切换至 "中华医学电子期刊资源库"

中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (05) : 284 -291. doi: 10.3877/cma.j.issn.2095-1221.2021.05.005

论著

circSERPINE2靶向miR-106b-5p对缺氧/复氧诱导的心肌细胞损伤的影响
魏薇1, 王文斌1,(), 刘秀梅1   
  1. 1. 102600 北京市大兴区人民医院心内科
  • 收稿日期:2021-05-06 出版日期:2021-10-01
  • 通信作者: 王文斌
  • 基金资助:
    首都卫生发展科研项目(2020-3-7122)

Effects of circSERPINE2 targeting miR-106b-5p on cardiomyocyte damage induced by hypoxia /reoxygenation

wei Wei1, Wenbin Wang1,(), Xiumei Liu1   

  1. 1. Department of Cardiology, Beijing Daxing District People’s Hospital, Beijing 102600, China
  • Received:2021-05-06 Published:2021-10-01
  • Corresponding author: Wenbin Wang
引用本文:

魏薇, 王文斌, 刘秀梅. circSERPINE2靶向miR-106b-5p对缺氧/复氧诱导的心肌细胞损伤的影响[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(05): 284-291.

wei Wei, Wenbin Wang, Xiumei Liu. Effects of circSERPINE2 targeting miR-106b-5p on cardiomyocyte damage induced by hypoxia /reoxygenation[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(05): 284-291.

目的

探讨circSERPINE2对缺氧/复氧(H/R)诱导的心肌细胞损伤的影响及机制。

方法

体外培养大鼠心肌细胞H9c2,分为对照组(用常规培养基置于常规培养箱中培养7 h)、H/R组[无血清低糖DMEM培养基缺氧(不含氧)培养3 h后,10﹪ FBS的DMEM培养基复氧(19.95﹪氧)培养4 h]、H/R+pcDNA组(转染pcDNA至H9c2细胞后进行H/R处理)、H/R+pcDNA-circSERPINE2组(转染pcDNA-circSERPINE2至H9c2细胞后进行H/R处理)、H/ R+anti-miR-NC组(转染anti-miR-NC至H9c2细胞后进行H/R处理)、H/R+anti-miR- 106b- 5p组(转染anti-miR-106b-5p至H9c2细胞后进行H/R处理)、H/R+pcDNA- circSERPINE2+ miR-NC组(共转染pcDNA-circSERPINE2和miR-NC至H9c2细胞后进行H/R处理)和H/ R+pcDNA-circSERPINE2+miR-106b-5p组(共转染pcDNA- circSERPINE2和miR- 106b- 5p mimics至H9c2细胞后进行H/R处理)。用RT-qPCR法检测细胞中circSERPINE2和miR- 106b- 5p表达,流式细胞术检测细胞凋亡,免疫印迹法检测细胞中cleaved caspase-3和cleaved caspase-9蛋白表达,试剂盒检测细胞中MDA含量、SOD和GSH-Px活性。双荧光素酶报告基因实验检测circSERPINE2和miR-106b-5p调控关系。方差齐性,两组间比较用t检验;方差不齐,两组间比较用t’检验。

