Silencing LncRNA 93358 Inhibits The Apoptosis Of Myocardial Cells In Myocardial Infarction Rats By Inducing The Expression Of SLC8A1

Background and objectiveMyocardial infarction(MI)is a fatal cardiovascular disease with millions of death cases annually all over the world,the morbidity and mortality of which remains high recently years.It is reported that physiological and pathological processes,such as inflammatory reactions,cell apoptosis,fibrosis,play important roles in impacting the prognosis and survival rate of MI patients.Long non-coding RNAs have no coding function and greater than 200 nucleotides in length,and play a critical role in multiple cellular progressions,such as cell proliferation,differentiation,and apoptosis.Studies have found that in the heart,LncRNAs participate in the processes of cardiac development differentiation,myocardial infarction and postinfarction myocardial fibrosis,cardiac remodeling,and heart failure through regulating transcription,post-transcriptional gene regulation,competitive endogenous RNA,and protein translation.However,not sufficient importance has been attached on the function of LncRNA in the regulation of myocardial apoptosis post MI.The present study will investigate the differentially expressed LncRNAs in MI through the high-throughput sequencing assay and explore the potential function of differentially expressed LncRNAs utilizing the methods of bioinformatics,co-expression network analysis,and fluorescence quantitative PCR(RT-PCR).Materials and methods1.The rat acute myocardial infarction(AMI)model was established by ligating the left anterior descending coronary artery.LncRNA 93358 was screened out using a high-throughput sequencing assay which was confirmed by RT-qPCR.The samples were myocardial tissue of AMI rats and sham-operated control groups.2.LncRNA 93358,one differentially expressed lncRNA,which was selected by high-throughput RNA sequencing in myocardial tissue of AMI rats.MiR-34a-3p,miR-125b-2-3p,and miR-466c-3p were predicted to interact with LncRNA 93358 by miRDB network.We further predicted the potential myocardial diseases related genes that interacted with these miRNAs,which were SLC8A1 and TRPS1.MS2-RIP experiments were used to verify an interaction relationship with miRNAs and LncRNA93358,and dual-luciferase experiments were used to verify an interaction relationship with miRNAs and target genes.3.At animals experiment level,to explore the possible molecular mechanisms for LncRNA93358 promoting the apoptosis of myocardial cells in AMI rats.Heart structure and function were measured by cardiac ultrasound,the degree of cardiac infarction was determined by TTC staining,cardiomyocyte pathological state was confirmed by HE staining and cell apoptosis was measured by TUNEL assay.LncRNA93358 knockdown lentivirus was packaged.The expression levels of Bax,Bcl-2,and SLC8A1 were determined using RT-PCR and Western blotting.Results1.A total of 241-differentially expressed lncRNAs were observed in myocardial tissue of AMI rats(|log2 Foldchange|>1 and P<0.05),of which 111 lncRNAs expressions were upregulated and 130 RNAs were downregulated,according to the results of high-throughput sequencing assay.We selected LncRNA 93358,a apoptosis-related upregulated lncRNA,which was abundant in AMI rats with seven-fold higher expression than in sham-operated control rats.Further more,the expression of LncRNA93358 in infarcted myocardial tissue of AMI group was higer than control group by using RT-PCR.2.After MS2-RIP assay,genes expression level were conducted by RT-PCR.The results indicated that LncRNA 93358 was found significantly upregulated in cells after transfected with LncRNA 93358-12*MS2.In addition,according to the results of RIP assay,the amplification efficiency of miR-466c-3p and miR-1306-5p were relatively high,and the expression level of miR-466c-3p and miR-125b-2-3p in the LncRNA 93358-12*MS2 group were significantly higher than in the MS2 group.It indicated that LncRNA 93358 might interact with these two miRNAs.In addition,the expression level of SLC8A1 was also detected.We suspected that miR-466c-3p and miR-125b-2-3p might be interactions with LncRNA 93358,and also these two miRNAs might be interactions with SLC8A1.Results of dual luciferase assay,compared to the WT-SLC8al and WT-SLC8a1+mimic NC groups,the fluorescence value in the WT-SLC8a1+miR-466c group declined significantly.After mutation induced on the binding site,there were no significant difference in the fluorescence value compared to WT-SLC8a1 and WT-SLC8a1+mimic NC groups.These data indicated that miR-466c interacted with SLC8a1,and miR-466c-3p could target with SLC8a1.3.Rats echocardiographic results showed that compared to the Model and NC groups,the ⅣSs were significantly increasing in shRNA group.Compared to NC groups,the LVIDs significantly reduced in the shRNA group.Compared to Model and NC group,LVPWd significantly decreased in shRNA group,while LVPWd was significantly increasing in model group compared with control group.Left ventricular EF and FS were significantly decreased in model group compared to control group,while left ventricular EF and FS were significantly higher in shRNA group compared with Model and NC group.HR and CO were no significant difference among the groups.Massive myocardial necrosis was observed in model rats according to the results of TTC staining,HE staining,and TUNEL assay.However,myocardial necrosis declined significantly in the LncRNA 93358-shRNA group.The expressions of LncRNA 93358 and Bax were significantly upregulated and the experssion of Bcl-2 was greatly downregulated in model group.While the resluts were dramatically reversed by the knockdown of LncRNA 93358,accompanied by the declined area of myocardial necrosis and decreased apoptotic myocardial cells.ConclusionSilencing LncRNA 93358 inhibits the apoptosis of myocardial cells in myocardial infarction rats by inducing the expression of SLC8A1.