结直肠癌中TOP2A与ZNF148的竞争性内源性RNA调控机制和功能研究

Competing endogenous RNA（ceRNA）regulation mechanism, in which different mRNA molecules can regulate each other by competitive binding to identical microRNA(miRNA) molecules, is a totally new regulation mechanism. Our previous studies have demonstrated that Topoisomerase II alpha (TOP2A) is overexpressed in colorectal cancer and exhibits tumor suppressing activity, but the underlying mechanism is still unknown. ZNF148 is a well-known tumor suppressor gene, and the mechanism of tumor suppressing is well studied. Bioinformatics analysis showes that both TOP2A mRNA and ZNF148 mRNA could bind to 13 identical miRNA molecules. Among them, 7 miRNAs (including miR-101) have been reported to be detected in colorectal cancer. So, it is probable that TOP2A exhibits tumor suppressing activity by regulating the expression of ZNF148 through the ceRNA regulation mechanism. Moreover, we had proved the role of miR-101 in the ceRNA regulation between TOP2A and ZNF148. This study aims to further elucidate the role of miR-101 and 6 other miRNA molecules in the ceRNA regulation mechanism in a larger sample, and then to clarify the similarities and differences of the 7 miRNAs in the ceRNA regulation; and to verify whether the regulation is reciprocal; and to clarify the alteration of signal transduction pathways and biological effects by in vitro and in vivo studies; and finally to further elucidate the ceRNA regulation mechanism between TOP2A and ZNF148.

竞争性内源性RNA（ceRNA）调控是一种全新的调控机制，即不同基因的mRNA可以通过竞争性结合相同的microRNA（miRNA）而相互调控。我们前期的研究表明TOP2A在结直肠癌中表达增高，但是却发挥抑癌作用，其发挥抑癌作用的机制还不清楚。ZNF148是一种公认的抑癌基因，而且抑癌机制比较明确。生物信息学预测显示：TOP2A和ZNF148可与13个相同的miRNA结合，其中7个（含miR-101）已有报道在结直肠癌中表达。所以，TOP2A可能通过ceRNA机制调控ZNF148的表达，进而发挥抑癌作用。我们已经证实miR-101在ceRNA调控中的作用，本项目拟扩大样本量进一步研究miR-101和其它6个miRNA在ceRNA调控中的作用，并比较分析它们的作用异同；然后研究反向调控是否存在；接着在体内外研究信号通路和功能的变化情况；进一步完善TOP2A与ZNF148的ceRNA调控机制。

竞争性内源性RNA（ceRNA）调控是一种全新的调控机制，即不同基因的mRNA可以通过竞争性结合相同的microRNA（miRNA）而相互调控。本项目在前期研究工作的基础上，进一步研究了结直肠癌中TOP2A 和ZNF148 的ceRNA 调控机制及其对信号通路和功能的影响。我们首先检测了ZNF148在结直肠癌不同发展阶段中的表达情况，发现ZNF148的表达水平与淋巴结转移、TNM分期、分化程度、局部复发和生存时间密切相关。成功构建稳定表达ZNF148 siRNA 的慢病毒表达载体。 ZNF148下调之后，RKO细胞的细胞增殖能力和克隆形成能力显著下降。在结直肠癌标本中，采用Envision免疫组化法检测 TOP2A 和ZNF148 的蛋白表达水平，发现两者呈显著的正相关(rs = 0.431, P<0.001)。采用了RT-PCR法检测了53例新鲜冻存的结直肠癌组织中TOP2A mRNA和ZNF148mRNA的表达情况，发现两者呈显著正相关(r=0.591, P<0.001)。采用茎环 RT-PCR 法检测miR101、miR144、miR335和miR365 在结直肠癌不同发展阶段中的表达情况。相关性分析显示，ZNF148 mRNA 的水平与miR101 (r=-0.237, P=0.126)、miR144 (r=-0.326, P=0.017)、 miR335 (r=-0.406, P=0.003)和miR365(r=-0.350, P=0.010)呈负相关。TOP2A mRNA 的水平与miR101 (r=-0.119, P=0.448)、miR144(r=-0.202, P=0.147、 miR335 (r=-0.343, P=0.012)和miR 365(r=-0.338, P=0.013)呈负相关。采用锁定核苷酸原位杂交技术(LNA-ISH)检测miR101在组织芯片中的表达情况。发现miR101高表达者淋巴结转移率低（P=0.001）、远处转移率低（P<0.001）、TNM分期低（P<0.001）、分化程度高（P<0.001）、复发率低（P<0.001）。发现miR101的表达水平与CRC患者的总生存时间（OS，P<0.001）和无瘤生存时间（DFS，P<0.001）呈正相关。