lncRNA-HOTAIR通过靶向负调控miR-1-3p抑制脂多糖诱导的H9c2大鼠心肌细胞增殖并促进炎症反应

lncRNA-HOTAIR inhibits the proliferation of H9c2 rat cardiomyocytes induced by lipopolysaccharide and promotes inflammation through targeting negative regulation of miR-1-3p

目的探讨长链非编码RNA-HOX转录本反义基因间RNA(lncRNA-HOTAIR)是否通过靶向微小RNA miR-1-3p调控脓毒症的炎症反应。方法利用0.5μg/mL脂多糖(LPS)诱导H9c2大鼠心肌细胞,建立脓毒症心肌细胞损伤模型,将H9c2细胞分为对照组、LPS组、LPS联合HOTAIR小干扰RNA阴性对照(si-NC)组、LPS联合HOTAIR小干扰RNA(si-HOTAIR)组、LPS联合miR-1-3p阴性对照(miR-NC)组、LPS联合miR-1-3p组、LPS联合si-HOTAIR和miR-1-3p拮抗剂阴性(anti-miR-NC)组、LPS联合siHOTAIR和anti-miR-1-3p组。实时定量PCR检测HOTAIR和miR-1-3p表达,CCK-8法检测细胞增殖,Western blot法分析KI-67抗原(ki67)、P21、B细胞淋巴瘤因子2(Bcl2)、Bcl2相关X蛋白(BAX)的蛋白表达,流式细胞术评估细胞凋亡,ELISA测定炎性因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平。双荧光素酶报告实验分析HOTAIR和miR-1-3p的靶向关系。结果LPS组H9c2细胞中HOTAIR表达量高于对照组,miR-1-3p表达量低于对照组。与LPS联合si-NC组比较,LPS联合si-HOTAIR组LPS处理H9c2细胞增殖活性、ki67、Bcl2表达量增多,P21表达量、凋亡率、BAX表达量、IL-6和TNF-α水平降低。HOTAIR靶向调控miR-1-3p的表达。LPS联合miR-1-3p组较LPS联合miR-NC组的H9c2细胞增殖活性、ki67、Bcl2表达量升高,P21表达量、凋亡率、BAX表达量、IL-6、TNF-α水平降低。与LPS联合si-HOTAIR和anti-miR-NC组比较,LPS联合si-HOTAIR和anti-miR-1-3p组H9c2细胞增殖活性降低、ki67、Bcl2表达减少,P21、BAX表达,细胞凋亡、IL-6、TNF-α水平增加。结论HOTAIR通过靶向负调控miR-1-3p的表达,抑制LPS诱导的H9c2心肌细胞增殖,并诱导细胞凋亡和炎症反应。

Objective To investigate whether long non-coding RNA-HOX transcript antisense intergenic RNA(lncRNA-HOTAIR)regulates the inflammatory response of sepsis by targeting microRNA miR-1-3p.Methods 0.5μg/mL lipopolysaccharide(LPS)was used to induce cardiomyocytes of H9c2 rats to establish a septic cardiomyocyte injury model.H9c2 cells were divided into control group,LPS group,and LPS combined with HOTAIR small interfering RNA negative control(si-NC)group,LPS combined with HOTAIR small interfering RNA(si-HOTAIR)group,LPS combined with miR-1-3p negative control(miR-NC)group,LPS combined with miR-1-3p group,LPS combined with si-HOTAIR and miR-1-3p inhibitor negative(anti-miR-NC)group,and LPS combined with siHOTAIR and anti-miR-1-3p group.Real-time quantitative PCR was used to determine the expression of HOTAIR and miR-1-3p,CCK-8 assay was used to detect cell proliferation,and Western blotting was used to analyze antigen KI-67(ki67),P21,B-cell lymphoma 2(Bcl2),Bcl2-related X protein(BAX)protein expression.Flow cytometry was performed to evaluate cell apoptosis.ELISA was used to determine the inflammatory factor interleukin 6(IL-6)and tumor necrosis factor-α(TNF-α)levels.The dual luciferase report experiment analyzes the targeting relationship between HOTAIR and miR-1-3p.Results The expression of HOTAIR in H9c2 cells of the LPS group was higher than that of the control group,and the expression of miR-1-3p was lower than that of the control group.Compared with the LPS combined with si-NC group,the LPS combined with si-HOTAIR treated H9c2 cell proliferation activity,presenting increased ki67 and Bcl2 expression,as well as decreased level of P21 expression,apoptosis rate,BAX expression,IL-6 and TNF-α.HOTAIR targets and regulates the expression of miR-1-3p.Compared with the LPS combined miR-1-3p group,the proliferation activity,ki67,Bcl2 expression of H9c2 cells increased in the LPS combined miR-NC group,and the P21 expression,apoptosis rate,BAX expression,IL-6,and TNF-α levels decreased.Compared with the LPS combined with si-HOTAIR and anti-miR-NC group,the H9c2 cell proliferation activity in the LPS combined with siHOTAIR and anti-miR-1-3p group descended,and the expression of ki67 and Bcl2 decreased,while the expression of P21,BAX,apoptosis,IL-6 and TNF-α levels increased.Conclusion HOTAIR negatively regulates the expression of miR-1-3p,inhibits LPS-induced cardiomyocyte H9c2 proliferation,and induces cell apoptosis and inflammation.