ZNF326通过BMI-1增强胶质瘤细胞恶性表型的机制研究

The molecular mechanism, expression and clinical pathological significance of Zinc finger protein ZNF326 in human tumor is unclear. We found that ZNF326 was highly expressed in glioma and correlated with its grade. Up- or down-regulating ZNF326 could respectively increase or decrease the proliferation and the related proteins expression. ZNF326 could interact with BMI-1, whereas, the proliferation of glioma enhanced by ZNF326 was abolished when we interfered BMI-1 by siRNA. Therefore, we propose that ZNF326 could form transcriptional complex by interacting with BMI-1, and bound with promote regions of P16 and NF-κB respectively via the ring finger domain of BMI-1 (or via the zinc finger domain of ZNF326), to enhance proliferation and invasion of glioma cells by repressing the transcription of P16 and activating NF-κB. In this research, to investigate the mechanism in which ZNF326 promotes the proliferation and invasion of glioma both in vivo and in vitro, we will up- and down-regulate the expression of ZNF326 and BMI-1 to construct the mutant plasmids in which ZNF326 could not bind with BMI-1, as well as the ones that both ZNF326 and BMI-1 could not bind with the promoter of P16 and NF-κB respectively.

锌指蛋白ZNF326在肿瘤组织中的表达、意义和作用机制尚无报道。我们发现ZNF326在胶质瘤中高表达且随着分级增加而增高，双向调控其表达可增强或下调其增殖能力和相关蛋白的表达，ZNF326与BMI-1存在相互作用，敲除BMI-1可消除ZNF326促进增殖的功能。结合文献我们提出ZNF326与BMI-1结合形成转录复合体，通过BMI-1的环指结构（或同时通过自身的锌指结构）分别与P16和NF-κB的启动子区结合，抑制P16转录活性和激活NF-κB，促进胶质瘤细胞的增殖和侵袭能力。本研究拟采用双向调控ZNF326和BMI-1，构建ZNF326与BMI-1不能结合的突变质粒，同时分别构建二者不能与P16和NF-κB启动子区结合的突变质粒等策略，揭示ZNF326促进胶质瘤增殖和侵袭的分子机制，并结合体外细胞功能试验和裸鼠移植试验进行验证。

ZNF326在胶质瘤中高表达，并与肿瘤的高分级密切相关；体内外实验均证明ZNF326可以促进胶质瘤细胞的增殖和侵袭能力。我们利用ChIP-seq和ChIP-qPCR的方法，筛选并证明了ZNF326作为起动因子上调HDAC7转录活性（Wu J, Zhang X, Han Q, Han X, Rong X, Wang M, et al. ZNF326 promotes proliferation of non-small cell lung cancer cells by regulating ERCC1 expression. Lab Invest. 2018. https://doi.org/10.1038/s41374-018-0148-y.）。在胶质瘤细胞中转染ZNF326的同时干扰HDAC7的表达可以消减ZNF326的促癌作用。我们通过构建ZNF326分子的不同剪切体，证明了ZNF326是通过自身的激活区域和锌指结构与HDAC7启动子区（-1552—-1301和-1073—-788）结合并启动HDAC7的转录活性。而转染不能够与HDAC7启动子区结合的ZNF326突变体则没有此作用。转染ZNF326后在上调HDAC7转录的同时伴随Wnt信号通路的激活。HDAC7与β-catenin相互作用并降低β-catenin第49赖氨酸位点的乙酰化水平，导致β-catenin第45丝氨酸位点磷酸化水平降低，促进β-catenin蓄积和入核，激活Wnt信号通路活性。而核内的β-catenin 与“扮演”共转录激活因子角色的ZNF326结合，又促进了β-catenin与TCF4的结合，促进了Wnt靶基因的转录。这样，ZNF326-HDAC7-β-catenin三者形成了激活和加强Wnt通路活性的体系，从而促进胶质瘤细胞的恶性表型。这些研究结果不仅揭示了ZNF326在胶质瘤的发生和发展中的作用和机制，也为开发新的靶向药物和解释肿瘤耐药机制等提供了重要的参考。