| Backgrounds:Doxorubicin(Dox)is a classic anthracycline antitumor drug,but its clinical application is limited due to its severe cardiotoxic side effects.Therefore,it is of great significance to elucidate the mechanism of doxorubicin-induced cardiotoxicity.Recent studies have found that non-codingRNAs(ncRNAs)such as miRNAs,lncRNAs and circRNAs are closely related to Dox-induced cardiotoxicity.The development of whole-transcriptome sequencing technology has enabled the identification of many ncRNAs,which has facilitated the study of the roles of ncRNAs in various diseases.At present,the role of ncRNAs in Dox-induced cardiotoxicity lacks systematic research,so it is important to analyze the role of ncRNAs in Dox-induced cardiotoxicity based on whole transcriptome sequencing technology.Objectives:Whole transcriptome sequencing technology was used to screen ncRNAs related to Dox-induced cardiotoxicity,and to construct differentially expressed lncRNA-miRNA-mRNA regulatory network.Further,to explore the role and mechanism of the screened lncRNA—lncRNA Gm16701 in Dox-induced cardiotoxicity.This study provides a theoretical basis for elucidating the specific molecular mechanism of cardiotoxicity induced by antitumor drugs.Methods:(1)C57BL/6 mice were given intraperitoneal injection of Dox to establish a cardiotoxicity model.The cardiac function,biochemical indexes and pathophysiological morphological changes of the mice were investigated.(2)We used whole transcriptome sequencing technology to screen the differentially expressed ncRNAs in the hearts of mice between the control group and the Dox group.Cytoscape software was used to construct a specific ceRNA regulatory network for the differentially expressed lncRNAs,miRNAs and mRNAs.The regulatory network was subjected to GO functional annotation and KEGG metabolic pathway enrichment analysis.q RT-PCR was used to verify the sequencing results and identify the target lncRNA—lncRNA Gm16701.(3)siRNA technology was used to silence the lncRNA Gm16701.The proliferation and viability of cardiomyocytes was detected by CCK-8,and the apoptosis of cardiomyocytes was detected by flow cytometry,hochest staining and western blot to investigate whether silencing lncRNA Gm16701 could alleviate Dox-induced cardiotoxicity.Results:(1)The left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were significantly decreased and serum myocardial enzyme levels were significantly increased in Dox-treated mice.In addition,myocardial tissue was pathologically changed,and myocardial fibers were disordered,some cardiomyocytes appeared hypertrophy;and cardiomyocyte apoptosis were increased.(2)The results of whole transcriptome sequencing showed that there were 217 differentially expressed lncRNAs(91 up-regulated,126 down-regulated),and 41 differentially expressed circRNAs(17up-regulated,24 down-regulated),11 differentially expressed miRNAs(2 up-regulated and 9 down-regulated),and 3633 differentially expressed mRNAs(1904 up-regulated and 1729down-regulated)between the control group and the Dox group.(3)Bioinformatics technology was used to screen lncRNAs and mRNAs that could bind to differentially expressed miRNAs,and then compared with the sequencing results to construct specific lncRNA-miRNA-mRNA ceRNA regulatory network related to Dox-induced cardiotoxicity.The network contains 7 lncRNAs,7miRNAs and 15 mRNAs.The GO functional analysis of the ceRNA network showed that it was mainly enriched in cell migration,intracellular protein modification process,intracellular components and protein binding.Comparative analysis of signaling pathways with KEGG database showed that ceRNA network was closely related to mitophagy,MAPK signaling pathway,cancer pathway,etc.(4)q RT-PCR validation results showed that the expression of lncRNA Gm16701 in the heart and cardiomyocytes of the Dox group was significantly higher than that of the control group,which was consistent with the sequencing results.Silencing lncRNA Gm16701 in HL-1 cardiomyocytes increased the cardiomyocyte viability,decreased the cardiomyocyte apoptosis,and alleviated doxorubicin-induced cardiotoxicity to some extent.Conclusions:The differential expression profile of ncRNAs related to doxorubicin-induced cardiotoxicity was established.The ceRNA regulatory network of specific lncRNA-miRNA-mRNA related to Dox-induced cardiotoxicity was constructed.It was identified that the expression of lncRNA Gm16701 was up-regulated in the Dox group,and silencing lncRNA Gm16701 on cells could alleviate doxorubicin-induced cardiomyocytes injury.Figure: 14,Table: 14,Reference: 92... |
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