PIWIL2基因异化激活蛋白PL2L60在肿瘤干细胞启动中的作用与机理研究

The activation and mutation of oncogenes change normal cells into cancer cells. Cancer genes can be categorized into cancer-causing genes and cancer-contributing genes. Piwil2 is a new cancer gene which was discovered in recent years. Our previous results showed that Piwil2 is a tumor-barrier gene. However, PL2L60, a product of alienation-activated Piwil2, promotes tumor growth. After the exon 3 of Piwil2 was knocked out at the 5' end, PL2L60 was activated and induced mice fibroblasts to reprogram into cancer stem cells. These results indicate that PL2L60 can contribute to tumorigenesis. Therefore, we assume that PL2L60 may play important roles in cancer stem cells development. In carcinogenic environmental, Piwil2 might be alienation-activated to product PL2L60. PL2L60 might interfere with the chromatin remodeling and induce cells to reprogram into cancer stem cells. Thus, it is essential to conduct this important project with the following aims: ⅰ) to clarify the mechanism of Piwil2’s alienation-activation in the carcinogenic environment, ⅱ) to explain effects of PL2L60 on the stability of the genome, ⅲ) to reveal the molecular mechanism of PL2L60 in cell transformation and reprogramming. The project is expected to clarify the regulatory mechanism of Piwil2 in tumor development and could provide novel theoretical basis for the development of effective cancer prevention strategies.

肿瘤细胞是由正常细胞因癌基因激活、突变而来。Piwil2是近年发现的一个新型癌基因。我们团队前期研究表明，Piwil2是一个屏障基因，它的异化激活产物PL2L60可以促进肿瘤生长。小鼠成纤维细胞在5′端敲除Piwil2的外显子3后，Piwil2异化激活产生PL2L60，促使正常细胞转化为肿瘤干细胞，提示PL2L60具有启动肿瘤发生的功能。因此，我们假设PL2L60可能在肿瘤干细胞启动中起重要作用：在致癌环境中异化激活蛋白PL2L60，干扰染色质重塑，诱导细胞转化、重编程为肿瘤干细胞。为此，本项目将解决如下关键科学问题：1）Piwil2在致癌环境中被异化激活的机制；2）Piwil 2异化激活产物PL2L60对基因组稳定性的影响；3）PL2L60诱导细胞转化、重编程的分子机制。本研究将阐明PL2L60在肿瘤发生中的关键作用，为发展有效的肿瘤防治策略提供新的理论基础。

本课题的研究目标是确定PL2L60在肿瘤干细胞启动中的重要作用：在致癌环境中异化激活蛋白PL2L60诱导细胞转化、重编程为肿瘤干细胞。主要有以下发现：.（1）克隆Piwil2基因内PL2l60的特异性启动子.前期研究发现，Piwil2主要在凋亡细胞以及原位癌中表达。PL2L60是Piwil2的变异体，在多种肿瘤细胞中表达，促进肿瘤生长。利用Gene2Promoter软件预测，PL2L60启动子位于Piwil2基因的第十个内含子和第十一个外显子之间，可翻译分子量约为60 kDa的蛋白。我们克隆了Piwil2基因内PL2L60特异性启动子，进一步阐明了PL2L60的表达机制。.（2）PL2L60蛋白可以成为一个癌干细胞标记物.我们选择了两个干细胞标记物CD44和EpCAM，分别和PL2L60抗体共染，结果表明在PL2L60蛋白过表达的癌细胞中CD44+KAO3+和EpCAM+KAO3+双阳性细胞群的比例明显升高，这一点充分证明PL2L60蛋白可以作为一个候选的肿瘤干细胞标记物。.（3）PL2L60蛋白对癌细胞功能的影响.PL2L60蛋白在癌细胞中的表达高低直接影响肿瘤细胞的功能。高表达PL2L60蛋白的癌细胞的增殖能力和致瘤能力较强，干扰掉PL2L60的表达后，癌细胞增殖被抑制，周期发生阻滞，体内外的致瘤能力减弱。癌细胞恶性显著减弱，进一步证明PL2L60蛋白在肿瘤的发生发展中发挥着重要的作用。.通过本课题的研究，进一步加深对肿瘤发生发展机制的认识，特别是对PIWIL2在肿瘤发生发展中的认识，有助于发展新型肿瘤防治方略。