Wang Jing, Zhu Xiaoli, Qin Xiujuan, Jiang Hui, Gao Yachen, Gao Jiarong
First Affiliated Hospital, Anhui University of Chinese Medicine, Hefei 230012, China.
College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Nov 30;40(11):1571-1578. doi: 10.12122/j.issn.1673-4254.2020.11.06.
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.
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.
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 ( < 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.
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.
探讨miR-324-5p对大鼠肾小球系膜（HBZY-1）细胞增殖的影响以及Syk/Ras/c-fos信号通路在介导该作用中的作用。
体外培养的HBZY-1细胞用miR-324-5p模拟物或miR-324-5p模拟物阴性对照进行瞬时转染，随后用脂多糖（LPS）处理。采用MTT法检测HBZY-1细胞的增殖活性，采用RT-qPCR检测miR-324-5p的表达以及Syk、Ras、MEK1/2、ERK1/2和c-fos mRNA的表达。通过蛋白质印迹法和免疫荧光法检测p-Syk、Ras、p-MEK1/2、p-ERK1/2和c-Fos的蛋白表达。
MTT法显示，暴露于LPS可显著增强HBZY-1细胞的增殖活性。与LPS和LPS +模拟物阴性对照处理的细胞相比，在LPS暴露前用miR-324-5p模拟物转染的细胞增殖活性显著降低。用miR-324-5p模拟物转染显著降低了Syk、Ras、MEK1/2、ERK1/2和c-fos的mRNA表达以及p-Syk、Ras、MEK1/2、ERK1/2和c-Fos的蛋白表达（<0.05），并减少了LPS暴露后p-Syk、Ras、p-MEK1/2、p-ERK1/2和c-Fos蛋白阳性细胞的数量。
miR-324-5p可通过抑制Syk/Ras/c-fos信号通路抑制LPS诱导的大鼠慢性肾小球肾炎细胞的增殖，并可能作为慢性肾小球肾炎的诊断指标和治疗靶点。