结果

与对照组比较,H/R组H9c2细胞中circSERPINE2表达(1.00±0.06比0.43±0.04)、SOD[(77.84±6.49)U/mL比(23.19±2.21) U/ mL]和GSH- Px活性[(59.21±5.31) U/mL比(17.62±1.53)U/mL]均降低,而miR-106b- 5p表达(1.00±0.07比2.66±0.23)、细胞凋亡率[(6.53±0.52)﹪比(24.21±2.18)﹪]、cleaved caspase-3(0.24±0.02比0.72±0.05)、cleaved caspase-9蛋白表达(0.13±0.02比0.65±0.04)和MDA含量[(5.23±0.47)nmol/L比(45.56±4.33)nmol/L]均升高,差异有统计学意义(P均< 0.05)。与H/R+pcDNA组比较,H/R+pcDNA-circSERPINE2组H9c2细胞凋亡率[(26.34±2.23)﹪比(10.08±0.81)﹪]、cleaved caspase-3 (0.75±0.05比0.32±0.04)和cleaved caspase-9蛋白表达(0.67±0.05比0.25±0.03)及MDA含量[(49.18±4.89) nmol/L比(6.79±0.59) nmol/L]均降低,而SOD[(21.42±2.03) U/ mL比(63.57±6.44)U/mL]和GSH-Px活性[(16.06±1.42) U/ mL比(47.94±5.52)U/mL]均升高,差异有统计学意义(P均< 0.05)。与H/R+anti-miR-NC组比较,H/R+anti-miR-106b-5p组H9c2细胞凋亡率[(26.23±2.15)﹪比(13.87±1.26)﹪]、cleaved caspase-3 (0.78±0.06比0.38±0.03)和cleaved caspase-9蛋白表达(0.68±0.05比0.31±0.03)及MDA含量[(48.38±4.21)nmol/L比(11.39± 1.42)nmol/L]均降低,而SOD[(22.19±2.21) U/ mL比(54.33±5.25)U/mL]和GSH- Px活性[(15.84±1.68) U/ mL比(40.42±3.51)U/ mL]均升高,差异有统计学意义(P均< 0.05)。circSERPINE2靶向结合并负调控miR-106b-5p表达。与H/R+pcDNA-circSERPINE2+miR-NC组比较,H/R+pcDNA- circSERPINE2+miR-106b-5p组H9c2细胞细胞凋亡率[(9.86±1.04)﹪比(18.47±1.24)﹪]、cleaved caspase-3 (0.31±0.03比0.66±0.04)、cleaved caspase-9蛋白表达(0.23±0.02比0.56±0.06)及MDA含量[(6.56±0.61)nmol/L比(36.97±3.14) nmol/ L,P < 0.05]均升高,而SOD[(64.14±6.32)U/ mL比(32.26±3.58)U/mL]和GSH-Px活性[(48.40±5.06)U/mL比(25.58±3.11)U/mL]均降低,差异有统计学意义(P均< 0.05)。

结论

上调circSERPINE2可抑制H/R诱导的大鼠心肌细胞凋亡及氧化应激反应,其作用机制可能通过靶向下调miR-106b-5p表达有关。

Objective

To investigate the effects of circSERPINE2 on hypoxia/reoxygenation (H/R) -induced cardiomyocyte damage and the mechanism.

Methods

Rat cardiomyocytes H9c2 were cultured in vitro and divided into control group (cells were cultured in a conventional incubator for 7 hours) H/R group (cells were cultured in serum-free low-glucose DMEM under hypoxia [no oxygen] for 3 hours, and then cultured in DMEM containing 10﹪ FBS under reoxygenation [19.95﹪ oxygen] for 4 hours) , H/R+pcDNA group (H9c2 cells transfected with pcDNA then treated by H/ R) , H/R+pcDNA-circSERPINE2 group (H9c2 cells transfected with pcDNA-circSERPINE2 then treated by H/R) , H/R+anti-miR-NC group (H9c2 cells transfected with anti-miR-NC then treated by H/R) , H/R+anti-miR-106b- 5p group (H9c2 cells transfected with anti-miR-106b-5p then treated by H/R) , H/R+pcDNA- circSERPINE2+miR- NC group (H9c2 cells co-transfected with pcDNA-circSERPINE2+miR-NC then treated by H/ R) and H/R+pcDNA- circSERPINE2+miR-106b-5p group (H9c2 cells transfected with pcDNA- circSERPINE2+miR-106b-5p then treated by H/R) . RT- qPCR was used to detect the expression of circSERPINE2 and miR-106b-5p. Flow cytometry was used to detect cell apoptosis. Western blotting was used to detect the protein expression of cleaved caspase-3 and cleaved caspase-9 in cells. The kits were used to detect the content of MDA and the activity of SOD and GSH-Px. The regulatory relationship between circSERPINE2 and miR- 106b- 5p was detected by dual luciferase reporter assay. The homogeneity of variance was compared between the two groups by t-test. The variance was uneven, and t' test was used.

