长链非编码RNA调控的凋亡通路在ERS抗口腔癌中的作用及其分子机制研究

Long non-coding RNA (lncRNA) plays a comprehensive and complicated regulatory role in the process of gene expression. It is a new strategy for cancer therapy by disturbing the expression of some lncRNA and inducing apoptosis through inducing environmental stimuli or homeostatic imbalance. The accumulation of unfolded protein during rapid tumor proliferation activates endoplasmic reticulum stress (ERS) and downstream apoptotic pathway. However, the effects of ERS on lncRNA expression are still unclear. The applicant firstly discovered characteristic changes in the lncRNA expression profile in oral cancer cell after inducing ERS condition in our research. Two key regulatory lncRNA MIR22HG and RP11-325K4.2 were identified by bioinformatics analysis. We propose the hypothesis that MIR22HG and RP11-325K4.2 activate numerous downstream apoptotic pathways and induce apoptosis in oral cancer cells through competitively binding with let-7 or up-regulating the expression of ERS-related apoptotic factors. We planned in this project to identify the relationship between lncRNA expression and the anti-cancer activity of ERS by detecting changes in histone modification and upstream transcriptional regulatory factors expression, conducting luciferase reporter assay, RNA-pull down assay, evaluating downstream apoptotic pathway and biological effects. These results will provide new targets for cancer therapy.

长链非编码RNA(lncRNA)在基因表达过程中具有广泛复杂的调控作用。通过外界刺激或干扰内稳态影响某些lncRNA的表达，诱导细胞凋亡，代表着肿瘤治疗的新策略。肿瘤快速增殖过程中蓄积的未折叠蛋白可启动内质网应激(ERS)和下游凋亡通路，然而其对lncRNA的影响尚不明确。申请者前期研究首次发现ERS诱导口腔癌细胞lncRNA表达谱出现特征性改变，并通过生物信息学分析筛选出MIR22HG和RP11-325K4.2两个关键lncRNA。我们提出：MIR22HG和RP11-325K4.2通过竞争性结合let-7或上调ERS相关凋亡因子表达，激活下游广泛的凋亡通路并诱导口腔癌细胞凋亡。本课题拟通过组蛋白修饰和上游转录调控因子检测、荧光素酶报告分析、RNA-pull down、下游凋亡通路和生物学效应评估，从分子、细胞和整体水平探讨lncRNA介导ERS抗肿瘤效应的分子机制，为肿瘤治疗提供新靶点。

长链非编码RNA(lncRNA)是一类长度超过200个核苷酸的非编码RNA，近年来研究发现其可在表观遗传学水平、转录水平和转录后水平调控基因表达。内质网是蛋白合成和修饰的场所，干扰内质网的正常功能可引起内质网应激，进而诱导细胞凋亡。目前关于lncRNA与内质网应激的调控关系尚不清楚，筛选出在内质网应激中发挥关键作用的lncRNA具有重要意义。.在本项目中，我们采用新一代lncRNA基因芯片，系统地比较了口腔癌细胞中对照组和内质网应激组的lncRNA表达谱改变，发现激活内质网应激后，许多lncRNA表达均出现明显变化。通过生物信息学手段，我们筛选出HITTERS和MIR22HG两个潜在的关键lncRNA。针对HITTERS这个全新的lncRNA，我们分析了它的基本生物信息，发现在多种细胞中内质网应激均可诱导HITTERS表达，HITTERS具有两个转录变异体，其中转录变异体1是主要的转录形式，转录体均具有polyA尾结构，主要定位于细胞核中。HITTERS与HERPUD1共用相同的启动子区。我们进一步分析HITTERS在内质网应激中的作用，发现HITTERS抑制内质网应激诱导的细胞凋亡，降低凋亡蛋白的表达。动物实验发现HITTERS抑制内质网应激的抗肿瘤活性。临床样本分析发现HITTERS高表达的口腔癌患者的临床预后较差。这些研究结果提示HITTERS可抑制内质网应激的凋亡效应。.根据既定的研究计划，我们还分析了另一个潜在的关键lncRNA MIR22HG。我们发现内质网应激诱导MIR22HG表达，其主要定位于细胞质中。在内质网应激状态下，干扰MIR22HG可提高肿瘤细胞活力，并抑制细胞凋亡。此外，MIR22HG具有独立的抗肿瘤活性，细胞实验和动物实验证实干扰MIR22HG表达后口腔癌的增殖和侵袭能力降低，并影响多条肿瘤相关信号通路。