摘要
目的:探讨热休克70 kDa蛋白1A(HSPA1A)受长链非编码RNA(lncRNA)高温转录物反义RNA(HOTAIR)/微小RNA(miR)-590-3p轴的调控,对心肌缺血/再灌注(I/R)诱导的急性心肌梗死大鼠模型的调控作用。方法:建立大鼠心肌I/R损伤模型,将SD大鼠随机分为6组,分别为假手术组、I/R组、I/R联合siRNA沉默的HSPA1A组(I/R+si-HSPA1A组)、I/R联合siRNA沉默的HOTAIR组(I/R+si-HOTAIR组)、I/R联合si-HSPA1A的阴性对照组(I/R+si-NC组)、I/R联合siRNA沉默的HOTAIR的阴性对照组(I/R+si-NC2组)。建立H9C2细胞缺氧/复氧(H/R)模型,将H9C2细胞随机分为Ctrl组、H/R组、H/R联合si-HOTAIR组(H/R+si-HOTAIR组)、H/R联合si-HOTAIR和过表达HSPA1A组(I/R+si-HOTAIR+pc-HSPA1A组)4组。将常规培养的H9C2细胞分为miR-590-3p的拟似物组(mimic组)、mimic阴性对照组(mimic-NC组)、si-NC2组和si-HOTAIR组4组。用苏木素-伊红(HE)染色进行心肌组织病理学检查。用酶联免疫吸附实验(ELISA)检测心肌组织中肌酸激酶MB(CK-MB)、心肌肌钙蛋白I(cTnI)的表达以及血清中白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)的浓度。四甲基偶氮唑蓝(MTT)法检测H9C2细胞的活力。用Western blot分析HSPA1A、核因子-κB(NF-κB)P65、磷酸化的NF-κB P65(p-NF-κB P65)、IL-6、IL-1β、TNF-α的表达。用实时定量PCR(RT-qPCR)法分析HOTAIR和miR-590-3p的表达。利用双荧光素酶报告实验检测miR-590-3p与HSPA1A的3′非翻译区(UTR)以及HOTAIR与miR-590-3p的结合作用。结果:与假手术组比,I/R诱导后明显增加了心肌组织损伤和炎症(均P<0.05)。与I/R+si-NC组比,I/R+si-HSPA1A组的HSPA1A、NF-κB P65、p-NF-κB P65、IL-6、IL-1β、TNF-α表达水平都下调(均P<0.05),缓解了心肌损伤和炎症反应。与假手术组比,I/R诱导后明显增加了HOTAIR表达并下调了miR-590-3p的表达(均P<0.05)。与I/R+si-NC2组比,I/R+si-HOTAIR组的HSPA1A、NF-κB P65、p-NF-κB P65、IL-6、IL-1β、TNF-α表达水平都下调(均P<0.05),miR-590-3p的表达水平上调(P<0.05),缓解了心肌损伤和炎症反应。与H/R组比,H/R+si-HOTAIR组上调了H/R诱导下的H9C2细胞的增殖率(P<0.05),而H/R+si-HOTAIR+pc-HSPA1A组的细胞增殖率明显低于H/R+si-HOTAIR组(P<0.05)。与H/R组比,H/R+si-HOTAIR组H/R诱导的H9C2细胞中HOTAIR、HSPA1A、IL-1β、IL-6和TNF-α的表达显著降低(均P<0.05),而H/R+si-HOTAIR+pc-HSPA1A组IL-1β、IL-6和TNF-α的表达则显著增加(均P<0.05)。双荧光素酶报告实验检测发现HSPA1A的3′-UTR可直接结合miR-590-3p(P<0.05),且在H9C2细胞中,与mimic-NC组比,mimic组中的HSPA1A蛋白表达明显下调(P<0.05)。另外,HOTAIR也可直接结合miR-590-3p(P<0.05)。在H9C2细胞中,与si-NC2组相比,si-HOTAIR组的miR-590-3p表达水平上调(P<0.05)。结论:HSPA1A受HOTAIR/miR-590-3p轴调控促进I/R诱导的心肌梗死大鼠体内NF-κB介导的炎症反应。
Objective:To investigate the regulatory role of heat shock 70 kDa protein 1A(HSPA1A)under the control of long non-coding RNA(lncRNA)HOTAIR/microRNA(miR)-590-3p axis in a rat model of acute myocardial infarction induced by myocardial ischemia/reperfusion(I/R).Methods:A rat model of myocardial I/R injury was established,and SD rats were randomly divided into six groups:sham surgery group,I/R group,I/R combined with siRNA-silenced HSPA1A group(I/R+si-HSPA1A group),I/R combined with si-NC negative control of HSPA1A group(I/R+si-NC group),I/R combined with si-HOTAIR group(I/R+si-HOTAIR group),and I/R combined with si-NC negative control of HOTAIR group(I/R+si-NC2 group).An H9C2 cell line was used to establish an H/R injury model,and the cells were randomly divided into four groups:Ctrl group,H/R group,H/R combined with si-HOTAIR group(H/R+si-HOTAIR group),and H/R combined with si-HOTAIR and overexpressed HSPA1A group(I/R+si-HOTAIR+pc-HSPA1A group).In addition,regular cultured H9C2 cells were divided into four groups:miR-590-3p mimic group(mimic group),mimic negative control group(mimic-NC group),si-NC2 group,and si-HOTAIR group.Histopathological examination of myocardial