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MiR-499靶向HMGB1保护缺氧复氧心肌细胞损伤的分子机制探讨

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目的 研究miR-499对缺氧复氧心肌细胞H9c2损伤的影响,并探讨其作用机制。方法 运
用免疫印迹法(Western blot)检测缺氧复氧心肌细胞中高迁移率族蛋白1(HMGB1)、B细胞淋巴瘤/白
血病-2(Bcl-2)、免抗人单克隆抗体(Bax)、caspase-3的蛋白表达;将H/R+miR-con组(转染miRcon)
、H/R+miR-499组(转染miR-499 mimics)、miR-con组(转染miR-con)、miR-499组(转染miR-499
mimics)、anti-miR-con组(转染anti-miR-con)、anti-miR-499组(转染anti-miR-499)、H/R+si-con组
(转染si-con)、H/R+si-HMGB1组(转染si-HMGB1)、H/R+miR-499+Ctrl组(miR-499 mimics和pcDNA3.1
共转染)、H/R+miR-499+HMGB1组(miR-499 mimics和pcDNA3.1-HMGB1共转染),均以脂质体法转染至
H9c2细胞;实时荧光定量聚合酶链锁反应(qRT-PCR)检测各组细胞中miR-499的表达;流式细胞术检测
各组细胞的凋亡;双荧光素酶报告基因检测实验检测各组细胞的荧光活性。结果 与NC组相比,H/R组细
胞中HMGB1蛋白表达显著升高,miR-499表达显著降低(P<0.05);过表达miR-499或敲减HMGB1均可显
著下调H/R H9c2细胞凋亡率,显著下调Bax、caspase-3的蛋白表达,上调Bcl-2的蛋白表达(P均<0.05);
过表达HMGB1可逆转miR-499对H/R H9c2细胞凋亡的抑制作用。结论 MiR-499可抑制缺氧复氧心肌细胞
H9c2凋亡,保护心肌细胞,其机制与靶向HMGB1有关,将可为心脏病的治疗提供新靶点。

Abstract:

Objective To study the influence of microRNA-499 (miR-499) on hypoxia/reoxygenation
cardiomyocyte H9c2 (H/R-H9c2) and discuss the effective mechanism. Methods The protein expressions of high
mobility group Box-1 protein (HMGB1), Bcl-2, Bax and caspase-3 in H/R-H9c2 were detected by using Western
blotting assay. H/R-H9c2 were transfected by using liposome method in H/R+miR-con group (transfected with miRcon),
H/R+miR-499 group (transfected with miR-499 mimics), miR-con group (transfected with miR-con), miR-499
group (transfected with miR-499 mimics), anti-miR-con group (transfected with anti-miR-con), anti-miR-499 group
(transfected with anti-miR-499), H/R+si-con group (transfected with si-con), H/R+si-HMGB1 group (transfected
with si-HMGB1), H/R+miR-499+Ctrl group (co-transfected with miR-499 mimics and pcDNA3.1), and H/R+miR-
499+HMGB1 group (co-transfected with miR-499 mimics and pcDNA3.1-HMGB1). The expression of miR-499
in H/R-H9c2 was detected by using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in
all groups. The apoptosis of H/R-H9c2 was detected by using flow cytometry (FCM), and fluorescence activity of H/
R-H9c2 was detected by using dual-luciferase reporter gene assay in all groups. Results Compared with NC group,
the protein expression of HMGB1 increased significantly and expression of miR-499 decreased significantly in H/R
group (P<0.05). The over-expression of miR-499 or knock-down of HMGB1 can significantly down-regulated H/
R-H9c2 apoptosis rate and protein expressions of Bax and caspase-3, and up-regulated protein expression of Bcl-2
(all P<0.05). The over-expression of HMGB1 could reverse the inhibitive effect of miR-499 on H/R-H9c2 apoptosis.
Conclusion MiR-499 can inhibit the apoptosis of H/R-H9c2 and protect cardiomyocytes, and the mechanism is
correlated to targeting HMGB1, which can provide a new target spot for heart disease treatment.

