PDSS2新的转录变异体PDSS2-D2促进肝癌细胞播散和干性增强及分子机制研究

In our previous study we found that PDSS2 (prenyl diphosphate synthase subunit 2) could regulate metabolism reprogramming in liver cancer cells. Knocking-down PDSS2 in an immortalized human liver cell line MIHA was able to induce malignant transformation. Our findings indicated that PDSS2 deficiency might be a novel driving factor in HCC (hepatocellular carcinoma ) development by reprograming glucose metabolism from mitochondrial respiration to aerobic glycolysis, and increasing chromosomal instability and finally transforms a normal cell into a malignant cell. RACE (rapid amplification of cDNA ends) results indicated that there are different transcript variants in the HCC cells. An unreported transcript variant, PDSS2-D2, caused our interest. We found that D2 increased in HCC tumor tissues compared with corresponding non-tumor tissues. D2 is devoid of the tumor-suppressive ability of PDSS2, increases motility and stemness of HCC cells. We also established xenograft spontaneous metastasis model. In this research, we will: 1) perform correlation analysis of D2 with clinical characteristics in HCC samples; 2) investigate whether D2 promotes HCC cells migration, invasion and stemness; 3) investigate the molecular mechanism of D2 promoting HCC cells motility and stemness; 4) explore the intrinsical mechanism of HCC metastasis by using the xenograft spontaneous metastasis model. This study is important for identifying new target molecules in liver cancer metastasis.

前期我们发现敲低PDSS2（异戊烯二磷酸合成酶亚基2）导致肝癌细胞发生代谢重编程以及染色体不稳定发生，最终导致肝永生化细胞MIHA发生恶性转化。RACE结果提示PDSS2在肝癌细胞中存在不同的转录变异体，其中，PDSS2-D2，一个未被报道的转录变异体引起了我们的兴趣。我们发现PDSS2-D2在肝癌组织里显著升高，且丧失了PDSS2（全长）抑制肝癌细胞增生的能力；并且使肝癌细胞转移能力显著增强，据此我们建立了皮下移植瘤自发转移模型；此外，过表达D2使肝癌细胞干性明显增强。本研究中将：1）进行D2在肝癌组织中临床意义分析； 2）研究D2促进肝癌细胞转移、侵袭以及增强干性；3）深入研究D2促进肝癌细胞转移、侵袭以及增强干性的分子机制；4）利用已建成的皮下移植瘤自发转移模型深入探讨肝癌转移机制。研究对发现新的肝癌转移靶标分子具有重要意义。

本课题按照研究计划执行。我们提取肝癌细胞的RNA，采取RACE方法并克隆测序，从测序结果得知PDSS2存在多个不同的转录变异体，其中的转录变异体PDSS2-Del2 (PDSS2-D2)引起了我们的兴趣。通过克隆测序分析，我们发现PDSS2-D2在多个肝癌细胞中可以检测到，荧光定量PCR法可以看到PDSS2-D2在肿瘤组织中显著升高。后续实验结果表明，在肝癌细胞里过表达PDSS2-D2不能增多CoQ10水平，并且PDSS2-D2丧失了PDSS2抑制肝癌细胞生长的能力，而且PDSS2-D2可以通过促进肝癌细胞发生EMT，从而使肝癌细胞获得了更强的移动能力，并且干性成球能力增强。进一步研究发现，PDSS2-D2能够活化NF-κB，并激活肝癌细胞的WNT/b-catenin通路，增加肿瘤细胞转移和血管生成。体外和体内实验表明过表达PDSS2-D2可显著提高细胞活动能力，促进肝癌细胞转移。