糖尿病性心肌纤维化中上调表达长链非编码RNA(lncRNA)的鉴定

目的鉴定在糖尿病性小鼠心肌和糖尿病性心肌纤维化细胞模型中一致上调表达的长链非编码RNA(lncRNA)。方法通过Masson染色检测糖尿病db/db小鼠和db/m对照小鼠心肌中的胶原含量。用小鼠lncRNA表达谱芯片检测小鼠心肌中lncRNA表达,用荧光定量PCR鉴定6个代表性的在糖尿病性心肌中高表达的lncRNAs。用33mmol/L葡萄糖分别和0.1、0.2、0.4、0.6 mU葡萄糖氧化酶处理小鼠心肌成纤维细胞,通过检测纤维化相关基因α-SMA、Col1a1和Col3a1表达来确定合适的葡萄糖/葡萄糖氧化酶(HG/Go)处理条件。用确定条件的HG/Go处理小鼠心肌成纤维细胞,用荧光定量PCR鉴定在糖尿病性心肌和糖尿病性心肌纤维化细胞模型中一致上调表达的lncRNA。结果Masson染色显示,糖尿病db/db小鼠心肌的胶原含量显著升高,与对照小鼠比较差异有统计学意义(P0.01)。同db/m对照小鼠相比,糖尿病db/db小鼠心肌中lncRNA出现差异性表达,其中354个lncRNAs呈1.5倍以上高表达,292个lncRNAs呈1.5倍以上下调表达。检测的6个代表性上调表达的lncRNAs中,AK014842、AK076377和BF607975表达在糖尿病性心肌中显著上调。33 mmol/L葡萄糖结合0.2、0.4 mU Go可以有效上调小鼠心肌成纤维细胞中α-SMA和Col3a1表达。荧光定量PCR结果显示,AK014842和BF607975在33mmol/L葡萄糖结合0.2 mU Go处理的小鼠心肌成纤维细胞中表达显著升高。结论LncRNA AK014842和BF607975在糖尿病性心肌和糖尿病性心肌纤维化细胞模型中一致上调表达。     

Upregulation of miR-29b contributes to the anti-fibrotic effect of macrophage migration inhibitory factor in diabetic myocardial fibrosis

Background Macrophage migration inhibitory factor(MIF) possesses proinflammatory function when secreted from the cells, and it also exhibits antioxidant properties based on its intrinsic oxidoreductase activity.However, the role of MIF in cardiac fibrosis is not well known. In the present study, the effect of MIF on fibrosis-associated gene expression and the underlying mechanism were examined. Methods The collagen content in mouse myocardium was detected by Masson staining. Expressions of MIF and fibrosis-associated Col1a1, Col3a1 and α-SMA in mouse myocardium or mouse cardiac fibroblasts were detected by quantitative real-time PCR and Western blot assay, respectively. Mature miR-29b expressions in mouse myocardium and cardiac fibroblasts were determined by real-time PCR. Smad3 activation in MIF-treated cardiac fibroblasts was also detected by Western blot assay. Results Compared with the db / m control mice, the collagen content was significantly increased in the myocardium of diabetic db / db mice. MIF was up-regulated, but miR-29b was down-regulated in the diabetic myocardium. Quantitative real-time PCR and Western blot assay showed that MIF could inhibit fibrosis-associated Col1a1, Col3a1 and α-SMA expressions in mouse cardiac fibroblasts.Smad3 activation was inhibited, but miR-29b was up-regulated in MIF-treated cardiac fibroblasts. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA mRNA and 1protein expressions in cardiac fibroblasts. Conclusions MIF possesses the anti-fibrosis activity through inhibiting Smad3activation and through up-regulating miR-29b expression, and miR-29b can inhibit fibrosis-associated Col1a1,Col3a1 and α-SMA expressions in cardiac fibroblasts.     

糖尿病性心肌纤维化中上调表达长链非编码RNA（1ncRNA）的鉴定

目的鉴定在糖尿病性小鼠心肌和糖尿病性心肌纤维化细胞模型中一致上调表达的长链非编码RNA（1ncRNA）。方法通过Masson染色检测糖尿病db／db小鼠和db／m对照小鼠心肌中的胶原含量。用小鼠lncRNA表达谱芯片检测小鼠心肌中lncRNA表达,用荧光定量PCR鉴定6个代表性的在糖尿病性心肌中高表达的lncRNAs。用33mmol／L葡萄糖分别和0．1、0．2、0．4、0．6mU葡萄糖氧化酶处理小鼠心肌成纤维细胞,通过检测纤维化相关基因仪．SMA、Collal和Col3al表达来确定合适的葡萄糖／葡萄糖氧化酶（HG／Go）处理条件。用确定条件的HG／Go处理小鼠心肌成纤维细胞,用荧光定量PCR鉴定在糖尿病性心肌和糖尿病性心肌纤维化细胞模型中一致上调表达的lncRNA。结果Masson染色显示,糖尿病db／db小鼠心肌的胶原含量显著升高,与对照小鼠比较差异有统计学意义（P〈0．01）。同db／m对照小鼠相比,糖尿病db／db小鼠心肌中lncRNA出现差异性表达,其中354个lncRNAs呈1．5倍以上高表达,292个lncRNAs呈1．5倍以上下调表达。检测的6个代表性上调表达的lncRNAs中,AK014842、AK076377和BF607975表达在糖尿病性心肌中显著上调。33mmol／L葡萄糖结合0．2、0．4mUGo可以有效上调小鼠心肌成纤维细胞中α—SMA和Col3al表达。荧光定量PCR结果显示,AK014842和BF607975在33mmol／L葡萄糖结合0．2mUGo处理的小鼠心肌成纤维细胞中表达显著升高。结论LncRNAAK014842和BF607975在糖尿病性心肌和糖尿病性心肌纤维化细胞模型中一致上调表达。     

miRNA-21 regulates proatherosclerotic gene expression in macrophages

Background MicroRNAs(miRNANAs) are endogenous, small non-coding RNAs that negatively regulate gene expression in diverse cardiovascular diseases. However, the roles of miRNANAs in atherosclerogenesis needs to be elucidated. In the present study, the effect of miRNA-21 on pro-atherosclerotic genes expression was examined. Methods The pro-atherosclerotic genes including COX2, VCAM1, ICAM1, MCP1 and miRNA-21 were detected in ox-LDL-treated mouse macrophage RAW264.7 cells. ApoE knock-out(ApoEKO) mice were fed with high-fat diet for 16 weeks, and the abdominal aorta were fixed and used for miRNA-21 hybridization. Lentivirus-based vectors for enforced expression of miRNA-21 and antisense miRNA-21were prepared. The expression of proatherosclerotic genes was determined in the RAW264.7 cells with lentivirusmediated up-regulation of miRNA-21. Results COX2, VCAM1, ICAM1 and MCP1 could be up-regulated by ox-LDL treatment, and 50 μg / mL ox-LDL could significantly increase the expression of above four genes in ox-LDL EAW264.7 cells. miRNA-21 could also be markedly up-regulated in ox-LDL-induced RAW264.7cells. The result of miRNANA hybridization showed that miRNA-21 was strongly expressed in atherosclerotic plaques but not in normal aorta. Lentivirus-mediated over-expression of miRNA-21 could significantly enhance expressions of COX2, VCAM1, ICAM1 and MCP1 in RAW264.7 cells, which could be reversed by antisense miRNA-21 mediated by lentivirus vector. Conclusions miRNA-21 could be modulated by ox-LDL in macrophage RAW264.7 cells, and miRNA-21 could enhance COX2, VCAM1, ICAM1 and MCP1expressions in macrophages.