miR-1322在低氧诱导动脉平滑肌细胞增殖中的作用及其机制

Role and mechanism of miR-1322 in proliferation of artery smooth muscle cells induced by hypoxia in vitro

目的研究miR-1322对低氧诱导人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,HPASMCs)增殖的调控作用并探讨其机制。方法 HPASMCs在1%O2低氧中分别暴露0、6、12、24、48 h,利用RT-qPCR检测各个时间点miR-1322的表达,Western blot检测骨形成蛋白受体2(bone morphogenetic protein receptor 2,BMPR2)、p-smad1/5/9、p21的蛋白表达,CCK-8检测细胞增殖。将miR-1322抑制物(inhibitor)和阴性对照(negative control,NC)转染HPASMCs细胞24 h后再行低氧处理48 h,采用CCK-8检测12、24、48 h时细胞的增殖水平。利用双荧光素酶报告基因检测分析miR-1322是否绑定BMPR2 3’非编码区(untranslated regions,UTR)。采用Western blot检测miR-1322对BMPR2蛋白的调控作用。转染BMPR2过表达质粒24 h后,再进行低氧处理48 h,采用CCK-8检测12、24、48 h时细胞的增殖水平,Western blot检测p-smad1/5/9和p21的蛋白表达。结果 HPASMCs低氧暴露12、24 h和48 h组miR-1322的表达均显著高于0 h组(P <0. 05)。miR-1322 inhibitor组miR-1322表达显著低于NC组(P <0. 05)。miR-1322 inhibitor/低氧组在24、48 h时细胞增殖水平显著低于相同时间点的低氧组(P <0. 05)。荧光素酶报告基因结果显示BMPR2是miR-1322的靶点。上调miR-1322的表达后,BMPR2蛋白表达较NC组有显著的下降(P <0. 05);相反地,抑制miR-1322后,BMPR2蛋白的表达显著高于NC组(P <0. 05)。低氧6、12 h和24 h时,BMPR2和p21的蛋白表达较0 h组显著降低(P <0. 05),而p-smad1/5/9的蛋白表达显著升高。低氧下BMPR2过表达组p-smad1/5/9和p21的蛋白表达较空载体显著升高(P <0. 05); CCK-8检测结果显示过表达BMPR2组的细胞增殖水平显著低于空载体组(P <0. 05)。结论低氧促进miR-1322在HPASMCs中的表达;上调miR-1322通过抑制BMPR2介导p-smad1/5/9-p21通路,促进细胞的增殖。

Objective To investigate the regulating effect of miR-1322 on the proliferation of human pulmonary artery smooth muscle cells（ HPASMCs） induced by hypoxia and study the possible mechanism.Methods After HPASMCs were exposed to hypoxia for 0,6,12,24 and 48 h respectively,the expression level of miR-1322 was evaluated using RT-qPCR,the protein levels of BMPR2,p-smad1/5/9 and p21 were detected by Western blotting,and cell proliferation was measured using CCK-8 assay. After HPASMCs were transfected respectively with miR-1322 inhibitor and negative control for 24 h,the cells were exposed to hypoxia for further 48 h. The proliferation of HPASMCs was detected by CCK-8 assay at 12,24 and 48 h.Luciferase reporter gene assay was used to evaluate whether miR-1322 binding BMPR2 3＇untranslated regions（ UTR）. The protein levels of p-smad1/5/9 and p21 were evaluated using Western blotting. HPASMCs were transfected with pcDNA3. 1-BMPR2 plasmid for 24 h,and then further exposed to 48 h. The cell proliferation and protein expression of p-smad1/5/9 and p21 were also evaluated. Results The level of miR-1322 was significantly higher in the HPASMCs after hypoxia exposure for 12,24 and 4 8 h than untreated cells（ P〈0. 05）. miR-1322 inhibitor suppressed the level of miR-1322 than the NC group of cells（ P〈0. 05）.The cell proliferation was significantly decreased in the cells of miR-1322 inhibitor/hypoxia group after 24 and48 h than the cells only exposed to hypoxia alone（ P〈0. 05）. Luciferase assay showed that BMPR2 was target of miR-1322. Furthermore,transfection of miR-1322 mimic decreased the expression of BMPR2 protein than the NC group（ P〈0. 05）,while miR-1322 inhibitor treatment improved the protein level than the NC group（ P〈0. 05）. The expression levels of BMPR2 and p21 were significant lower at 6,12 and 24 h after hypoxia than the NC group at same time points（ P〈0. 05）,whereas,the expression of p-smad1/5/9 was obvious higher（ P〈0. 05）. The protein levels of p-smad1/5/9 and p21 were higher in the BMPR2 overexpression/hypoxia group than the vector/hypoxia group（ P〈0. 05）. CCK-8 assay showed that the proliferation HAPSMCs was lower in BMPR2 overexpression group than in vector group（ P〈0. 05）.Conclusion Hypoxia improves the expression of miR-1322 in HPASMCs. Up-regulation of miR-1322 promotes HPASMCs proliferation through inhibiting BMPR2 to mediate p-smad1/5/9-p21 signal pathway.