基金项目:

十二五国家科技支撑计划(2011BAI11B01);咸宁市中心医院 湖北科技学院附属第一医院 项目资助(2018XYA006)

参考文献:

[1] Kim HM,Kim YM. HMGB1: LPS Delivery Vehicle for Caspase-11-
Mediated Pyroptosis[J]. Immunity,2018,49(4):582-4.
[2] Deng M,Tang Y,Li W,et al. The Endotoxin Delivery Protein HMGB1
Mediates Caspase-11-Dependent Lethality in Sepsis[J]. Immunity,
2018,49(4):740-53.
[3] Lu B,Wang H,Andersson U,et al. Regulation of HMGB1 release by
inflammasomes[J]. Protein Cell,2013,4(3):163-7.
[4] 鄂璐莎,南景龙. HMGB1对大鼠心肌缺血/再灌注损伤后内质网应
激的影响[J]. 中华危重病急救医学,2017,29(10):916-20.
[5] 赵延兵,张玮,苏瑜恒,等. 微小RNA30b3p在肝脏缺血再灌注损伤
小鼠肝组织中的表达及意义[J]. 新乡医学院学报,2018,5:361-7.
[6] 柳培雨. miRNA-214/Ca~(2+)信号通路在大鼠电针预处理心肌缺
血—再灌注损伤保护中的作用[D]. 南方医科大学,2016.
[7] 李雪,周春燕,贾竹青. 微小RNA-499的表达特征及生物学作用[J].
医学分子生物学杂志,2012,9(2):126-31.
[8] Halushka PV,Goodwin AJ,Halushka MK. Opportunities for microRNAs
in the Crowded Field of Cardiovascular Biomarkers[J]. Annu Rev
Pathol,2018,doi:10.1146/012418-012827.
[9] Bernardo BC,Ooi JY,Lin RC,et al. miRNA therapeutics: a new class
of drugs with potential therapeutic applications in the heart[J]. Future
Med Chem,2015,7(13):1771-92.
[10] 孙小琴,任玲,孙阳阳,等. microRNA-499通过靶向调节SOX6抵抗
缺氧诱导的神经细胞损伤[J]. 神经解剖学杂志,2018,34(1):53-9.
[11] 岳莹,吕风华,陈玉磊,等. miR-499对缺氧/复氧诱导的心肌细胞凋
亡的影响[J]. 郑州大学学报(医学版),2018,4,503-7.
[12] 郑重洲. miR-499在H9C2心肌细胞缺氧复氧损伤中的表达变化及
调控机制研究[D]. 广东医学院,2016.
[13] Yamada S,Maruyama I. HMGB1, a novel inflammatory cytokine[J]. Clin
Chim Acta,2007,375(1-2):36-42.
[14] Raucci A,Di Maggio S,Scavello F,et al. The Janus face of HMGB1
in heart disease: a necessary update[J]. Cell Mol Life Sci,
2018,76(2):211-29.
[15] Jiang W,Pisetsky DS. Mechanisms of Disease: the role of high mobility
group protein 1 in the pathogenesis of inflammatory arthritis[J]. Nat
Clin Pract Rheumatol,2007,3(1):52-8.
[16] 瓦哈甫·马木提,陆荣荣,阿布都乃孜尔·肉孜,等. 冠状动脉粥样
硬化斑块特性的早期识别及药物干预对血清学指标的影响[J].
中国医药,2019,14(2):175-8.
[17] Paudel YN,Shaikh MF,Chakraborti A,et al. HMGB1: A Common
Biomarker and Potential Target for TBI, Neuroinflammation, Epilepsy,
and Cognitive Dysfunction[J]. Front Neurosci, 2018,12:628.
[18] Musumeci D,Roviello GN,Montesarchio D. An overview on HMGB1
inhibitors as potential therapeutic agents in HMGB1-related
pathologies[J]. Pharmacol Ther,2014,141(3):347-57.
[19] 邓秋菊,李慧颖,向琳. HMGB1对缺氧复氧心肌细胞氧化损伤的影
响研究[J]. 中国循证心血管医学杂志,2018,10(08):89-92.
[20] 赵丽华,尹宏磊,吕风华,等. HMGB1对缺氧复氧环境下心肌细胞凋
亡的影响及机制研究[J]. 实用预防医学,2018,25(6):746-50.
[21] 邵忠华,冯斌. 针刺内关穴对大鼠心肌缺血再灌注损伤后HMGB1
表达的影响[J]. 中国中医药科技,2014,21(3):243-5.
[22] 黄仁发,高玉,梁群卿,等. 缺氧-复氧诱导HK2细胞HMGB1/TLR-4
信号通路活化调控TNF-α、IL-1β表达的意义[J]. 中国中西医
结合肾病杂志,2017,18(9):766-70.
[23] 薛晋红,刘艳,王丽月,等. 橙皮素对H2O2诱导H9c2心肌细胞凋亡
的抑制效应研究[J]. 中国心血管病研究,2017,15(3):276-9.
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