Results

Compared with the control group, circSERPINE2 expression in H9c2 cells of H/R group was decreased (1.00±0.06 vs 0.43±0.04) , SOD[ (77.84±6.49) U/ mL vs (23.19±2.21) U/mL] and GSH-Px activity [ (59.21±5.31) U/mL vs (17.62±1.53) U/ mL] decreased, while miR-106b- 5p expression (1.00±0.07 vs 2.66±0.23) , apoptosis rate [ (6.53±0.52) ﹪ vs (24.21±2.18) ﹪], cleaved caspase-3 (0.24±0.02 vs 0.72± 0.05) and cleaved caspase-9 protein expression (0.13±0.02 vs 0.65±0.04) were increased. The content of MDA (5.23±0.47) nmol/L was higher than (45.56±4.33) nmol/L, and the difference was statistically significant (P < 0.05) . Compared with H/R + pcDNA group, the apoptosis rate of H9c2 cells [ (26.34±2.23) ﹪ vs (10.08±0.81) ﹪], cleaved caspase-3 (0.75±0.05 vs 0.32±0.04) , cleaved caspase-9 protein expression (0.67±0.05 vs 0.25±0.03) and MDA content [ (49.18±4.89) nmol/L vs (6.79±0.59) nmol/ L] in H/R + pcDNA-circSERPINE2 group were lower, However, SOD [ (21.42 ± 2.03) U/mL vs (63.57± 6.44) U/ mL] and GSH-Px activity [ (16.06 ± 1.42) U/ mL vs (47.94±5.52) U/mL] were increased, and the difference was statistically significant (P < 0.05) . Compared with H/R + anti-miR-NC group, the apoptosis rate of H9c2 cells[ (26.23± 2.15) ﹪ vs (13.87±1.26) ﹪], cleaved caspase- 3 (0.78±0.06 vs 0.38±0.03) , cleaved caspase-9 protein expression (0.68±0.05 vs 0.31±0.03) and MDA content [ (48.38±4.21) nmol/L vs (11.39±1.42) nmol/ L] in H/R + anti-miR-NC group were lower. However, SOD [ (22.19±2.21) U/ mL vs (54.33 ± 5.25) U/mL] and GSH- Px activity [ (15.84±1.68) U/mL vs (40.42±3.51) U/mL]were increased, and the difference was statistically significant (P < 0.05) . circSERPINE2 targeted and negatively regulated the expression of miR-106b-5p. Compared with the H/R+pcDNA-circSERPINE2+miR- NC group, the apoptosis rate [ (9.86±1.04) ﹪vs (18.47±1.24) ﹪], the protein expression of cleaved caspase-3 (0.31±0.03 vs 0.66±0.04) and cleaved caspase-9 (0.23±0.02 vs 0.56±0.06) , and the content of MDA [ (6.56±0.61) nmol/ L vs (36.97±3.14) nmol/L] of H9c2 cells in the H/ R+pcDNA-circSERPINE2+miR- 106b- 5p group were increased, but the SOD[ (64.14±6.32) U/ mL vs (32.26±3.58) U/mL] and GSH- Px activity [ (48.40±5.06) U/mL vs (25.58±3.11) U/mL] were decreased (P < 0.05) .

Conclusion

Up-regulation of circSERPINE2 can inhibit H/R-induced cardiomyocyte apoptosis and oxidative stress in rats, and its mechanism may be related to the targeted down-regulation of miR- 106b-5p expression.

