抑制LncRNA-VSTM4介导的异常自噬在逆转胆囊癌化疗耐药中的机制研究

Abnormal autophagy has been proposed as one of the main factors that contributed to the chemoresistance of carcinomas and it is of great significance to suppress autophagy in order to revserse chemoresistnace in cancer. Our previous research demonstrated that chemoresistant gallbladder cancer cell lines had an increased autophagic flux and suppression of autophagy could chemosensitize chemoresistant (CR) cell lines. CR cells harbored an ehanced level of lncRNA-VSTM4, which plays an important role in regulation of autophagy, while the precise molecular mechanism remains unknown. We hypothesize that lncRNA-VSTM4 may enhance the binding of transcriptional factor FOXO3 to the promoter of autophagy-related factor ATG10 and activate its transcription; the activation of lncRNA-VSTM4-FOXO3-ATG10 axis may contribute to the enhanced autophagy and then chemoresistance. The research was conducted both in vitro and in vivo to study the role of lncRNA-VSTM4, FOXO3 and ATG10 in autophagy and chemoresistnace. Our study aims to clarify the molecular mechanism of the abnormal activated lncRNA-VSTM4-FOXO3-ATG10 axis-mediated enhanced autophagy and chemoresistance in gallbladder cancer. We also sought to determine the possibility of regulating the expression levels of lncRNA-VSTM4, ATG10 and FOXO3 to reverse the chemoresistance, which provides valid experimental data for improving clinical chemotherapeutic efficiency.

自噬异常是诱导肿瘤化疗耐药的机制之一，干预关键靶点以抑制自噬对逆转肿瘤耐药意义重大。前期研究发现胆囊癌耐药细胞株的自噬水平异常增高，抑制自噬可提高化疗敏感性；耐药株中lncRNA-VSTM4表达异常升高，且在介导自噬异常中发挥关键作用，但其中具体分子机制尚不明了。我们假设lncRNA-VSTM4促进转录因子FOXO3结合至自噬相关蛋白ATG10启动子区域从而激活ATG10的转录；lncRNA-VSTM4/FOXO3/ATG10信号轴表达异常可能导致胆囊癌细胞自噬异常激活，进而诱导化疗耐药。本课题拟通过体内外实验研究lncRNA-VSTM4与FOXO3以及ATG10在胆囊癌自噬过程中的作用和关系，阐明lncRNA-VSTM4/ FOXO3/ ATG10信号轴表达异常，进而激活胆囊癌细胞自噬以及诱导化疗耐药的分子机制，探讨干预lncRNA-VSTM4等基因表达以逆转化疗耐药的可行性。

胆囊癌是最常见的胆道恶性肿瘤，对化疗不敏感，既往研究显示自噬是延长肿瘤细胞在化疗应激下生存的重要因素。同时长非编码rna（lncRNAs）在胆囊癌细胞自噬和耐药中的作用及机制不甚明确。我们通过建立多柔比星（Dox）耐药胆囊癌细胞株，用基因芯片技术比较lncRNAs在Dox耐药胆囊癌细胞及其亲本细胞中的表达。通过基因敲除或外源性表达结合体内外实验，证实了lncRNA的功能意义。用stubRFP-sensGFP-LC3和western blot检测lncRNA对自噬的影响。我们用RNA下拉和质谱分析鉴定了lncRNA的靶蛋白。结果显示胆囊癌细胞的耐药性与其自噬活性增强有关。我们发现了一种称为胆囊癌耐药相关lncRNA1（GBCDRlnc1）的lncRNA ENST0000425894，它是胆囊癌化疗耐药的关键调节因子。此外，我们发现胆囊癌组织中GBCDRlnc1表达上调。GBCDRlnc1基因的敲除，通过在体外和体内抑制自噬，增强Dox耐药胆囊癌细胞对Dox的敏感性。我们研究证实了GBCDRlnc1与磷酸甘油酸激酶1相互作用，抑制其在Dox耐药胆囊癌细胞中的泛素化，从而导致自噬启动子ATG5-ATG12结合物的下调。因此，GBCDRlnc1可能是化疗药物治疗晚期胆囊癌的候选靶点。