LncRNA-LINC00221/CPSF6激活内质网应激-自噬通路在肺腺癌化疗耐药中的作用及机制研究

Chemo-resistance is one of the major causes of the treatment failure in patients with lung adenocarcinoma (LUAD). The formation of chemo-resistant phenotypes is closely related to the epigenetic regulation of drug-resistant genes in tumors. Previous studies suggested that the activation of endoplasmic reticulum stress (ERs)-autophagy pathway involved in the chemo-resistance of LUAD cells. Moreover, we found that activation of ERs-related protein XBP1 mediated by LINC00221 was closely correlated with ERs-autophagy pathway activation. Mass spectrometry and RNA pull down assay revealed that CPSF6 involved in the mRNA selective polyadenylation could interact with LINC00221. And bioinformatics analysis indicates the presence of CPSF6 action site in XBP1 mRNA 3'UTR. Therefore, we propose LINC00221/CPSF6/XBP1 mechanism axis .Based on the results of our previous studies, we propose to carry out experiments such as co-IP, RNA-EMSA, to prove that LINC00221 recruited CPSF6 to regulate the alternative splicing of the XBP1 mRNA 3'UTR poly(A) sites, so as to activate ERs-autophagy pathway and aggravate the multidrug-resistant phenotype in LUAD cells. This discovery represents a further explanation on the mechanism of chemo-resistance in LUAD cells, providing new thoughts for clinical reversal of chemo-resistance in LUAD.

化疗耐药是导致人肺腺癌治疗失败的重要原因，其形成与肿瘤耐药相关基因的表观遗传调控密切相关。前期发现，内质网应激-自噬通路参与调控肺腺癌细胞化疗耐药，该通路的激活与LINC00221介导的内质网应激相关蛋白XBP1的活化密切相关。RNA pulldown及质谱鉴定发现LINC00221可与CPSF6（mRNA选择性多聚腺苷酸化相关蛋白）结合，生物信息学分析提示XBP1 mRNA 3'UTR存在CPSF6的作用位点。故提出LINC00221/CPSF6/XBP1机制轴设想。在此基础上，课题组拟采用免疫共沉淀、RNA-EMSA等实验技术验证LINC00221/CPSF6调控XBP1 mRNA 3'UTR poly(A)位点的选择性多聚腺苷酸化，揭示内质网应激-自噬通路参与调控肺腺癌化疗耐药表型形成的分子机制。该调控机制的发现有利于进一步阐明肺腺癌化疗耐药机制，为临床逆转肺腺癌化疗耐药提供新策略。

化疗耐药是导致人肺腺癌治疗失败的重要原因，其形成与多种肿瘤耐药相关基因的表观遗传学调控密切相关。越来越多的证据表明，长链非编码RNA（LncRNA）参与多种肿瘤的发展进程。在本研究中，我们探讨了长非编码RNA LINC00221在肺腺癌细胞化疗耐药中的作用及其潜在的分子机制。首先我们在体外细胞水平证实了干扰LINC00221能够抑制肺腺癌耐药株的增殖，促进细胞凋亡从而逆转肺腺癌多西他赛耐药细胞株的化疗耐药表型。随后我们通过RNA pulldown、RNA免疫共沉淀、EMSA等实验技术，发现LINC00221可与选择性多聚腺苷酸化相关蛋白CPSF6结合。该项目首次证实了LINC00221能够招募CPSF6到XBP1 3’UTR，通过调控XBP1 3’UTR poly (a)位点的选择性多聚腺苷化来影响XBP1蛋白翻译，而XBP1表达上调可以促进BECN1转录表达，进而激活自噬通路，证实了内质网应激-自噬通路的激活与LINC00221介导的内质网应激相关蛋白XBP1的活化密切相关。本项目通过揭示LINC00221/CPSF6/XBP1/BECN1信号轴对肺腺癌耐药表型的作用，明确了内质网应激-自噬通路参与调控肺腺癌化疗耐药表型形成的分子机制，该调控机制的发现有利于进一步阐明肺腺癌化疗耐药机制，为临床逆转肺腺癌化疗耐药提供新策略。