内质网应激特异miRNAs对肝癌细胞自噬的调控及其对化疗药物敏感性影响的机制

Drug resistance in hepatocellular carcinoma is an obstacle issue in clinical chemotherapy. It is well believed that autophagy induced by endoplasmic reticulum stress plays an important role in this process. However, the molecular and cellular mechanisms underlying autophagy induced by endoplasmic reticulum stress are largely unknown . Currently, the relationship between miRNA and chemotherapeutic drug sensitiveness is becoming a hot topic in cancer area. Based on our promising preliminary experiment results, the overall goal of this project is to identify key specific miRNAs involved in autophagy induced by endoplasmic reticulum stress in hepatoma carcinoma cell and investigate their effects on chemotherapy sensitiveness. To achieve this goal, we will first use high-flux sequence platform to perform depth sequencing on hepatoma cell lines before and after endoplasmic reticulum stress stimulation. Then these results will be evaluated and analyzed by custom-tailor high-capacity chip; and verification will be performed using qRT-PCR method in clinical hepatocellular carcinoma specimens and in vitro cell culture system. Secondly, specific miRNA expression profiling will be screened correlates closely with autophagy in hepatoma carcinoma cell. Thirdly, the functions of this miRNA spectrum including their target genes and related signal pathways will be predicted and confirmed. Furthermore, the most important key miRNA related to autophagy and drug resistance will be identified and characterized. Lastly, preclinical studies including in vitro cell culture system and in vivo tumor-bearing mouse models will be performed to test the effects of these obtained miRNAs on autophagy and drug resistance. The proposed studies above not only could further precisely demonstrate the molecular mechanisms of autophagy induced by endoplasmic reticulum stress , but also highlight the potential uses of miRNA and miRNA-based drug target for enhancing the sensitiveness of hepatocellular carcinoma therapy.

肝癌细胞的抗药性是困扰临床的重要问题，内质网应激介导的自噬产生保护机制可能是其重要原因，但其机制尚不清楚。研究表明miRNA能以网络的形式参与信号通路的调控作用，有望成为肿瘤治疗新策略。前期工作中,我们利用芯片技术并经生物信息学分析,发现肝癌细胞发生内质网应激后有8条特异miRNA与自噬相关,本研究拟在此基础上利用qRT-PCR对临床肝癌标本和应激后的肝癌细胞进行验证，将miRNA mimics/inhibitors转染入肝癌细胞，观察肝癌细胞自噬及药物敏感性变化，进一步筛选与自噬及与药物敏感性相关的特异miRNA。通过分析并验证其上游调控机制及下游自噬相关靶基因，以阐明相关信号通路。在此基础上，通过临床标本和荷瘤小鼠动物模型研究内质网应激特异miRNAs对自噬及化疗药物敏感性的影响，以期进一步阐明内质网应激介导肝癌细胞自噬产生耐药的分子机制，为提高肝癌药物治疗的敏感性提供高效的作用靶点。

肝癌细胞的抗药性是困扰临床的重要问题，内质网应激介导的自噬产生保护机制可能是其重要原因，但其机制尚不清楚。研究表明mi RNA能以网络的形式参与信号通路的调控作用，有望成为肿瘤治疗新策略。前期工作中,我们利用芯片技术并经生物信息学分析,发现肝癌细胞发生内质网应激后有18条特异miRNA，其中 miR-103a 的表达水平在肝癌组织中明显下调，mi R-663在肝癌组织中表达水平发生显著上调，提示 miR-103a、miR-663 与肝癌耐药可能存在一定的联系。同时我们对应激后的培养细胞进行验证，发现肝癌细胞内质网应激后 miR-663 表达明显升高，miR-103a 的表达明显下调，同时调控了 miR-663 及 miR-103a 的表达水平，观察了 miR-663 及 miR-103a 的表达对细胞自噬、凋亡及药物敏感性变化，从而进一步发现与内质网应激耐受的特异 mi R-663及 miR-103a 可能与药物敏感性相关。通过进一步分析并验证了 miR-663 调控的下游靶基因，阐明了相关信号通路。以期进一步阐明内质网应激介导肝癌细胞自噬产生耐药的分子机制，为提高肝癌药物治疗的敏感性提供高效的作用靶点。