Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Nov 30;40(11):1571-1578.
doi: 10.12122/j.issn.1673-4254.2020.11.06.

[miR-324-5p inhibits lipopolysaccharide-induced proliferation of rat glomerular mesangial cells by regulating the Syk/Ras/c-fos pathway]

[Article in Chinese]
Jing Wang  1   2 , Xiaoli Zhu  1   2 , Xiujuan Qin  1 , Hui Jiang  1   3 , Yachen Gao  1 , Jiarong Gao  1   3
Affiliations

[miR-324-5p inhibits lipopolysaccharide-induced proliferation of rat glomerular mesangial cells by regulating the Syk/Ras/c-fos pathway]

[Article in Chinese]
Jing Wang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

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.

PubMed Disclaimer

Figures

1
1
各组HBZY-1细胞增殖活性 Proliferative activity of HBZY-1 cells in different groups. **P < 0.01 vs normal group after 48 h of treatment. ##P < 0.01 vs LPS group after 48 h of treatment.
2
2
miR-324-5p在各组HBZY-1细胞中的表达水平 Expression level of miR-324-5p in HBZY-1 cells in different groups. **P < 0.01 vs normal group. ##P < 0.01 vs LPS group.
3
3
过表达miR-324-5p对LPS诱导的HBZY-1细胞中Syk表达的影响 Effect of overexpression of miR- 324-5p on Syk expression in LPS- induced HBZY-1 cells. A: Immunofluorescence assay of p-Syk protein in HBZY-1 cells (Original magnification: ×200). a: Normal group; b: LPS group; c: LPS+mimics group; d: LPS+mimics-NC group. B: RT-qPCR for detecting mRNA changes of Syk in HBZY-1 cells. C: Western blotting for detecting protein expression of p-Syk. D: Semi-quantitative analysis of p-Syk protein expression. **P < 0.01 vs normal group. ##P < 0.01 vs LPS group; #P < 0.05 vs LPS group.
4
4
过表达miR-324-5p对LPS诱导的HBZY-1细胞中Ras表达的影响 Effect of overexpression of miR-324-5p on Ras expression in LPS-induced HBZY-1 cells. A: Immunofluorescence method was used to observe the changes of Ras in HBZY-1 (×200). a: Normal group, b: LPS group, c: LPS+mimics group, d: LPS+mimics-NC group; B: RTqPCR was used to detect the mRNA changes of Ras in HBZY-1; C: Western Blot was used to detect the protein expression of Ras; D: Semi quantitative analysis of Ras **P < 0.01 vs normal group. ##P < 0.01 vs LPS group.
5
5
过表达miR-324-5p对LPS诱导的HBZY-1细胞中MEK1/2表达的影响 Effect of overexpression of miR-324-5p on MEK1/2 expression in HBZY-1 cells induced by LPS. A: Immunofluorescence method was used to observe the changes of MEK1/2 in HBZY-1 (×200). a: Normal group, b: LPS group, c: LPS+mimics group, d: LPS+ mimicsNC group; B: RT-qPCR was used to detect the mRNA changes of MEK1/2 in HBZY-1 cells; C: Western blotting was used to detect the protein expression of MEK1/2; D: Semi quantitative analysis of p-MEK expression. **P < 0.01 vs normal group. ##P < 0.01 vs LPS group.
6
6
过表达miR-324-5p对LPS诱导的HBZY-1细胞中ERK1/2表达的影响 Effect of overexpression of miR-324-5p on ERK1/2 expression in HBZY-1 cells induced by LPS. A: Immunofluorescence method was used to observe the changes of ERK1/2 in HBZY-1 (×200). a: Normal group, b: LPS group, c: LPS+mimics group, d: LPS+mimics-NC group; B: RT-qPCR was used to detect the mRNA changes of ERK1/2 in HBZY-1 cells; C: Western blotting was used to detect the protein expression of ERK1/2; D: Semi-quantitative analysis of p-ERK expression. **P < 0.01 vs normal group. ##P < 0.01 vs LPS group.
7
7
过表达miR-324-5p对LPS诱导的HBZY-1细胞中c-fos表达的影响 Effect of overexpression of miR-324-5p on c-fos expression in LPS-induced HBZY-1 cells. A: Immunofluorescence method was used to observe the changes of c-fos in HBZY-1 (×200). a: Normal group, b: LPS group, c: LPS+mimics group, d: LPS+ mimics-NC group; B: RT-qPCR was used to detect the mRNA changes of c-fos in HBZY-1 cells; C: Western blotting was used to detect the protein expression of c-fos; D: Semi-quantitative analysis of c-fos. **P < 0.01 vs normal group. ##P < 0.01 vs LPS group.
See this image and copyright information in PMC

References

    1. 杨 倩春, 杨 霓芝. 急性肾小球肾炎病因病机探讨. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=lnzyxyxb2.... 辽宁中医学院学报. 2003;1:13. [杨倩春, 杨霓芝.急性肾小球肾炎病因病机探讨[J].辽宁中医学院学报, 2003, 1: 13.]
    1. 卢 作奎, 许 为佳, 史 秀岩. 基于WNT/β-catenin信号通路研究DMBT1调控慢性肾小球肾炎发生发展的作用机制. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=lcszbzz20.... 临床肾脏病杂志. 2020;20(4):297–302. [卢作奎, 许为佳, 史秀岩.基于WNT/β-catenin信号通路研究DMBT1调控慢性肾小球肾炎发生发展的作用机制[J].临床肾脏病杂志, 2020, 20(4): 297-302.]
    1. Decleves A, Sharma K. Novel targets of antifibrotic and antiinflammatory treatment in CKD. Nat Rev Nephrol. 2014;10(5):257–67. doi: 10.1038/nrneph.2014.31. [Decleves A, Sharma K. Novel targets of antifibrotic and antiinflammatory treatment in CKD[J]. Nat Rev Nephrol, 2014, 10(5): 257-67.] - DOI - PMC - PubMed

    1. 杨莉.福辛普利对肾小球系膜细胞增殖的影响[D].中国医科大学, 2007.

    1. Dweep H, Sticht C, Pandey P, et al. miRWalk-Database: Prediction of possible miRNA binding sites by walking the genes of three genomes. http://dl.acm.org/citation.cfm?id=2304897. J Biomed Informatics. 2011;44(5):839–47. [Dweep H, Sticht C, Pandey P, et al. miRWalk-Database: Prediction of possible miRNA binding sites by walking the genes of three genomes [J]. J Biomed Informatics, 2011, 44(5): 839-47.] - PubMed

LinkOut - more resources