LncRNA PVT1调控miR-9-5p/HIPK2轴对缺氧复氧诱导的心肌细胞损伤的影响OA
The Effect of LncRNA PVT1 on Hypoxia/reoxygenation-Induced Cardiomyocyte Injury by Regulating the miR-9-5p/HIPK2 Axis
目的:探讨长链非编码 RNA(LncRNA)浆细胞瘤变异易位1(PVT1)调控微小 RNA-9-5p(miR-9-5p)/同源结构域相互作用蛋白激酶2(HIPK2)轴对缺氧复氧(H/R)诱导的心肌细胞损伤的影响.方法:将 AC16 细胞分为 CG 组、H/R 组、sh-NC 组、sh-PVT1 组、sh-PVT1+NC-inhibitor 组、sh-PVT1+miR-9-5p-inhibitor 组,除了 CG 组剩余各组均进行 H/R 处理,在 37℃缺氧环境(94%N2、5%CO2、1%O2)下8h,在常氧环境(95%O2 和 5%CO2)下再氧合 12h.qRT-PCR 法检测 AC16 细胞中 Ln-cRNA PVT1、miR-9-5p、HIPK2 表达;MTS 法和平板克隆实验检测 AC16 细胞增殖;流式细胞仪检测AC16 细胞凋亡;ELISA 法检测 AC16 细胞上清液心肌细胞损伤指标(LDH、CK)的释放水平;DCFH-DA检测 AC16 细胞中 ROS 水平;ELISA 法 AC16 细胞裂解液中氧化应激(OS)指标(SOD、MDA)和炎症因子(TNF-α、IL-6)的含量;Western blot 检测 AC16 细胞中 Ki67、HIPK2、p53、Cleaved Caspase-3 蛋白的表达.结果:LncRNA PVT1 可以靶向负调控 miR-9-5p、miR-9-5p 可以靶向负调控 HIPK2.H/R 组OD490 值、克隆数、miR-9-5p、SOD、Ki67、p53 低于 CG 组,凋亡率、ROS 平均荧光强度、LncRNA PVT1、HIPK2 mRNA 和蛋白、LDH、CK、MDA、TNF-α、IL-6、Cleaved Caspase-3 高于 CG 组(P<0.05);sh-PVT1组 OD490 值、克隆数、miR-9-5p、SOD、Ki67、p53 高于 H/R 组、sh-NC 组,凋亡率、ROS 平均荧光强度、Ln-c RNA PVT1、HIPK2 mRNA 和蛋白、LDH、CK、MDA、TNF-α、IL-6、Cleaved Caspase-3 低于 H/R 组、sh-NC 组(P<0.05);sh-PVT1+miR-9-5p-inhibitor 组 OD490 值、克隆数、miR-9-5p、SOD、Ki67、p53 低于 sh-PVT1 组、sh-PVT1+NC-inhibitor 组,凋亡率、ROS 平均荧光强度、HIPK2 mRNA 和蛋白、LDH、CK、MDA、TNF-α、IL-6、Cleaved Caspase-3 高于 sh-PVT1 组、sh-PVT1+NC-inhibitor 组(P<0.05).结论:敲低 LncRNA PVT1 可能通过调控 miR-9-5p/HIPK2 轴,减缓 H/R 诱导的心肌细胞损伤.
Objective:To discuss the effect of long non-coding RNA(LncRNA)plasmacytoma variant translocation 1(PVT1)on hypoxia/reoxygenation(H/R)-induced cardiomyocyte injury by regulating the microrNA-9-5p(miR-9-5p)/homeodomain-interacting protein kinase 2(HIPK2)axis.Methods:AC16 cells were separated into the CG group,H/R group,sh-NC group,sh-PVT1 group,sh-PVT1+NC-inhibitor group,and sh-PVT1+miR-9-5p-inhibitor group.Except for the CG group,the remaining groups were all treated with H/R:8 hours in a hypoxic environment(94%N2,5%CO2,1%O2)at 37℃,followed by 12 hours of reoxygenation uder normoxic environment(95%O2 and 5%CO2).The expression levels of LncRNA PVT1,miR-9-5p and HIPK2 in AC16 cells were detected by qRT-PCR.The proliferation of AC16 cells was detected by MTS assay and colony formation assay.The apoptosis of AC16 cells was detected by flow cytome-try.The release levels of myocardial cell injury indicators(LDH,CK)in the supernatant of AC16 cells were detected by ELISA.DCFH-DA was used to detect the ROS levels in AC16 cells.The contents of oxidative stress(OS)indicators(SOD,MDA)and inflammatory factors(TNF-α,IL-6)in the lysate of AC16 cells were determined by ELISA.Moreover,the protein expression levels of Ki67,HIPK2,p53 and Cleaved Caspase-3 in AC16 cells were detected by Western blot.Results:LncRNA PVT1 could target and negatively regulate miR-9-5p,and miR-9-5p could target and negatively regulate HIPK2.Compared with the CG group,the H/R group had lower OD490 value,clone number,miR-9-5p,SOD,Ki67 and p53,as well as higher apoptosis rate,average fluorescence intensity of ROS,the LncRNA PVT1,HIPK2 at mRNA and pro-tein levels,LDH,CK,MDA,TNF-α,IL-6,and Cleaved Caspase-3(P<0.05).Compared with the H/R group and sh-NC group,the sh-PVT1 group had higher OD490 value,clone number,miR-9-5p,SOD,Ki67 and p53,as well as lower apoptosis rate,average fluorescence intensity of ROS,the LncRNA PVT1,HIPK2 at mRNA and protein levels,LDH,CK,MDA,TNF-α,IL-6,and Cleaved Caspase-3(P<0.05).Com-pared with the sh-PVT1 group and sh-PVT1+NC-inhibitor group,the sh-PVT1+miR-9-5p-inhibitor group had lower OD490 value,clone number,miR-9-5p,SOD,Ki67 and p53,as well as higher apoptosis rate,av-erage fluorescence intensity of ROS,the LncRNA PVT1,HIPK2 at mRNA and protein levels,LDH,CK,MDA,TNF-α,IL-6,and Cleaved Caspase-3(P<0.05).Conclusion:Knockdown of LncRNA PVT1 may alleviated H/R-induced cardiomyocyte injury by regulating the miR-9-5p/HIPK2 axis.
刘芳;刘欣;刘沙沙;解娟
河北省保定市第一中心医院心内科,河北 保定 071000河北省保定市第一中心医院心内科,河北 保定 071000河北省保定市第一中心医院心内科,河北 保定 071000河北省保定市第一中心医院心内科,河北 保定 071000
心肌细胞损伤长链非编码 RNA浆细胞瘤变异易位1微小 RNA-9-5p同源结构域相互作用蛋白激酶2缺氧复氧
Cardiomyocyte injuryLong non-coding RNAPlasmacytoma variant translocation 1MicroRNA-9-5pHomeodomain interacting protein kinase 2Hypoxia/reoxygenation
《河北医学》 2026 (5)
720-728,9
河北省医学科学研究课题(编号:20261384)
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