circ_LAS1L调控SFRP5抑制心肌细胞铁死亡

Circ_LAS1L Affects Cardiomyocyte Ferroptosis by Regulating SFRP5 Expression

目的探讨心肌缺血-再灌注损伤进展中circ_LAS1L调控分泌型卷曲相关蛋白5(SFRP5)表达的潜在机制和circ_LAS1L/SFRP5通路在大鼠心肌细胞铁死亡中的作用。方法传代培养大鼠胚胎心肌细胞株H9c2细胞,H9c2心肌细胞分别转染或共转染circ_LAS1L过表达载体、circ_LAS1L siRNA、miR-125b mimic、miR-125b siRNA和SFRP5 siRNA,然后进行缺氧/复氧处理。RT-qPCR检测circ_LAS1L和miR-125b的表达,RT-qPCR和Western blot检测SFRP5和GPX4的表达,电镜观察线粒体结构变化,CCK-8法检测细胞存活力,同时检测乳酸脱氢酶(LDH)、活性氧(ROS)、Fe^(2+)和丙二醛(MDA)含量。结果circ_LAS1L-O可增加SFRP5的mRNA和蛋白表达,而抑制circ_LAS1L表达可降低SFRP5的mRNA和蛋白表达(P均<0.05)。miR-125b mimic抑制而miR-125b siRNA促进SFRP5的RNA和蛋白表达,circ_LAS1L-O能减弱miR-125b mimic调控SFRP5表达的作用(P均<0.05);circ_LAS1L siRNA也能逆转miR-125b siRNA对SFRP5的表达调控(P均<0.05)。circ_LAS1L和SFRP5在缺氧/复氧处理的心肌细胞中表达升高,而GPX4表达降低(P均<0.05);SFRP5 siRNA可抵消circ_LAS1L过表达对GPX4表达的调控作用(P均<0.05)。缺氧/复氧导致线粒体明显皱缩和线粒体嵴减少,降低细胞存活力和增强LDH释放,并且促进ROS、Fe^(2+)和MDA的累积,而circ_LAS1L-O明显减弱缺氧/复氧导致的上述变化,si-SFRP5则拮抗circ_LAS1L过表达的作用。结论circ_LAS1L促进SFRP5的表达而抑制铁死亡关键调控因子GPX4的表达,进而参与心肌细胞铁死亡过程;miR-125b能抑制SFRP5的表达,且减弱circ_LAS1L调控SFRP5表达的作用。

Objective To study the mechanism of circ_LAS1L regulating the expression of SFRP5 and the role of circ_LAS1L/SFRP5 pathway in ferroptosis of rat cardiomyocytes in the progression of myocardial ischemia-reperfusion injury.Methods Rat embryonic myocardial cells H9c2 cardiomyocytes were cultured and transfected or co-transfected with circ_LAS1L overexpression vector(circ_LAS1L-O),circ_LAS1L siRNA,miR-125b mimic,miR-125b siRNA and SFRP5 siRNA,and then subjected to hypoxia/reoxygenation(H/R)treatment.The expression of circ_LAS1L and miR-125b was detected by RT-qPCR,and the expression of SFRP5 and GPX4 was detected by RT-qPCR and Western blot.Electron microscopy was used to observe changes in mitochondrial structure,and the CCK-8 method was used to detect cell viability.LDH release,ROS,Fe^(2+)and MDA contents were detected via the reagent kits.Results circ_LAS1L overexpression promoted the RNA and protein expression of SFRP5,but circ_LAS1L siRNA had the opposite effect(P all<0.05).miR-125b mimic inhibited but miR-125b siRNA promoted the RNA and protein expression of SFRP5,and circ_LAS1L overexpression blocked the role of miR-125b mimic in regulating SFRP5 expression(P all<0.05);Similarly,circ_LAS1L siRNA could also reverse the regulation of miR-125b siRNA on SFRP5 expression(P all<0.05).The expression of circ_LAS1L and SFRP5 was increased in H/R-treated cardiomyocytes,while the expression of GPX4 was decreased(P all<0.05);SFRP5 siRNA could counteract the regulatory effect of circ_LAS1L overexpression on GPX4 expression(P all<0.05).H/R led to significant mitochondrial shrinkage and reduced mitochondrial cristae,reduced cell viability,enhanced LDH release,and promoted ROS,Fe^(2+)and MDA accumulation,while circ_LAS1L-O significantly attenuated the above changes caused by H/R,but si-SFRP5 antagonized the effect of circ_LAS1L-O.Conclusion circ_LAS1L can promote the expression of SFRP5 and inhibit the expression of GPX4,the key regulatory factor in ferroptosis,thereby participating in the process of myocardial cell ferroptosis miR-125b can inhibit the expression of SFRP5 and significantly weaken the effect of circ-LAS1L on promoting SFRP5 expression.