Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK

Purpose: Our laboratory has recently reported novel tumor-suppressive roles of PANX1 in both embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma, but the underlying molecular mechanism remains to be elucidated. Methods: PANX1-induced differential gene expression profile in alveolar rhabdomyosarcoma (RMS) cell line, Rh30, was generated by deep sequencing using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed by BOWTIE/TOPHAT pipeline with accessary programs SAMTOOLS (v1.0) and CUTADAPT (v1.7.1) followed by Cufflinks (v2.2.1). The protein expression of the differentially regulated genes was validated by Western blotting. Results: Using RNA-seq and DAVID analysis, we showed that the Gene Ontology (GO) terms such as apoptosis and migration, and KEGG pathways such as MAPK and Rap1 Signaling pathways were amongst the most highly enriched processes in Rh30 (aRMS) cells with ectopic PANX1 expression. We found that both GJA1 and its encoded protein connexin 43 (Cx43) were significantly increased in PANX1 expressing Rh30 (aRMS) cells. Conclusion: PANX1 transcriptome and proteome databases hold great potential for discovery of novel PANX1 tumor-suppressive mechanisms and therapeutic targets for RMS. PANX1-induced differential gene expression profile in alveolar rhabdomyosarcoma (RMS) cell line, Rh30, was generated by deep sequencing using Illumina HiSeq2000.

目的：本实验室近期报道了PANX1在胚胎型横纹肌肉瘤（embryonal rhabdomyosarcoma, eRMS）与腺泡型横纹肌肉瘤（alveolar rhabdomyosarcoma, aRMS）中发挥的全新抑癌作用，但具体分子机制仍有待阐明。
方法：本研究通过Illumina HiSeq2000进行深度测序，构建了过表达PANX1的腺泡型横纹肌肉瘤（rhabdomyosarcoma, RMS）细胞系Rh30中PANX1诱导的差异基因表达谱。对通过质量过滤的序列读数，采用BOWTIE/TOPHAT分析流程结合辅助工具SAMTOOLS（v1.0）与CUTADAPT（v1.7.1）进行处理，后续再使用Cufflinks（v2.2.1）完成后续分析。差异调控基因的蛋白表达水平通过蛋白质印迹法（Western blotting）进行验证。
结果：通过RNA测序（RNA-seq）与DAVID分析，我们发现，在异位表达PANX1的Rh30（aRMS）细胞中，凋亡、迁移等基因本体论（Gene Ontology, GO）条目，以及丝裂原活化蛋白激酶（MAPK）、Rap1信号通路等京都基因与基因组百科全书（KEGG）通路均为显著富集的生物学过程。我们观察到，GJA1及其编码的连接蛋白43（connexin 43, Cx43）在表达PANX1的Rh30（aRMS）细胞中均显著上调。
结论：PANX1转录组与蛋白质组数据库为挖掘PANX1全新的抑癌分子机制以及横纹肌肉瘤的治疗靶点提供了重要潜力。本研究中，过表达PANX1的腺泡型横纹肌肉瘤（rhabdomyosarcoma, RMS）细胞系Rh30的差异基因表达谱，同样通过Illumina HiSeq2000深度测序构建完成。