表1 引物序列信息
表2 circSERPINE2和miR-106b-5p在H/R诱导的心肌细胞中的表达( ± s
图1 上调circSERPINE2后H/R诱导的心肌细胞中cleaved caspase-3和cleaved caspase-9蛋白表达
图2 上调circSERPINE2后H/R诱导的心肌细胞凋亡情况
表3 上调circSERPINE2对H/R诱导的心肌细胞损伤的影响( ± s
图3 circSERPINE2的序列中含有与miR-106b-5p互补的核苷酸序列
表4 双荧光素酶报告实验( ± s
图4 下调miR-106b-5p对H/R诱导的心肌细胞凋亡的影响
表5 下调miR-106b-5p对H/R诱导的心肌细胞损伤的影响( ± s
图5 上调miR-106b-5p逆转上调circSERPINE2对H/R诱导的心肌细胞凋亡的作用
表6 上调miR-106b-5p逆转上调circSERPINE2对H/R诱导的心肌细胞损伤的作用( ± s
1
张旭,陈旺盛,冯健, 等. 银杏内酯B保护缺氧/复氧诱导心肌细胞损伤模型的作用及机制[J]. 中国组织工程研究, 2019, 23(19):3097-3101.
2
Zou JF, Wu XN, Shi RH, et al. Inhibition of microRNA-184 reduces H2O2-mediated cardiomyocyte injury via targeting FBXO28[J]. Eur Rev Med Pharmacol Sci, 2020, 24(21):11251-11258.
3
Zhu YH, Zou CW, Jia YT, et al. Knockdown of circular RNA circMAT2B reduces oxygen-glucose deprivation-induced inflammatory injury in H9c2 cells through up-regulating miR-133[J]. Cell Cycle, 2020, 19(20):2622-2630.
4
Zhang QP, Qiao XM, Xia WW. CircSERPINE2 weakens IL-1β-caused apoptosis and extracellular matrix degradation of chondrocytes by regulating miR-495/TGFBR2 axis[J]. Biosci Rep, 2020, 40(11):BSR20201601. doi:10.1042/BSR20201601.
5
Li PF, Shen MH, Gao F, et al. An antagomir to microRNA-106b- 5p ameliorates cerebral ischemia and reperfusion injury in rats via inhibiting apoptosis and oxidative stress[J]. Mol Neurobiol, 2017, 54(4):2901-2921.
6
Zheng DL, Li MM, Li GF, et al. Circular RNA circ_DROSHA alleviates the neural damage in a cell model of temporal lobe epilepsy through regulating miR-106b-5p/MEF2C axis[J]. Cell Signal, 2021, 80:109901.
7
Xu GX, Zhang W, Wang ZL, et al. Matrine regulates H2O2-induced oxidative stress through long non-coding RNA HOTAIR/miR-106b-5p axis via AKT and STAT3 pathways[J]. Biosci Rep, 2020, 40(5):BSR20192560. doi:10.1042/BSR20192560.
8
张秀娥,成蓓,彭雯, 等. p38MAPK信号通路在钙调磷酸酶促心肌凋亡中的作用[J]. 中国病理生理杂志, 2008, 24(2):266-269.
9
王彦利,李纪明,罗进光. miR-137靶向下调SETD7表达对缺氧复氧诱导的心肌细胞氧化应激的影响研究[J]. 分子诊断与治疗杂志, 2019, 11(6):462-467.
10
张会超,徐里,芮浩淼, 等. 苦豆碱对缺血再灌注引起的H9c2心肌细胞损伤和炎症应答的作用[J]. 中国病理生理杂志, 2018, 34(2):281-286.
11
Zou JF, Wu XN, Shi RH, et al. Inhibition of microRNA-184 reduces H2O2-mediated cardiomyocyte injury via targeting FBXO28[J]. Eur Rev Med Pharmacol Sci, 2020, 24(21):11251-11258.
12
Cai XY, Zhang P, Wang S, et al. lncRNA FGD5 antisense RNA 1 upregulates RORA to suppress hypoxic injury of human cardiomyocyte cells by inhibiting oxidative stress and apoptosis via miR-195[J]. Mol Med Rep, 2020, 22(6):4579-4588.
13
Lu M, Qin XL, Yao JG, et al. MiR-134-5p targeting XIAP modulates oxidative stress and apoptosis in cardiomyocytes under hypoxia/reperfusion-induced injury[J]. IUBMB Life, 2020, 72(10):2154-2166.
14
Zheng TT, Chen KK, Zhang X, et al. Knockdown of TXNDC9 induces apoptosis and autophagy in glioma and mediates cell differentiation by p53 activation[J]. Aging (Albany NY), 2020, 12(18):18649-18659.
