肝癌细胞内质网应激耐受特异miRNAs鉴定及其对化疗敏感性影响的机制

Drug resistance of hepatic carcinoma is the obstacle and important issue in clinical chemotherapy. It is well believed that endocytoplasmic reticulum stress resistance plays an important role in this process. However, the molecular and cellular mechanisms underlying endocytoplasmic reticulum stress resistance are largely unknown and still needed further investigation. Currently, the relationship between miRNA and endoplasmic reticlum stress resisitance is becoming a hot topic in cancer area. Using high-flux sequence platform, we observed that 18 miRNA expressions significantly changed after endocytoplasmic reticulum stress stimulation in hepatoma cell lines and normal hepatocytes. Based on these promising data, verification will be performed using qRT-PCR and Northern blot method in clinical hepatoma carcinoma specimens and in in vitro cell culture system. We will transfect miRNA mimics/inhibitors into cell, examine cell proliferation, apoptosis and change of drug sensitivity. Then the most important key miRNA related to endocytoplasmic reticulum stress resistance and drug resistance will be identified and characterized. Furthermore, the functions of this miRNA spectrum including their target genes and related signal pathways will be predicted and confirmed. Lastly, preclinical studies including in vitro cell culture system, in vivo tumor-bearing mouse models and clinical specimen will be performed to test the effects of these obtained miRNAs on drug resistance. The proposed studies above not only could further precisely demonstrate the molecular mechanisms of endocytoplasmic reticulum stress resistance, but also highlight the potential uses of miRNA and miRNA-based drug target for enhancing the sensitiveness of hepatoma carcinoma therapy. Thus, this project will provide solid evidence and basis for developing novel miRNA drug or miRNA-based drug in the future.

肝癌细胞的抗药性是困扰临床的重要问题，对内质网应激（ERS）的耐受可能是其重要原因，但机制尚有待研究。miRNA与ERS耐受的关系鲜见报道。我们已在证实肝癌细胞对ERS耐受的基础上，利用芯片技术，发现肝癌细胞和正常肝细胞在内质网应激后，有18条miRNA呈差异表达。本项目将在前期基础上，利用qRT-PCR和Northern blot对临床肝癌标本和应激后的培养细胞进行验证和筛选；将miRNA mimics/inhibitors转染不同细胞，观察其增殖、凋亡及药物敏感性变化，进一步筛选与ERS耐受及药物敏感性相关的特异miRNA,进而分析并验证其调控的下游靶基因及上游调控机制，以阐明相关信号通路;通过荷瘤小鼠动物模型和临床标本研究miRNAs对药物敏感性的影响。本研究不仅将进一步阐明肝癌细胞ERS耐受的分子机制，也将为提高肝癌药物治疗的敏感性提供高效的作用靶点，并为开发创新核酸药物打下基础。

原发性肝癌的耐药与其在发生发展过程中所产生的对内质网应激的耐受有密切关系，而miRNAs可能在其中发挥了重要的调控作用。我们利用芯片技术，发现肝癌细胞和正常肝细胞在内质网应激后，有18条miRNA呈差异表达，其中miR-107的表达水平在肝癌组织中明显下调，miR-663在肝癌组织中表达水平发生显著上调，提示miR-107、miR-663与肝癌可能存在一定的联系。同时我们对临床肝癌标本和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-107的表达明显下调，将miRNA mimics/inhibitors转染不同细胞，观察了细胞增殖、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miR-663及miR-107可能与药物敏感性相关。进一步分析并验证miR-663调控的下游靶基因调控机制，阐明相关信号通路。本研究不仅将进一步阐明肝癌细胞内质网应激耐受的分子机制，也将为提高肝癌药物治疗的敏感性提供高效的作用靶点，并为开发创新核酸药物打下基础。