Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity
Cardiac Expression of Esophageal Cancer-Related Gene-4 is Regulated by Sp1 and is a Potential Early Target of Doxorubicin-Induced Cardiotoxicity
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食管癌相关基因4的心脏表达受Sp1调控，是阿霉素诱导心脏毒性的潜在早期靶点
DOI：
10.1007/s12012-022-09722-0
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发表时间：
2022-02-07
影响因子：
3.2
通讯作者：
Dang, Xitong
中科院分区：
文献类型：
--
作者：
Long, Dandan;Chen, Chunyue;Dang, Xitong
食管癌相关基因4 （Ecrg4）在心肌细胞和心传导系统表达下调在心脏缺血和房颤。探讨Ecrg4是否在阿霉素（DOX）诱导的心脏毒性中起作用。采用大鼠和新生大鼠心肌细胞（NRCMs）研究DOX对Ecrg4转录的影响。采用生物信息学与启动子分析相结合的方法对大鼠Ecrg4启动子进行定位。ChIP法评估Sp1与Ecrg4启动子的结合。采用瞬时转染法研究Sp1对内源性Ecrg4表达的影响。DOX降低内源性Ecrg4基因在心脏和培养的nrcm中的表达。计算机分析表明，位于起始密码子ATG上游的5 ' UTR含有一个假定的富含gc的启动子，并包含CpG岛，多个重叠的sp1位点。转录主要在- 15的“C”上开始。序列5 '缺失结合双荧光素酶检测显示，大鼠Ecrg4核心启动子位于- 1/- 800。Sp1转激活了Ecrg4基因，该基因几乎被DOX消除。此外，ChIP实验显示特异性结合Ecrg4启动子的Sp1被DOX打断。最后，DOX抑制Sp1蛋白的表达，Sp1的恢复增加了Ecrg4的表达，对DOX诱导的Ecrg4下调具有抗性。重要的是，心肌细胞特异性的Ecrg4缺失显著富集了dox诱导的心脏毒性信号通路中的差异表达蛋白。我们的研究结果表明Sp1介导dox诱导的Ecrg4的抑制，这可能间接导致其心脏毒性。
Esophageal Cancer-Related Gene 4 (Ecrg4) expressed in cardiomyocytes and the cardiac conduction system is downregulated during cardiac ischemia and atrial fibrillation. To explore whether Ecrg4 plays any role in doxorubicin (DOX)-induced cardiotoxicity. Rats and neonatal rat cardiomyocytes (NRCMs) were employed to study the effect of DOX on Ecrg4 transcription. Bioinformatics combined with promoter analysis were used to map the rat Ecrg4 promoter. ChIP assay was used to evaluate the binding of Sp1 to the Ecrg4 promoter. Transient transfection was used to study the effect of Sp1 on the expression of endogenous Ecrg4. DOX decreased endogenous Ecrg4 gene expression in the heart and cultured NRCMs. In silico analysis showed that the 5 ' UTR immediately upstream of the start codon ATG, harbors a putative promoter that is GC-rich, and contains CpG islands, multiple overlapping Sp1sites. Transcription is initiated mainly on the 'C' at - 15. Serial 5 '-deletion combined with dual-luciferase assays showed that the rat Ecrg4 core promoter resides at - 1/- 800. Sp1 transactivated Ecrg4 gene, which was almost abolished by DOX. Furthermore, ChIP assay showed that Sp1 specifically bound to the Ecrg4 promoter was interrupted by DOX. Finally, DOX suppressed Sp1 protein expression, and restoration of Sp1 increased Ecrg4 expression that was resistant to DOX-induced Ecrg4 downregulation. Importantly, cardiomyocyte-specific loss of Ecrg4 significantly enriched the differentially expressed proteins in the signaling pathways commonly involved in DOX-induced cardiotoxicity. Our results indicate that Sp1 mediates DOX-induced suppression of Ecrg4, which may contribute indirectly to its cardiotoxicity.