MEIS2通过NF-κB/UXT/p65复合体调节H2AFJ转录参与直肠癌放化疗抵抗的作用和机制研究

The individual treatment responses to neoadjuvant chemoradiotherapy (CRT) for locally advanced rectal cancer vary widely, and approximately 15% to 45% of rectal cancers remain resistant. We previously found that MEIS2 was down-regulated and hypermethylated in CRT-resistant tissues through multi-omic analysis and immunohistochemical analysis. Furthermore, the MEIS2 expression was increased after the administration of 5-aza-2-deoxycytidine (DAC) in vitro as a demethylating agent. By pretreating with DAC at a non-toxic level, we found the restoration of oxaliplatin chemosensitivity. In addition, our previous research indicated that the upregulation of another gene, H2AFJ, could cause radioresistance via MAPK7 pathway. However, the upstream regulation mechanism of H2AFJ expression remains unclear. Through correlation analysis and ChIP-Seq analysis, we hypothesized that MEIS2 may participate in the transcriptional regulation of H2AFJ by regulating the NF-κB/UXT/P65 complex. Thus, we intend to test this hypothesis by performing: (1) gain- and loss-of-function experiments and xenograft animal studies to demonstrate the role of MEIS2 in chemoradiotherapy resistant phenotype of colorectal cancer cells; (2) methylation-specific PCR to verify the upstream regulation mechanism of MEIS2; (3) cell counting kit-8 (CCK-8) assays after different treatment combination to confirm the CRT sensitization effect of DAC; (4) ChIP-PCR and luciferase reporter assays to explore the regulatory mechanism of MEIS2 on NF-kappa B/UXT, and NF-kappa B/P65 on H2AFJ. This study may provide a new potential target for reversing CRT resistance, and provide a theoretical basis for CRT sensitization of DAC for rectal cancer.

新辅助放化疗（CRT）对局部进展期直肠癌的疗效不一，课题组前期基于多组学技术和免疫组化发现MEIS2在CRT抵抗组下调，其启动子区高甲基化，且体外小剂量甲基化酶抑制剂（DAC）预处理肠癌细胞系可上调其表达，并对奥沙利铂起增敏作用；此外，课题组前期发现H2AFJ上调可通过MAPK7通路产生放疗抗性，但H2AFJ的上游调控机制不明。通过相关性分析、ChIP-Seq，我们提出假说：MEIS2可能通过调节NF-κB/UXT/p65复合体参与H2AFJ的转录调控。据此假说，本课题组拟：①沉默和过表达MEIS2，从细胞和动物水平验证其放疗抵抗和耐药表型；②上游：甲基化PCR验证MEIS2下调机制，探索DAC的CRT增敏作用；③下游：ChIP-PCR和Lucifrase报告基因检测MEIS2对UXT及p65对H2AFJ的调控机制。本研究将为逆转CRT抵抗寻找新靶点，为DAC的CRT增敏作用提供理论基础。

本研究：①对MEIS2在结直肠癌含奥沙利铂（L-OHP）放疗方案耐药中的机制进行研究，发现MEIS2表达与结直肠癌细胞的肿瘤干性和肿瘤干性标记物表达呈负相关；过表达MEIS2可抑制结直肠癌细胞增殖，并降低其肿瘤干性，具有抗肿瘤效果。此外，过表达MEIS2可通过提高其凋亡水平，逆转结直肠癌细胞株对L-OHP的耐药性，具有增敏作用；②下游：MEIS2通过Bcl-2/Bax/Caspase-3通路抑制结直肠癌细胞凋亡，并通过二代测序鉴定MEIS2下游作用通路；③上游：发现MEIS2在结直肠癌组织中启动子区为不完全甲基化，进一步按课题计划探索去甲基酶抑制剂对奥沙利铂的增敏作用时，意外发现低浓度地西他滨（DAC）对结肠癌细胞系HT29有促增殖效果，课题组进一步研究其促癌机制，发现HT29结肠癌细胞中BCL2启动子区的高甲基化本身是保护机制，起抑癌效果。低浓度DAC处理后，BCL2启动子区DNA甲基化程度降低，而其表达增加，从而阻断HT29细胞的内源性凋亡，反而起促癌效果。据此提出了结直肠癌耐药新机制，即“癌基因启动子区高甲基化保护”机制；④基于单细胞测序数据发现了耐药细胞在肿瘤进化树（早、中、晚期）中分布的时间不均一性和MAPK通路活性的空间不均一性，提出耐药的肿瘤生态学原理；⑤鉴定一系列结直肠癌预后预测和新辅助治疗疗效预测新型分子标记物。报道了SPINK4在结直肠癌中的抑癌基因角色，其下调通过改变细胞周期通路导致CRT抵抗；报道了NLGN1在结直肠癌的的癌基因角色，结直肠癌细胞可通过NLGN1相关配受体作用，促进其沿神经束侵袭。本项目共发表SCI论文15篇。

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