miR-203-3p靶向TREM1基因调控TGF-β1/p38MAPK信号通路对狼疮性肾炎小鼠肾小管上皮细胞增殖和凋亡的影响

doi:10.6040/j.issn.1671-7554.0.2020.1135

摘要/Abstract

摘要： 目的探讨miR-203-3p对狼疮性肾炎(LN)小鼠肾小管上皮细胞增殖凋亡的影响及其可能的分子机制。方法选取自发性LN小鼠(LN组)和野生型C57BL/6小鼠(NC组)各8只,应用qRT-PCR法检测肾组织miR-203-3p表达水平,Western blotting法检测肾组织中髓细胞触发受体1基因(TREM1)蛋白表达水平。分离LN小鼠肾小管上皮细胞,并将miR-203-3p模拟物(miR-203-3p mimic)及其阴性对照(miR-NC)转染至肾小管上皮细胞中,采用qRT-PCR法检测转染后肾小管上皮细胞中miR-203-3p表达水平;CCK-8法检测细胞增殖能力;流式细胞术检测细胞凋亡情况;ELISA法检测细胞中炎症因子肿瘤坏死因子-α(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6)的表达水平;Western blotting法检测细胞中TREM1、Bax、Bcl-2、转化生长因子-β1(TGF-β1)、磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)和p38MAPK等蛋白表达水平;双荧光素酶报告基因实验检测miR-203-3p与TREM1之间的靶向关系。结果与NC组小鼠比较,LN组小鼠肾组织中miR-203-3p表达水平降低(P<0.001),而TREM1蛋白表达水平升高(P<0.001)。miR-203-3p过表达可显著抑制LN小鼠肾小管上皮细胞中TREM1蛋白表达(F=366.230,P<0.001),并且双荧光素酶报告基因实验证实TREM1是miR-203-3p的靶基因。此外,miR-203-3p过表达还可显著抑制LN小鼠肾小管上皮细胞中TNF-α、IL-1β和IL-6等炎症因子的表达(P均<0.01),并促进细胞增殖( F_{24 h}=14.841,F_{48 h}=21.701,F_{72 h}=29.893,P<0.001),抑制其凋亡(F=238.700,P<0.001),同时上调细胞中Bcl-2蛋白表达水平(F=371.820,P<0.001),而下调Bax、TGF-β1和p-p38MAPK等蛋白表达水平(F_Bax=225.640,F_TGF-β1=27.090,F_p-p38MAPK=103.250,P<0.001)。结论 miR-203-3p通过靶向下调TREM1蛋白表达促进LN小鼠肾小管上皮细胞的增殖,抑制其凋亡,其机制可能与TGF-β1/p38MAPK信号通路的抑制有关。

关键词: 狼疮性肾炎, miR-203-3p, 髓细胞触发受体1基因, TGF-β1/p38MAPK信号通路

Abstract: Objective To investigate the effect of miR-203-3p on proliferation and apoptosis of renal tubular epithelial cells in lupus nephritis(LN)mice and its possible molecular mechanism. Methods Spontaneous LN mice(LN group)and wild-type C57BL/6 mice(NC group)were selected, 8 in each group, and the expression of miR-203-3p and triggering receptor expressed on myeloid cells-1(TREM1)protein in renal tissue were detected by qRT-PCR and Western blotting, respectively. The renal tubular epithelial cells of LN mice were isolated and transfected with miR-203-3p mimic and its negative control(miR-NC), then the expression of miR-203-3p in transfected renal tubular epithelial cells was detected by qRT-PCR; cell proliferation and apoptosis were detected by CCK-8 and flow cytometry; the levels of inflammatory factors TNF-α, IL-1β and IL-6 were detected by ELISA; the proteins expressions of TREM1, Bax, Bcl-2, TGF-β1, p-p38MAPK and p38MAPK were evaluated by Western blotting; the target relationship between miR-203-3p and TREM1 was assessed by dual luciferase reporter gene assay. Results Compared with NC group mice, the expression level of miR-203-3p in renal tissue of LN mice was significantly decreased(P<0.001), while the expression of TREM1 protein was significantly increased(P<0.001). Overexpression of miR-203-3p markedly inhibited the expression of TREM1 protein in renal tubular epithelial cells of LN mice(F=366.230, P<0.001), and the dual luciferase reporter gene experiment confirmed that TREM1 was the target gene of miR-203-3p. In addition, overexpression of miR-203-3p significantly inhibited the levels of TNF-α, IL-1β and IL-6 in tubule epithelial cells of LN mice(all P<0.01), promoted the cell proliferation(F_{24 h}=14.841, F_{48 h}=21.701, F_{72 h}=29.893, P<0.001), inhibited its apoptosis(F=238.700, P<0.001), and up-regulated the expression of Bcl-2 protein(F=371.820, P<0.001), while down-regulated the proteins expression of Bax, TGF-β1 and p-p38MAPK(F_Bax=225.640, F_TGF-β1=27.090, F_p-p38MAPK=103.250, P<0.001). Conclusion miR-203-3p can promote the proliferation and inhibit apoptosis of LN mice glomerular epithelial cells by targeting the down-regulation of TREM1 protein expression, which may be related to inhibition of TGF-β1/p38MAPK signaling pathway.

Key words: Lupus nephritis, miR-203-3p, Triggering receptor expressed on myeloid cells-1, TGF-β1/p38MAPK signaling pathway

中图分类号:

罗慧臣,胡丹慧,张济. miR-203-3p靶向TREM1基因调控TGF-β1/p38MAPK信号通路对狼疮性肾炎小鼠肾小管上皮细胞增殖和凋亡的影响[J]. 山东大学学报 (医学版), 2021, 59(3): 18-25.

LUO Huichen, HU Danhui, ZHANG Ji. Effect of miR-203-3p targeted TREM1 gene on the regulation of TGF-β1/p38MAPK signaling pathway on the proliferation and apoptosis of renal tubular epithelial cells in lupus nephritis mice[J]. Journal of Shandong University (Health Sciences), 2021, 59(3): 18-25.

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