hPOT1分子调控端粒长度影响肝癌细胞恶性生物学行为的分子作用机制

Telomere can protect chromosome stability from nucleolytic degradation, chromosome ends infusion, and chromosome aberrant recombination. Telomeres shortening is a major mechanism leading to genetic instability that accompany the carcinogenesis and progression of tumors. The human protection of telomeres 1 (hPOT1) protein, a single-strand telomeric DNA binding protein, plays an important role in telomere length (TL) regulation and chromosome stability. In our previous study, we have reported that leukocyte TL may serve as an independent prognostic marker for hepatocellular carcinoma (HCC) patients treated with transarterial chemoembolisation.The results have been published on Feb. 8, 2012 in Carcinogenesis. Moreover, results of our preliminary experiments showed that hPOT1 protein in HCC tissues was significantly higher than in adjacent noncancerous tissues.Further analysis showed a significant positive correlation between hPOT1 and TL(r = 0.840, P＜0.001). However, the role of hPOT1 in malignant behavior of HCC cells by regulating TL remains unclear. In this study, through transfection of pcDNA3.0-hPOT1 and/or hPOT1-shRNA into HCC cell lines, a stable expression of hPOT1 / gene silencing cell model will be constructed to confirm our hypothesis that hPOT1 can regulate the TL, then affect the malignant behavior of cancer cell (proliferation, migration, adhesion, invasion, apoptosis and et al.), and in vivo assay will be further used to verify this process. Furthermore, we will evaluate the relationship between hPOT1 and TL and clinical pathological characteristics of HCC(tissue type, depth of invasion, lymph node metastasis,tumour size, TNM stage, recurrence and et al.), and further investigate whether hPOT1 is associated with the prognosis of HCC patients. These findings across this study can help to elucidate that hPOT1 may play an key role in the development and progression of HCC through regulation of TL and may be a useful prognostic marker for HCC patients, and may have wide implications for prevention and treatment of HCC in the future.

端粒长度(TL)异常所致的基因组不稳定参与了肿瘤的发生及进展。人端粒保护蛋白1(hPOT1) 是新发现的一种端粒单链DNA结合蛋白，其在TL调控及染色体稳定中起重要作用。我们前期研究发现外周血TL是影响肝癌患者预后的独立因子(Carciongenesis杂志)。预实验表明肝癌组织中hPOT1的表达显著高于癌旁，并与TL呈正相关。为进一步阐明hPOT1是否通过调控TL影响肝癌细胞的恶性生物学行为，是否与肝癌患者的临床病理学特征及预后相关，其分子机制是什么？本研究首先构建hPOT1稳定过表达/基因沉默的肝癌细胞株，试图证实通过调节hPOT1的表达，能够改变TL，继而影响肝癌细胞的增殖及侵袭等恶性生物学行为；并经荷瘤裸鼠模型加以验证；在肝癌组织标本中研究hPOT1与TL、患者临床病理学特征及预后的关系。通过上述探索性研究，将有助于阐明hPOT1在肝癌发生进展中的作用,并为肝癌的防治提供分子靶标。

端粒长度(TL)异常所致的基因组不稳定参与了肝癌的发生及进展，我们研究表明TL是肝癌患者预后的独立因子。人端粒保护蛋白1(hPOT1) 是新发现的一种端粒单链DNA结合蛋白，其在TL调控及染色体稳定中起重要作用；然而其在肝癌中的作用以及临床意义尚不清楚。. 本研究的主要研究内容（1）首先构建hPOT1稳定过表达/基因沉默的肝癌细胞株，试图通过调节hPOT1的表达，证实hPOT1是否调控TL，继而影响肝癌细胞的增殖及侵袭转移等恶性生物学行为；并经荷瘤裸鼠模型加以验证；（2）在肝癌组织标本中研究hPOT1与TL、患者临床病理学特征及预后的关系。. 第一部分结果（1）首先建立了TL的实时定量PCR检测方法，并检测了8株肝癌细胞的TL，Pearson相关性分析表明 hPOT1与TL呈正相关（P<0.05），提示hPOT1可能是调控TL的关键因子；（2）hPOT1显著抑制肝癌细胞的生长增殖，但不影响其凋亡；（3）hPOT1通过调控EMT抑制肝癌细胞的侵袭转移；（4）初步证实了hPOT1参与了肿瘤干细胞特性的维持；（5）EPA能显著上调hPOT1的表达，抑制肝癌细胞的侵袭转移。. 第二部分结果（1）与癌旁组织相比较，hPOT1在癌组织中的表达显著降低（n=66），其表达水平与TL呈正相关关系（P<0.05）；（2）hPOT1与肿瘤大小、TNM分级显著相关。. 总之，我们初步证实了hPOT1在肝癌中显著低表达，其通过调控EMT抑制肝癌细胞的侵袭转移，本课题的研究将有助于阐明肝癌的发生机制并为建立新的肝癌防治策略奠定基础。