MiR-495在GRP78介导的自噬参与胃癌多药耐药中的作用及机制的研究

multi-drug resistance( MDR) is a major clinical obstacle in the treatment of gastric cancer (GC) and accounts for the majority of cancer-related mortality. MicroRNAs have been recently emerged as regulators of MDR through acting on multiple signaling pathways. We have identified miR-495 that diffential expressed between ADR GC cells using RT-PCR. And miR-495 showed the most strong effect on inhibiting GC cell autophagy and MDR. An MTT assay and xenograft model and Immunofluorescence staining demonstrated that the transfection of miR-495 induced autophagic activity and cell death in gastric cancer cells. We further investigated the underlying mechanism of mir-495 in GC autophagy and MDR. bioinformatics and proteomics will be used to predict and screen the potential target gene that MDR regulates. ChIP and Luciferase reporter assays will be used to identify the direct target gene. At last, expression of and mir-495 itself and target gene will be testified in human GC tissues. To reveal the mechanisms of miR-495-GRP78 contributes to autophagy-induced MDR in gastric cancer, and would be helpful in the therapeutic application to block GC MDR.

胃癌多药耐药是导致胃癌病人死亡的主要原因，其具体机制尚不清楚，miR-495已被发现参与调节多种肿瘤的发生发展，但其参与调节胃癌多药耐药的具体机制尚未阐明。前期我们发现miR-495在胃癌亲本细胞及耐药细胞中表达差异明显，并通过MTT，平板克隆、裸鼠成瘤实验、免疫化学染色等体内外实验验证其可能是通过调节自噬这条途径参与调节胃癌耐药细胞的生长及凋亡，为进一步研究其具体机制，我们通过生物信息学方法预测其下游靶基因并通过基因报告实验及免疫共沉淀对其进行验证，最后在胃癌临床标本中验证其表达及相关性，本研究想通过探讨miR-495-GRP78通过自噬参与调节胃癌多药耐药中的作用及机制，为临床胃癌多药耐药的发生发展提供一个新的治疗策略。

多药耐药胃癌患者5年生存率低及预后不良的主要原因。MicroRNAs (miRNAs) 是多药耐药的关键调控因素，MiR-495已被发现参与调节多种肿瘤的发生发展，但其参与调节胃癌多药耐药的具体机制尚未阐明主要通过目标基因的调节其耐药过程。目前的研究表明，miR-495-3p通过一个目标基因GRP78来调节自噬过程并抑制胃癌多药耐药过程。根据体外和体内功能性实验验证，我们发现，在体外实验中，miR-495-3p的过度表达足以逆转为四种化疗药物的耐药性，而在体内实验中其也可以抑制小鼠体内肿瘤生长。此外，GRP78 与自噬的发生呈正相关性。因此，当我们下调GRP78表达后，导致了类似于Mir 495-3p的自噬活性，同时恢复GRP78的表达则减弱了miR-495-3p引起的反自噬效应。除此以外，我们发现，在临床上胃癌患者的Mir-495-3p水平的下调或GRP78水平的升高都与与胃癌的恶性表型有关。总之，这些发现表明，miR-495-3p/GRP78/自噬调节环在胃癌的多药耐药过程中起着关键作用。除此以外，miR-495-3p和GRP78可以作为胃癌整体生存率的预后因素，这意味着miR-495-3p是癌症的治疗靶点。

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