Role of mast cell-miR-490-5p in irritable bowel syndrome
Role of mast cell-miR-490-5p in irritable bowel syndrome
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DOI：
10.3748/wjg.v23.i1.93
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发表时间：
2017-01-07
影响因子：
4.3
通讯作者：
Liang, Lie-Xin
中科院分区：
文献类型：
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作者：
Ren, Hai-Xia;Zhang, Fa-Can;Liang, Lie-Xin
目的探讨miR-490- 5 p在肥大细胞增殖、凋亡及肥大细胞类胰蛋白酶/PAR-2信号通路中的作用。Shanghai，China）作为报告基因构建mmu-miR-490- 5 p慢病毒表达载体pEGFP-miR-4905 p，慢病毒载体pEGFP阴性作为阴性对照。然后构建稳定转染的肥大细胞系p815。GFP阳性细胞为成功转染的细胞。我们使用定量实时PCR（qRT-PCR）测定转染前后p815肥大细胞中miR-490- 5 p的表达。此外，在用慢病毒载体转导后，分别使用CCK-8测定和流式细胞术研究miR-490- 5 p在肥大细胞增殖和凋亡中的作用。qRT-PCR检测类胰蛋白酶和PAR-2的mRNA水平，Western blot检测蛋白水平。结论抑制miR-490- 5 p表达可促进p815肥大细胞凋亡，抑制其增殖。与对照组相比，转染后类胰蛋白酶和PAR-2的mRNA水平显著升高，类胰蛋白酶（P = 0.721，normal vs null; P = 0.001，siRNA vs normal; P = 0.002，siRNA vs null）和PAR-2（P = 0.027，siRNA vs null; P = 0.353，normal vs null; P = 0.105，siRNA vs normal）的mRNA水平显著升高。siRNA组类胰蛋白酶和PAR 2蛋白水平略高于对照组，但差异无统计学意义结论miR-490- 5 p通过影响肥大细胞增殖和凋亡在肠易激综合征的发病中起重要作用;随着miR-490- 5 p的下调，肥大细胞类胰蛋白酶和PAR-2的mRNA水平升高，蛋白水平升高，但差异无统计学意义。
AIMTo determine the functional role of miR-490-5p in mast cell proliferation and apoptosis, and in the mast cell tryptase/PAR-2 signal pathway.METHODSThe 3rd generation of lentivirus vector systems containing enhanced green fluorescent protein (EGFP) (Ruisai Inc., Shanghai, China), which acts as a reporter gene was used to construct the mmu-miR-490-5p lentivirus expression vector pEGFP-antagomiR-4905p, and the lentivirus vector pEGFP-negative was used as a negative control. The stably transfected mast cell line p815 was then constructed. GFP positive cells were successfully transfected cells. We determined the expression of miR-490-5p in p815 mast cells before and after transfection using quantitative real-time PCR (qRT-PCR). In addition, after transduction with the lentivirus vectors, the role of miR-490-5p in mast cell proliferation and apoptosis was investigated using the CCK-8 assay and flow cytometry, respectively. The mRNA levels of tryptase and PAR-2 were detected by qRT-PCR and the protein levels were detected by Western blot.RESULTSThe inhibition of miR-490-5p expression promoted apoptosis and inhibited proliferation of p815 mast cells. The mRNA levels of tryptase and PAR-2 were significantly increased after transfection compared with the control group, tryptase (P = 0.721, normal vs null; P = 0.001, siRNA vs normal; P = 0.002, siRNA vs null) and PAR-2 (P = 0.027, siRNA vs null; P = 0.353, normal vs null; P = 0.105, siRNA vs normal). The protein levels of tryptase and PAR2 were slightly higher in the siRNA group than those in the control group, but the difference was not statistically significant (P > 0.05).CONCLUSIONmiR-490-5p plays a vital role in the pathogenesis of irritable bowel syndrome by affecting mast cell proliferation and apoptosis; with down-regulation of miR-490-5p, the mRNA level of mast cell tryptase and PAR-2 increased, and the protein level increased, but the difference was not statistically significant.