HOXA10-ALKBH5基因环路通过mRNA m6A去甲基化促进卵巢子宫内膜样癌进展的机制研究

The mechanism of ovarian endometrioid cancer tumorigenesis is unknown. Our research group has confirmed that HOXA10 gene overexpression is the key to the ovarian endometrioid cancer tumorigenesis. We recently found HOXA10 mRNA m6A demethylation in the ovarian cancer, HOXA10 can directly induce RNA demethylase ALKBH5 high expression, and ALKBH5 can reversely promote the HOXA10 m6A demethylation, resulting in HOXA10 protein high expression, forming a gene regulatory loop. After raising expression of ALKBH5 in tumor cells, the cell proliferation activity was enhanced. The signal pathway chip revealed that the WNT signal pathway was activated, and the expression of WNT5a was increased. RNA methylation sequencing demonstrated that the WNT5a mRNA was demethylated and the expression of WNT5a protein was elevated. Therefore, we proposed the following hypothesis: the HOXA10 and ALKBH5 genes form a regulatory loop to maintain the high expression of both proteins, high expression level of ALKBH5 results in WNT5a protein expression increase through the m6A demethylation mechanism, then activates of WNT signaling pathway to promote ovarian endometrioid cancer progression. We intend to confirm this hypothesis from several experimental levels in order to provide a potential new target for the accurate treatment of ovarian cancer.

卵巢子宫内膜样癌发生机制不明。课题组已证实HOXA10基因高表达是卵巢子宫内膜样癌发生的关键。我们近期发现HOXA10 mRNA在卵巢癌中存在m6A去甲基化改变，HOXA10可直接诱导RNA去甲基化酶ALKBH5高表达，而ALKBH5可反向促进HOXA10发生m6A去甲基化改变，导致HOXA10蛋白高表达，形成基因调控环路。增强ALKBH5在肿瘤细胞中表达后，细胞增殖活力增强，信号通路芯片显示WNT信号通路激活，WNT5a表达升高；RNA甲基化测序证实WNT5a mRNA发生m6A去甲基化改变，WNT5a蛋白表达升高。因此，课题组提出如下假说：HOXA10与ALKBH5基因形成调控环路维持两者蛋白高表达；高水平的ALKBH5通过m6A去甲基化机制，导致WNT5a蛋白表达升高，激活WNT信号通路，促进卵巢子宫内膜样癌进展。课题组拟从多个实验层面证实此假说，以期为卵巢癌精准治疗提供潜在新靶标。

卵巢癌是死亡率第一的妇科恶性肿瘤，卵巢癌最主要的病理类型是卵巢上皮性癌，铂耐药是导致卵巢癌预后不良的重要原因，淋巴结转移是影响卵巢癌预后的关键因素，而铂耐药和淋巴结转移的发生机制目前未明。本研究从分子、细胞、组织、动物等多个水平，应用meRIP测序、mRNA测序、RIP-qPCR、MeRIP-qPCR、CO-IP、RNA pull-down、m6A dot blot、双荧光素酶报告基因实验及放线菌酮-D实验等方法，探究卵巢上皮性癌中ALKBH5的分子功能及调控机制。结果发现：（1）HOXA10基因与ALKBH5之间的环状调控关系在卵巢上皮性癌细胞增殖和铂耐药中起到了关键作用。其下游调控机制：ALKBH5 蛋白可靶向调控JAK2 mRNA m6A修饰，并通过抑制 m6A “Reader”蛋白YTHDF2介导的m6A-JAK2 mRNA 降解作用，促进 JAK2 表达。过表达ALKBH5-HOXA10 环路通过m6A依赖途径激活JAK2/STAT3信号通路，进而促进卵巢上皮性癌增殖及耐药。此发现为卵巢上皮性癌增殖和耐药的分子机制研究提供了新的线索，为肿瘤治疗提供了潜在的干预靶标。（2）卵巢上皮性癌中ALKBH5高表达促进肿瘤淋巴管生成和淋巴结转移，在卵巢上皮性癌细胞淋巴结转移中发挥了重要作用。其下游调控机制：ALKBH5 蛋白可靶向调控ITGB1 mRNA m6A修饰，并通过抑制m6A “Reader”蛋白YTHDF2介导的m6A-ITGB1 mRNA降解作用，维持ITGB1 mRNA稳定性及表达。ITGB1磷酸化激活下游FAK/Src通路，促进淋巴管生成和淋巴结转移。为卵巢上皮性癌淋巴结转移的分子机制研究提供了理论依据，为分子靶向药物的研发提供潜在新靶点。