circ_LAS1L通过调控分泌型卷曲相关蛋白5影响心肌细胞存活的机制

Mechanism of circ_LAS1L Effecting Cardiomyocyte Survival by Regulating Secreted Frizzled-Related Protein 5

目的探讨circ_LAS1L通过调控分泌型卷曲相关蛋白5(SFRP5)影响心肌细胞存活的机制。方法本实验时间为2023年1—5月。(1)将心肌细胞H9c2随机分为对照组(不进行转染)、NC组(转染空载体)、circ_LAS1L组(转染circ_LAS1L过表达载体),然后检测各组circ_LAS1L表达水平及m^(6)A修饰酶〔甲基转移酶3(METTL3)、甲基转移酶14(METTL14)、Wilms肿瘤1相关蛋白(WTAP)、脂肪和肥胖相关蛋白(FTO)、α-酮戊二酸依赖性双加氧酶alkB同系物5(ALKBH5)〕、SFRP5 mRNA、蛋白表达水平。(2)将心肌细胞H9c2随机分为对照组、si-NC组(转染siRNA)、si-circ_LAS1L组(转染circ_LAS1L siRNA),各组检测指标同上。(3)将心肌细胞H9c2随机分为对照组、NC组、si-circ_LAS1L组、si-METTL3组(转染METTL3 siRNA)、si-circ_LAS1L+METTL3组(共转染circ_LAS1L siRNA和METTL3过表达载体),检测各组circ_LAS1L表达水平和METTL3、SFRP5 mRNA、蛋白表达水平及N^(6)-甲基腺苷(m^(6)A)^(+)SFRP5 mRNA表达水平、心肌细胞存活指标〔细胞存活力、乳酸脱氢酶(LDH)释放率、细胞凋亡率〕。结果circ_LAS1L组circ_LASIL表达水平及METTL3、METTL14、WTAP、SFRP5 mRNA、蛋白表达水平高于对照组,FTO、ALKBH5 mRNA、蛋白表达水平低于对照组(P<0.05)。si-circ_LAS1L组circ_LASIL表达水平及METTL3、METTL14、WTAP、SFRP5 mRNA、蛋白表达水平低于对照组,FTO、ALKBH5 mRNA、蛋白表达水平高于对照组(P<0.05)。si-circ_LAS1L组、si-circ_LAS1L+METTL3组circ_LAS1L表达水平低于对照组,si-circ_LAS1L组、si-METTL3组METTL3、SFRP5 mRNA、蛋白表达水平及m6A+SFRP5 mRNA表达水平低于对照组、si-circ_LAS1L+METTL3组(P<0.05)。si-circ_LAS1L组、si-METTL3组细胞存活力低于对照组、si-circ_LAS1L+METTL3组,LDH释放率、细胞凋亡率高于对照组、si-circ_LAS1L+METTL3组(P<0.05)。结论circ_LAS1L通过METTL3提高SFRP5的m^(6)A修饰水平,从而促进SFRP5表达,进而维持心肌细胞存活,且circ_LAS1L/METTL3/SFRP5轴是与心肌细胞功能密切相关的重要信号通路。

Objective To explore the mechanism of circ_LAS1L effecting cardiomyocyte survival by regulating secreted frizzled-related protein 5(SFRP5).Methods This experiment time was from January to May 2023.(1)H9c2 cardiomyocytes were randomly divided into control group(without transfection),NC group(transfected with empty vector)and circ_LAS1L group(transfected with circ_LAS1L overexpression vector).The expression levels of circ_LAS1L,mRNA and protein expression levels of m^(6)A modifying enzymes[methyltransferase like 3(METTL3),methyltransferase like 14(METTL14),Wilms tumor 1-associating protein(WTAP),fat mass and obesity-associated protein(FTO),alpha-ketoglutarate-dependent dioxygenase alkB homolog 5(ALKBH5)]and SFRP5 were detected.(2)H9c2 cardiomyocytes were randomly divided into control group,si-NC group(transfected with siRNA)and si-circ_LAS1L group(transfected with circ_LAS1L siRNA).The detection indexes of each group were the same as above.(3)H9c2 cardiomyocytes were randomly divided into control group,NC group,si-circ_LAS1L group,si-METTL3 group(transfected with METTL3 siRNA),si-circ_LAS1L+METTL3 group(co-transfected with circ_LAS1L siRNA and METTL3 overexpression vector).The expression level of circ_LAS1L,the mRNA and protein expression levels of METTL3 and SFRP5,mRNA expression level of N^(6)-methyladenosine(m^(6)A)^(+)SFRP6 and the survival indexes of cardiomyocytes[cell viability,lactic dehydrogenase(LDH)release rate and apoptosis rate]were detected in each group.Results The expression level of circ_LAS1L and the mRNA and protein expression levels of METTL3,METTL14,WTAP and SFRP5 in circ_LAS1L group were higher than those in control group,while the mRNA and protein expression levels of FTO and ALKBH5 were lower than those in control group(P<0.05).The expression level of circ_LAS1L and the mRNA and protein expression levels of METTL3,METTL14,WTAP and SFRP5 in si-circ_LAS1L group were lower than those in control group,while the mRNA and protein expression levels of FTO and ALKBH5 were higher than those in control group(P<0.05).The expression level of circ_LAS1L of si-circ_LAS1L group and si-circ_LAS1L+METTL3 group was lower than that of the control group,and the mRNA and protein expression levels of METTL3 and SFRP5 and m6A+SFRP5 mRNA expression level in si-circ_LAS1L group and si-METTL3 group were lower than those in control group and si-circ_LAS1L+METTL3 group(P<0.05).The cell viability of si-circ_LAS1L group and si-METTL3 group was lower than that of the control group and si-circ_LAS1L+METTL3 group,and the LDH release rate and apoptosis rate were higher than those of the control group and si-circ_LAS1L+METTL3 group(P<0.05).Conclusion circ_LAS1L can increase the m^(6)A modification level of SFRP5 through METTL3,thereby promote the expression of SFRP5 and then maintain the survival of cardiomyocytes.circ_LAS1L/METTL3/SFRP5 axis is an important signaling pathway closely related to cardiomyocyte function.