15
李世勋,周凡,王岩. miR-155靶向SIRT1调控缺氧/复氧心肌细胞的活力、凋亡和氧化应激[J]. 中国老年学杂志, 2019, 39(24):6084-6090.
16
Zhou JL, Li LW, Hu H, et al. Circ-HIPK2 accelerates cell apoptosis and autophagy in myocardial oxidative injury by sponging miR-485-5p and targeting ATG101[J]. J Cardiovasc Pharmacol, 2020, 76(4):427-436.
17
Zhang CL, Long TY, Bi SS, et al. CircPAN3 ameliorates myocardial ischaemia/reperfusion injury by targeting miR-421/Pink1 axis-mediated autophagy suppression[J]. Lab Invest, 2021, 101(1):89-103.
18
Chai QY, Zheng MQ, Wang L, et al. Circ_0068655 promotes cardiomyocyte apoptosis via miR-498/PAWR axis[J]. Tissue Eng Regen Med, 2020, 17(5):659-670.
19
Shen SY, Wu YZ, Chen JX, et al. CircSERPINE2 protects against osteoarthritis by targeting miR-1271 and ETS-related gene[J]. Ann Rheum Dis, 2019, 78(6):826-836.
20
Pan HJ, Zhao FF, Yang YM, et al. Overexpression of long non-coding RNA SNHG16 against cerebral ischemia-reperfusion injury through miR-106b-5p/LIMK1 axis[J]. Life Sci, 2020, 254:117778. doi: 10.1016/j.lfs.2020.117778.
21
Wu HF, Wang J, Ma Z. Long noncoding RNA HOXA-AS2 mediates microRNA-106b-5p to repress sepsis-engendered acute kidney injury[J]. J Biochem Mol Toxicol, 2020, 34(4):e22453.doi: 10.1002/jbt.22453.
[1] 孔莹莹, 谢璐涛, 卢晓驰, 徐杰丰, 周光居, 张茂. 丁酸钠对猪心脏骤停复苏后心脑损伤的保护作用及机制研究[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 355-362.
[2] 张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[3] 钟轼, 李斌飞, 温君琳, 古晨, 廖小卒. 右美托咪定缓解神经病理性疼痛作用机制的研究进展[J]. 中华普通外科学文献(电子版), 2023, 17(03): 237-240.
[4] 刘硕儒, 王功炜, 张斌, 李书豪, 胡成. 新型溶瘤病毒M1激活内质网应激致前列腺癌细胞凋亡的机制[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(04): 388-393.
[5] 刘骏, 朱霁, 殷骏. 右美托咪定对腹股沟疝手术麻醉效果及安全性的影响[J]. 中华疝和腹壁外科杂志(电子版), 2023, 17(05): 570-573.
[6] 邓春文, 陈嵩, 钟裴, 闵师强, 万健. LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 129-136.
[7] 张师垚, 徐岩岩, 张琦, 李春强, 赵智成, 刘刚. m6A结合蛋白YTHDC2调节p38MAPK信号通路影响结直肠癌细胞凋亡[J]. 中华结直肠疾病电子杂志, 2023, 12(02): 117-124.
[8] 尚慧娟, 袁晓冬. 机械取栓术后应用依达拉奉右崁醇对急性缺血性脑卒中预后的改善[J]. 中华神经创伤外科电子杂志, 2023, 09(05): 295-301.
[9] 阿迪莱·阿卜杜热西提, 费奥, 邢晓雯, 谢胜强, 张睿, 兰晓娟, 程岗. 三种模拟创伤性脑损伤体外细胞模型的损伤特征比较[J]. 中华神经创伤外科电子杂志, 2023, 09(02): 69-75.
[10] 于迪, 于海波, 吴焕成, 李玉明, 苏彬, 陈馨. 发状分裂相关增强子1差异表达对胆固醇刺激下血管内皮细胞的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 264-270.
[11] 邹勇, 顾应江, 丁昊, 杨呈浩, 陈岷辉, 蔡昱. 基于Nrf2/HO-1及NF-κB信号通路探讨葛根素对大鼠脑出血后早期炎症反应及氧化应激反应的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 271-277.
[12] 张敏洁, 张小杉, 段莎莎, 施依璐, 赵捷, 白天昊, 王雅晳. 氢气治疗心肌缺血再灌注损伤的作用机制及展望[J]. 中华临床医师杂志(电子版), 2023, 17(06): 744-748.
[13] 孙凤兰, 周萍, 程兴璞, 张倩倩. 腹腔镜全子宫切除术对子宫肌瘤患者机体氧化应激损伤及术后并发症的影响[J]. 中华临床医师杂志(电子版), 2023, 17(02): 149-153.
[14] 买买提·依斯热依力, 依力汗·依明, 王永康, 阿巴伯克力·乌斯曼, 艾克拜尔·艾力, 李义亮, 克力木·阿不都热依木. 氧化应激对3T3-L1前脂肪细胞中GLP-1/DPP-4信号通路以及炎症因子表达的影响[J]. 中华肥胖与代谢病电子杂志, 2023, 09(03): 186-191.
[15] 邱甜, 杨苗娟, 胡波, 郭毅, 何奕涛. 亚低温治疗脑梗死机制的研究进展[J]. 中华脑血管病杂志(电子版), 2023, 17(05): 518-521.
阅读次数
全文


摘要