[關(guān)鍵詞]
[摘要]
目的 研究 microRNA-378b(miR-378b)對(duì)心臟成纖維細(xì)胞的纖維化水平的影響及分子機(jī)制。方法 小鼠行慢性心肌梗死手術(shù)構(gòu)建體內(nèi)心肌纖維化模型,利用轉(zhuǎn)化生長因子-β(TGF-β)誘導(dǎo)原代心臟成纖維細(xì)胞發(fā)生纖維化以構(gòu)建體外心肌纖維化模型;實(shí)時(shí)熒光定量 PCR(qRT-PCR)法檢測(cè) 2 種纖維化模型中 miR-378b 的表達(dá)水平;Western blotting 法檢測(cè) miR-378b 模擬物和抑制物對(duì)心臟成纖維細(xì)胞 α-平滑肌肌動(dòng)蛋白(α-SMA,心肌纖維化特異性指標(biāo))表達(dá)量的影響;TargetScan、miRDB 和 miRWalk 軟件預(yù)測(cè) miR-378b 的下游靶基因,雙熒光素酶實(shí)驗(yàn)驗(yàn)證 miR-378b 與生長相關(guān)蛋白 43(GAP43)的靶向關(guān)系;Western blotting 法檢測(cè) miR-378b 模擬物和抑制物對(duì)心臟成纖維細(xì)胞 GAP43 表達(dá)的影響;Western blotting 法檢測(cè)體內(nèi)和體外 2 種纖維化模型中 GAP43 的蛋白水平。結(jié)果 小鼠心肌纖維化模型組 miR-378b 表達(dá)量較假手術(shù)組顯著降低(P<0.01),心臟成纖維細(xì)胞 TGF-β處 理 組 miR-378b表達(dá)量較對(duì)照組顯著降低(P<0.001)。在基礎(chǔ)水平和 TGF-β 處理后,與對(duì)照模擬物組比較,miR-378b 模擬物顯著降低細(xì)胞的α-SMA蛋白水平(P<0.05);在基礎(chǔ)水平,與對(duì)照抑制物組比較,miR-378b抑制物可顯著升高細(xì)胞的α-SMA蛋白水平(P<0.05);但在 TGF-β 處理的細(xì)胞中,miR-378b 抑制物不能進(jìn)一步增加α-SMA蛋白表達(dá)量。GAP43 是 miR-378b 直接作用的下游靶基因,與對(duì)照組比較,miR-378b可負(fù)調(diào)控心臟成纖維細(xì)胞GAP43的蛋白表達(dá)水平(P<0.01)。心肌纖維化模型組小鼠心肌 GAP43 蛋白表達(dá)量較假手術(shù)組顯著升高(P<0.01),心臟成纖維細(xì)胞 TGF-β處理組GAP43蛋白表達(dá)量較對(duì)照組顯著升高(P<0.001)。結(jié)論 miR-378b 可抑制心臟成纖維細(xì)胞纖維化,該功能可能通過抑制其下游靶基因 GAP43 發(fā)揮作用。
[Key word]
[Abstract]
Objective To investigate the effect of microRNA-378b (miR-378b) on the fibrosis level of cardiac fibroblasts and its molecular mechanism. Methods The model of myocardial fibrosis in vivo was established by operation of chronic myocardial infarction in mice, transforming growth factor-β (TGF-β) was used to treat cardiac fibroblasts to construct myocardial fibrosis model in vitro. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels of miR-378b in two fibrosis models. The effects of miR-378b mimics and inhibitors on α -smooth muscle actin (α -SMA, a specific indicator of myocardial fibrosis) expression of cardiac fibroblasts were detected by Western blotting. The downstream target genes of miR-378b were predicted by three softwares (TargetScan, miRDB, and miRWalk), double luciferase experiment verified the targeting relationship between miR-378b and GAP43. Western blotting was used to detect the effects of miR-378b mimics and inhibitors on GAP43 in cardiac fibroblasts. The protein levels of GAP43 in two fibrosis models were detected by Western blotting. Results The expression of miR-378b in the mouse myocardial fibrosis model group was significantly decreased compared with the sham operation group (P<0.01), and the expression of miR-378b in the cardiac fibroblast TGF-β treatment group was significantly decreased compared with the control group (P<0.001). After treatment with TGF-β and in basal level, miR-378b mimics significantly decreased α-SMA protein level compared with control mimics group (P<0.05). At the basal level, compared with the control group, miR-378b inhibitors significantly increased the α-SMA protein level of cells (P<0.05). However, miR-378b inhibitors did not further increase α-SMA protein expression in TGF-β-treated cells. GAP43 was a downstream target gene directly affected by miR-378b. Compared with the control group, miR-378b can negatively regulate the protein expression level of GAP43 in cardiac fibroblasts (P<0.01). Compared with sham operation group, the expression level of GAP43 protein in myocardial fibrosis model group was significantly increased (P<0.01), and that in cardiac fibroblast TGF-β treatment group was significantly increased compared with control group (P<0.001). Conclusion miR-378b can inhibit fibrosis level of cardiac fibroblasts, which may play a role through its downstream target gene GAP43.
[中圖分類號(hào)]
R542.2
[基金項(xiàng)目]
南京市衛(wèi)生科技發(fā)展專項(xiàng)資金項(xiàng)目計(jì)劃(YKK19052)