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摘要：
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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-α was 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-α were 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-α were 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-α in 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. |
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参考文献：
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[1] BOARESCU P M,BOARESCU I,BULBOACA∨ A E,et al.Multi-organ protective effects of curcumin nanoparticles on drug-induced acute myocardial infarction in rats with type 1 diabetes mellitus[J].Appl Sci,2021,11(12):5497-5512.
[2] LIU Y,WANG H,WANG X,et al.MiR-29b inhibits ventricular remodeling by activating notch signaling pathway in the rat myocardial infarction model[J].Heart Surg Forum,2019,22(1):E019-E023.
[3] OZAKI Y,KATAGIRI Y,ONUMA Y,et al.CVIT expert consensus document on primary percutaneous coronary intervention(PCI) for acute myocardial infarction(AMI) in 2018[J].Cardiovasc Interv Ther,2018,33:178-203.
[4] ZENG G,AN H,FANG D,et al.Plantamajoside protects H9c2 cells against hypoxia/reoxygenation-induced injury through regulating the akt/Nrf2/HO-1 and NF-κB signaling pathways[J].J Recept Signal Transduct,2022,42(2):125-132.
[5] 李昊,贾孝,白雅琴,等.小鼠急性心肌梗死后不同时间点的基因表达谱[J].法医学杂志,2022,38(3):343.
[6] HUANG L,GUO B,LIU S,et al.Inhibition of the LncRNA Gpr19 attenuates ischemia-reperfusion injury after acute myocardial infarction by inhibiting apoptosis and oxidative stress via the miR-324-5p/Mtfr1 axis[J].IUBMB Life,2020,72(3):373-383.
[7] LIANG H,LI F,LI H,et al.Overexpression of lncRNA HULC attenuates myocardial ischemia/reperfusion injury in rat models and apoptosis of hypoxia/reoxygenation cardiomyocytes via targeting miR-377-5p through NLRP3/Caspase-1/IL-1β signaling pathway inhibition[J].Immunol Invest,2021,50(8):925-938.
[8] HUANG P,YANG D,YU L,et al.Downregulation of lncRNA ZFAS1 protects H9c2 cardiomyocytes from ischemia/reperfusion-induced apoptosis via the miR-590-3p/NF-κB signaling pathway[J].Mol Med Rep,2020,22(3):2300-2306.
[9] FRANGOGIANNIS N G.Cell biological mechanisms in regulation of the post-infarction inflammatory response[J].Curr Opin Physiol,2018,1:7-13.
[10] BIEMMI V,MILANO G,CIULLO A,et al.Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation[J].Theranostics,2020,10(6):2773.
[11] WANG S,YAO T,DENG F,et al.LncRNA MALAT1 pro- motes oxygen-glucose deprivation and reoxygenation induced cardiomyocytes injury through sponging miR-20b to enhance beclin1-mediated autophagy[J].Cardiovasc Drugs Ther,2019,33:675-686.
[12] BOTTI G,SCOGNAMIGLIO G,AQUINO G,et al.LncRNA HOTAIR in tumor microenvironment:what role?[J].Int J Mol Sci,2019,20(9):2279.
[13] PETKEVICIUS V,THON C,STEPONAITIENE R,et al.Differential expression of long noncoding RNA HOTAIR in intestinal metaplasia and gastric cancer[J].Clin Transl Gastroenterol,2022,13(5):e00483.
[14] GAO L,LIU Y,GUO S,et al.Circulating long noncoding RNA HOTAIR is an essential mediator of acute myocardial infarction[J].Cell Physiol Biochem,2017,44(4):1497-1508.
[15] ZHAO S,YANG G,LIU P N,et al.miR-590-3p is a novel MicroRNA in myocarditis by targeting nuclear factor Kappa-B in vivo[J].Cardiology,2015,132(3):182-188.
[16] BAO M H,LI J M,ZHOU Q L,et al.Effects of miR-590 on oxLDL-induced endothelial cell apoptosis:roles of p53 and NF-κB[J].Mol Med Rep,2016,13(1):867-873.
[17] SUZUKI H,KOSUGE Y,KOBAYASHI K,et al.Heat-shock protein 72 promotes platelet aggregation induced by various platelet activators in rats[J].Biomed Res,2017,38(3):175-182.
[18] WU F H,YUAN Y,LI D,et al.Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance[J].Cancer Lett,2012,317(2):157-164.欢迎订阅 欢迎投稿 |
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