Objective: To investigate the effect of miR-324-5p on the proliferation of rat glomerular mesangial (HBZY-1) cells and the role of Syk/Ras/c-fos signaling pathway in mediating this effect.
Methods: HBZY-1 cells cultured in vitro were transiently transfected with miR-324-5p mimics or miR-324-5p-mimics-NC followed by treatment with lipopolysaccharide (LPS). MTT assay was used to detect the proliferation activity of HBZY-1 cells, and RT-qPCR was used to detect the expressions of miR-324-5p and the mRNA expressions of Syk, Ras, MEK1/2, ERK1/2 and c-fos mRNA. The protein expressions of p-Syk, Ras, p-MEK1/2, p-ERK1/2 and c-Fos were detected by Western blotting and immunofluorescence assay.
Results: MTT assay showed that exposure to LPS significantly enhanced the proliferative activity of HBZY-1 cells. Compared with the cells treated with LPS and LPS + mimics NC, the cells transfected with miR-324-5p mimics prior to LPS exposure exhibited significantly lowered proliferative activity. Transfection with miR-324-5p mimics significantly lowered the mRNA expressions of Syk, Ras, MEK1/2, ERK1/2 and c-fos and the protein expressions of p-Syk, Ras, MEK1/2, ERK1/2 and c-Fos (P < 0.05), and reduced numbers of cells positive for p-Syk, Ras, p-MEK1/2, p-ERK1/2 and c-Fos proteins following LPS exposure.
Conclusions: miR-324-5p can inhibit the proliferation of rat chronic glomerulonephritis cells induced by LPS by inhibiting Syk/Ras/c-fos signaling pathway and may potentially serve as a diagnostic indicator and a therapeutic target for chronic glomerulonephritis.
目的: 探讨miR-324-5p调控Syk/Ras/c-fos信号通路对大鼠肾小球系膜（HBZY-1）细胞增殖能力的影响。
方法: 体外培养HBZY-1细胞；设计并合成miR-324-5p mimics，miR-324-5p mimics-NC片段；采用Lipo3000试剂盒瞬时转染miR-324-5p mimics，miR-324-5p mimics-NC片段至脂多糖（LPS）诱导的HBZY-1细胞内，RT-qPCR验证转染效率；将HBZY-1细胞分为对照组（生理盐水），LPS组（LPS 0.5 μg/mL），LPS+mimics组（LPS 0.5 μg/mL+miR-324-mimics），LPS+mimics-NC组（LPS 0.5 μg/mL+miR-324-mimics-NC）；MTT法检测各组HBZY-1细胞增殖活性；RT-qPCR法检测各组细胞miR-324-5p及Syk、Ras、MEK1/2、ERK1/2、c-fos mRNA的表达；Western blot和免疫荧光法检测各组细胞p-Syk、Ras、p-MEK1/2、p-ERK1/2、c-fos蛋白的表达。
结果: MTT结果显示，LPS组细胞增殖水平较正常组显著升高，与LPS组及LPS+mimics-NC组相比，LPS+mimics组细胞增殖能力降低；RT-qPCR结果显示，LPS+mimics组中miR-324-5p表达明显高于LPS组及LPS+mimics-NC组，且LPS+mimics组中Syk、Ras、MEK1/2、ERK1/2、c-fos mRNA的表达显著低于LPS组及LPS+mimics-NC组, 差异具有统计学意义（P < 0.05）；Western Blot结果显示，与LPS组及LPS+mimics-NC组相比LPS+mimics组中p-Syk、Ras、p-MEK1/2、p-ERK1/2、c-fos蛋白表达显著降低，差异具有统计学意义（P < 0.05）；免疫荧光结果显示，与LPS组及LPS+mimics-NC组相比，LPS+mimics组p-Syk、Ras、p-MEK1/2、p-ERK1/2、c-fos蛋白标记细胞数目明显减少。
结论: miR-324-5p可通过抑制Syk/Ras/c-fos信号通路降低LPS诱导的慢性肾小球肾炎细胞的增殖活性，miR-324-5p有望成为慢性肾小球肾炎诊断和治疗的潜在靶标。
Keywords: Chronic glomerulonephritis; Syk/Ras/c-fos signaling pathway; cell proliferation; miR-324-5p.