexpression of miR-106a-5p was able to reverse LN-IgG induced podocyte viability inhibition and apoptosis promotion,inhibit increases of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),as well as promote podocyte autophagy,and there were significant differences(P<0.05).CSF1 was a functional target of miR-106a-5p.The experiment results showed that upregulation of CSF1 was able to significantly inhibit miR-106a-5p overexpression-mediated cell viability enhancement,apoptosis inhibition,inflammatory response inhibition,and autophagy enhancement in LN-IgG induced podocytes(P<0.05).Conclusion The miR-106a-5p attenuates LN-IgG induced apoptosis,inflammation in podocytes and promotes podocyte autophagy by targeting CSF1,which suggests that miR-106a-5p may be an effective therapeutic target to delay or alleviate LN.