骨髓间充质干细胞来源外泌体中lncRNA SNHG12对心肌细胞的影响及其机制

Effect of lncRNA SNHG12 in exosomes derived from bone marrow mesenchymal stem cells on cardiomyocytes and its mechanism

目的探讨骨髓间充质干细胞(BMSCs)来源外泌体中长链非编码RNA(lncRNA)小核仁RNA宿主基因12(SNHG12)对心肌细胞的影响及其机制。方法贴壁法分离培养BMSCs并传代。取传3代BMSCs,转染SNHG1248 h,分离外泌体并鉴定。取H9c2细胞,随机分为阴性对照(NC)外泌体组与SNHG12外泌体组,分别转染NC外泌体、SNHG12外泌体;SNHG12外泌体+miR138-5p抑制剂组与SNHG12外泌体+NC抑制剂组,分别转染SNHG12外泌体+miR138-5p抑制剂及SNHG12外泌体+NC抑制剂;SNHG12外泌体+SH-NC组与SNHG12外泌体+SH-SIRT1组,分别转染SNHG12外泌体+SH-NC及SNHG12外泌体+SH-SIRT1。制备缺氧溶液,将上述各组细胞与缺氧溶液共孵育6 h,建立体外缺氧细胞模型。收集各组细胞培养液,离心留取上清液。采用ELISA法检测上清液IL-1β、IL-18含量。收集各组细胞,采用Western blotting法检测核苷酸结合寡聚化结构域样受体3(NLRP3)、凋亡相关斑点样蛋白(ASC)、剪切的半胱氨酸蛋白酶1(Cleaved-Caspase-1)、剪切的焦孔素D(Cleaved-GSDMD)蛋白表达,采用CCK-8法检测细胞活力,采用TUNEL法检测细胞凋亡率。结果经鉴定,成功获取BMSCs来源外泌体。在体外缺氧细胞中,SNHG12外泌体组细胞凋亡率以及NLRP3、ASC、Cleaved-Caspase-1、Cleaved-GSDMD蛋白表达和上清液IL-1β、IL-18水平均低于NC外泌体组(P均<0.05),细胞活力高于NC外泌体组(P<0.05)。下调miR-138-5p表达后,BMSCs来源外泌体中SNHG12可导致NLRP3、ASC、Cleaved-Caspase-1、Cleaved-GSDMD蛋白表达降低,细胞活力升高、细胞凋亡率降低(P均<0.05);下调SIRT1表达后,BMSCs来源外泌体中SNHG12可导致NLRP3、ASC、Cleaved-Caspase-1、Cleaved-GSDMD蛋白表达升高,细胞活力降低、细胞凋亡率升高(P均<0.05)。结论BMSCs来源外泌体中lncRNA SNHG12可能通过调节miR-138-5p/SIRT1轴抑制心肌细胞炎症反应及心肌细胞焦亡和凋亡,从而发挥对心肌细胞的保护作用。

Objective To investigate the effect of long non-coding RNA(lncRNA)small nucleolar RNA host gene 12(SNHG12)in exosomes from bone marrow mesenchymal stem cells(BMSCs)on cardiomyocytes and its mechanism.Methods BMSCs were isolated and cultured by adhesion method.BMSCs of the third generation were transfected with SNHG12 for 48 h,and exosomes were isolated and identified.H9c2 cells were randomly divided into the negative control(NC)exosome group and SNHG12 exosome group,and were transfected with equal volume of NC exosome and SNHG12 exosome,respectively;the SNHG12 exosome+miR138-5p inhibitor group and SNHG12 exosome+NC inhibitor group were transfected with equal volume of SNHG12 exosome+miR138-5p inhibitor and SNHG12 exosome+NC inhibitor group,respectively;the SNHG12 exosome+SH-NC group and SNHG12 exosome+SH-SIRT1 group were transfected with equal volume of SNHG12 exosome+SH-NC and SNHG12 exosome+SH-SIRT1 group,respectively.Anoxic solution was prepared,and cells of each group were incubated with anoxic solution for 6 h to establish anoxic cell models in vitro.The cell culture medium of each group was collected,and the supernatant was retained by centrifugation.The content of IL-1βand IL-18 in the supernatant was detected by ELISA.We collected cells of each group.Western blotting was used to detect nucleotide-binding oligomerized domain-like receptor 3(NLRP3),apoptosis-associated speck-like protein(ASC),Cleaved cysteine proteinase-1(Cleaved-Caspase-1),and Cleaved focal porin D(Cleaved-GSDMD)protein expression.CCK-8 assay was used to detect cell viability and TUNEL assay was used to detect the apoptosis rate.Results Exosomes from BMSCs were identified and obtained successfully.In hypoxic cells in vitro,the apoptosis rate,protein expression lev⁃els of NLRP3,ASC,Cleaved-Caspase-1,Cleaved-GSDMD,and levels of IL-1βand IL-18 in supernate of the SNHG12 exosome group were significantly lower than those of the NC exosome group(all P<0.05);the cell viability was significantly higher than that of the NC exosome group(P<0.05).After down-regulating the expression of miR-138-5p,SNHG12 in the exosomes of BMSCs significantly decreased the relative expression levels of NLRP3,ASC,Cleaved-Caspase-1,Cleaved-GSDMD(all P<0.05);the cell viability significantly increased and apoptosis rate significantly decreased(all P<0.05).After down-regulated SIRT1 expression,SNHG12 in exosomes from BMSCs significantly increased the relative protein expression levels of NLRP3,ASC,Cleaved-Caspase-1,and Cleaved-GSDMD(all P<0.05),significantly decreased the cell viability,and significantly increased the apoptosis rate(all P<0.05).Conclusions LncRNA SNHG12 in BMSCs-derived exosomes may inhibit the inflammatory response,pyroptosis and apoptosis of cardiomyocytes by regulating the miR-138-5p/SIRT1 axis,thus playing a protective role in cardiomyocytes.