假基因PTTG3P促进原发性肝细胞癌生长的机制及其临床诊断价值研究

Pseudogenes have been reported to play critical roles in the progression of various diseases through regulating their parental genes. However, the role and mechanism of pituitary tumor-transforming 3 pseudogene (PTTG3P) in hepatocellular carcinoma (HCC) remains largely unknown. We previously found that PTTG3P and its parental gene pituitary tumor-transforming 1 (PTTG1) were frequently up-regulated in HCC through microarray analysis, quantitative real-time polymerase chain reaction, in situ hybrization and western blot. Over-expression of PTTG3P elevated the level of PTTG1 and promoted HCC cell growth. Moreover, bioinformatic analysis revealed the binding sites of miR-186 and miR-655 in both PTTG3P and PTTG1 mRNA 3’UTR. Furthermore, the serum level of PTTG3P was higher in patients with HCC than healthy controls. Thus, we hypothesized that pseudogene PTTG3P could function as a competing endogenous RNA and up-regulate PTTG1 expression by competing for microRNA (miR-186 and miR-655), thereby promoting HCC tumor growth. And serum PTTG3P could help to diagnose HCC. To prove this hypothesis, we intend to investigate the role of PTTG3P on tumor growth, clarify the relationship and regulatory mechanism between PTTG3P and PTTG1, and illustrate the diagnostic value of serum PTTG3P for HCC through molecular, cellular and animal experiments and serum biomarker screening. Through our study, we hope to provide novel potential biomarkers for HCC diagnosis, and also lay the foundation for developing new therapeutic targets for HCC.

假基因可调控其亲本基因的表达，参与肝癌等众多疾病的发病，但假基因PTTG3P能否参与原发性肝细胞癌的发生尚不清楚。我们前期通过基因芯片等技术发现假基因PTTG3P及其亲本基因PTTG1在肝癌组织中表达均上调；过表达PTTG3P可上调PTTG1，促进肝癌细胞生长；PTTG3P和PTTG1均含有miR-186和miR-655的结合位点；肝癌患者血清PTTG3P水平有升高趋势。由此，我们提出假说：PTTG3P可作为竞争性内源RNA，与PTTG1mRNA竞争结合microRNA，保护PTTG1的表达水平，促进肝癌细胞生长，并且血清PTTG3P可协助诊断肝癌。本项目拟通过分子、细胞和动物实验、血清标志物筛选等方法，明确PTTG3P在肝癌生长中的作用，阐明PTTG3P与PTTG1的调控机制，探讨血清PTTG3P对肝癌的临床诊断价值，为原发性肝癌的诊断提供新的指标，亦为发展肝癌新的治疗靶标奠定基础。

长链非编码RNA（lncRNA）被报道与肝细胞癌（HCC）的发生发展密切相关，但是lncRNA PTTG3P在HCC进展中的作用和机制尚不明确。此外，研究已发现lncRNA可稳定存在于外周循环中，可作为HCC的诊断标志物。但是血清lncRNA PTTG3P能否协助诊断HCC有待进一步明确。.我们通过实时荧光定量PCR（qPCR）和原位杂交技术发现PTTG3P在HCC癌组织中高表达，PTTG3P表达量与患者的肿瘤大小和TNM分期正相关，生存分析结果显示PTTG3P高表达是HCC患者预后不良的指标，且PTTG3P表达量及肿瘤大小和TNM分期是HCC患者总生存率的独立影响因素。随后，利用慢病毒构建稳定过表达PTTG3P和稳定干扰PTTG3P的肝癌细胞系，通过CCK-8实验、平板克隆形成实验、EDU掺入实验和流式细胞术检测细胞周期和细胞凋亡，发现PTTG3P可促进肝癌细胞的生长，抑制肝癌细胞凋亡。Transwell小室迁移实验和Boyden小室侵袭实验结果表明PTTG3P显著促进肝癌细胞的迁移侵袭。裸鼠皮下成瘤实验和裸鼠脾注射肝定植模型结果也证实PTTG3P能够在体内促进肝癌细胞的生长和侵袭转移。在机制研究层面，通过Western blot和qPCR发现，PTTG3P可上调PTTG1，激活PI3K/AKT信号通路，影响其下游信号分子的表达，从而促进肝癌细胞的生长和转移。.在临床诊断价值研究方面，收集298位参与者（129例HCC患者、27例慢性乙型肝炎（CHB）患者、49例肝硬化患者和93例健康对照）的血清，通过qPCR技术检测血清PTTG3P的表达水平，结果显示HCC患者血清PTTG3P含量明显高于CHB患者、肝硬化患者和健康对照；ROC曲线分析结果显示，相对于健康对照，血清PTTG3P诊断HCC的AUC为0.785。相对于CHB和肝硬化患者，血清PTTG3P诊断HCC的AUC为0.768，说明血清PTTG3P对HCC具有一定的辅助诊断价值。.综上，我们的研究阐明了PTTG3P在肝癌生长和转移中的作用和具体分子机制，探讨了PTTG3P对HCC 的诊断和预后评估价值，为HCC的诊断和预后评估提供了新指标，亦为发展HCC新的治疗靶标奠定基础，具有重要的理论和实际意义。