lncRNAENST-hnRNPU-GSDMD轴抑制细胞焦亡在乳腺癌化疗耐药中的作用及机制研究

Chemotherapeutic resistance is a difficult problem to be solved in the treatment of cancer and pyroptosis is a kind of inflammatory cell necrosis that has been put forward in recent years. LncRNA ENST00000508435 (ENST) that we have obtained through screening has a high expression level in breast cancer. Our results showed that the expression of ENST was increased significantly in doxorubicin resistant MCF-7 cells, and the expression of GSDMD, the key molecule that leads to pyroptosis, was decreased after overexpression of ENST was overexpressed in MCF-7. It is suggested that ENST plays an important role in chemotherapeutic drug resistance and is related to the inhibition of pyroptosis. Our early data demonstrated the combination of ENST and hnRNPU, and it was reported that hnRNPU can bind to β-TrCP to inhibit NF-κB or combine with PRC2 to regulate the genes related to pyroptosis by chromatin remodeling. We hypothize that ENST inhibits cell pyroptosis through the ENST-hnRNPU-GSDMD axis to promote chemotherapeutic resistance. The aim of this project is to clarify the role of ENST in chemoresistance through in vivo and in vitro experiments. The mechanisms for ENST promotinged breast cancer chemotherapy resistance may involve ENST mediated PRC2 recruitment through hnRNPU to inhibit TRAF-MALT1-caspase 1 or ENST mediated of NF-κB inhibition and down regulation of GSDMD, which results in inhibition of pyroptosis and inducing breast cancer chemotherapy resistance. This study can provide new ideas for the prevention and treatment of chemotherapeutic drug resistance.

化疗耐药是癌症治疗亟待解决的难题；焦亡是近年提出的一种炎性细胞坏死。ENST00000508435（简称ENST）是我们通过芯片筛选获得的在乳腺癌高表达的lncRNA。实验发现：阿霉素耐药MCF7细胞ENST表达显著升高，亲本MCF-7过表达ENST后焦亡关键分子GSDMD表达下降。提示：ENST在化疗耐药中发挥重要作用并与抑制焦亡相关。前期发现ENST与hnRNPU结合。而hnRNPU与β-TrCP结合抑制NF-κB或与PRC2结合通过染色质重塑调控焦亡基因。设想ENST通过ENST-hnRNPU-GSDMD轴抑制细胞焦亡促进化疗耐药。本项目拟通过体内外实验，明确ENST在化疗耐药中的作用，阐明其机制是ENST介导hnRNPU招募PRC2抑制TRAF-MALT1-caspase 1或抑制NF-κB进而下调GSDMD，抑制细胞焦亡，导致乳腺癌化疗耐药。本研究可为防治化疗耐药提供新思路。

乳腺癌严重危害女性生命健康。近年研究表明lncRNA在多种肿瘤中表达异常，且在肿瘤进展中发挥重要作用。本课题组通过芯片技术首次在乳腺癌组织中筛选出高表达的lncRNA ENST00000508435（ENST，STMN1P2），并对其进行了深入的研究工作。.第一部分 ENST特性及表达与临床乳腺癌化疗耐药的相关性.1、通过数据库、RACE技术及核质分离等实验进一步确认了ENST的属性、全长序列以及核定位。.2、验证实验表明ENST在乳腺癌组织及细胞表达明显升高，尤其是在临床乳腺癌化疗耐药癌组织和耐药细胞中上调更为显著；并与化疗后肿瘤大小和淋巴结转移呈正相关。.第二部分 ENST在乳腺癌化疗耐药及转移中的作用.1、细胞实验及动物模型表明ENST促进乳腺癌细胞的化疗耐药。.2、多项细胞实验显示：干扰ENST表达可抑制细胞迁移；过表达ENST细胞迁移明显增强。.第三部分 ENST抑制细胞焦亡在乳腺癌化疗耐药中的作用.1、.焦亡相关实验结果显示：化疗药阿霉素能诱导乳腺癌细胞发生NLRP3-ASC-Caspase 1-GSDMD通路的细胞焦亡。.2、.干扰ENST细胞焦亡水平提高；过表达ENST使阿霉素诱导的细胞焦亡被抑制，促进了乳腺癌化疗耐药。.第四部分 进一步探究ENST抑制细胞焦亡促进乳腺癌化疗耐药的作用机制.1、RNA pull down及质谱结果显示：ENST与hnRNPU、FXR1结合。.2、 ENST能与hnRNPU相互作用且调节hnRNPU的表达。挽救实验结果显示：干扰hnRNPU能逆转ENST抑制焦亡、介导耐药的作用；抑制hnRNPU/ FXR1可逆转ENST的促细胞迁移作用。.3、 Co-IP和Western Blot实验发现ENST可能介导hnRNPU与EZH2结合，通过EZH2-TRAF6-MALT1-Caspase 1信号通路抑制乳腺癌细胞焦亡、促进化疗耐药。. 结论：① 首次发现ENST在乳腺癌表达上调，尤以化疗耐药乳腺癌组织及细胞显著，并与化疗后肿瘤大小和淋巴结转移呈正相关；②ENST结合hnRNPU，通过招募EZH2，抑制TRAF6-MALT1-Caspase 1信号通路，抑制细胞焦亡进而促进乳腺癌化疗耐药。③ ENST通过调控hnRNPU和FXR1促进乳腺癌转移。该研究为防治乳腺癌化疗耐药提供了新的潜在靶点。