tissues was performed using Hematoxylin-Eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was conducted to detect the expression of creatine kinase-MB(CK-MB),cardiac troponin I(cTnI),interleukin(IL)-6,IL-1β,and tumor necrosis factor-alpha(TNF-α)in myocardial tissues and the concentrations of these markers in serum.The viability of H9C2 cells was assessed using the Methyl Thiazolyl Tetrazolium(MTT)assay.The expression of HSPA1A,NF-κB P65,phosphorylated NF-κB P65(p-NF-κB P65),IL-6,IL-1β,and TNF-αwas analyzed by Western blot.The expression of miR-590-3p was analyzed by RT-qPCR.The binding interaction between miR-590-3p and the 3′UTR of HSPA1A,as well as the interaction between HOTAIR and miR-590-3p,was detected using a dual-luciferase reporter assay.Results:Compared with the sham surgery group,I/R induction significantly increased myocardial tissue damage and inflammation(all P<0.05).Compared with the I/R+si-NC group,the expression levels of HSPA1A,NF-κB P65,p-NF-κB P65,IL-6,IL-1β,and TNF-αwere downregulated in the I/R+si-HSPA1A group(all P<0.05),which alleviated myocardial injury and inflammation.Compared with the sham surgery group,HOTAIR expression was significantly increased and miR-590-3p expression was downregulated after I/R induction(all P<0.05).In the I/R+si-NC2 group,the expression levels of HSPA1A,NF-κB P65,p-NF-κB P65,IL-6,IL-1β,and TNF-αwere downregulated(all P<0.05),while miR-590-3p expression was upregulated(P<0.05),relieving myocardial injury and inflammation.Compared with the H/R group,the proliferation rate of H9C2 cells induced by H/R was upregulated in the H/R+si-HOTAIR group(P<0.05),while the proliferation rate in the H/R+si-HOTAIR+pc-HSPA1A group was significantly lower than that in the H/R+si-HOTAIR group(P<0.05).Compared with the H/R group,the H/R+si-HOTAIR group significantly reduced the expression of HOTAIR,HSPA1A,IL-1β,IL-6,and TNF-αin H/R-induced H9C2 cells(all P<0.05),whereas the H/R+si-HOTAIR+pc-HSPA1A group significantly increased the expression of IL-1β,IL-6,and TNF-α(all P<0.05).The dual-luciferase reporter assay showed that the 3′-UTR of HSPA1A could directly bind to miR-590-3p(P<0.05),and in H9C2 cells,the protein expression of HSPA1A in the mimic group was significantly downregulated compared with the mimic-NC group(P<0.05).Furthermore,HOTAIR could directly bind to miR-590-3p(P<0.05).In H9C2 cells,compared with the si-NC2 group,the expression level of miR-590-3p was upregulated in the si-HOTAIR group(P<0.05).Conclusion:HSPA1A,regulated by the HOTAIR/miR-590-3p axis,promotes NF-κB-mediated inflammation in rats with myocardial infarction induced by I/R.
作者
刘超群
梁丽艳
刘顺民
钟小兰
LIU Chaoqun;LIANG Liyan;LIU Shunmin;ZHONG Xiaolan(Department of Cardiology,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,China)
出处
《东南大学学报（医学版）》
CAS
2023年第5期696-705,共10页
Journal of Southeast University(Medical Science Edition)
