miRNA-186-5p通过METTL3在结直肠癌中调控HIF-1α mRNA m6A修饰的机制研究

Recent research hotspot——N6-methyladenine (m6A) is a new epigenetic modification of RNA and performs critical functions in many biological processes, such as cell survival, development and tumor formation. However, the function of m6A in colorectal carcinoma have not been reported yet. Our previous studies found that the expression of METTL3 (an important m6A methyltransferase) was obviously up-regulated in colorectal carcinoma. Using bioinformatics analysis, we predicted that miRNA-186-5p may target the METTL3, which was also verified in our preliminary experiment. Hypoxia exerts profound effects on the cellular microenvironment with impressive effects on cancer progression, we found the levels of METTL3 were positively correlated with those of HIF-1α by bioinformatics analysis. Furthermore, m6A is the common modification that occurs in non-coding regions and exons of HIF-1α. Based on the above, we presumed that miRNA-186-5p could regulate the expression of METTL3, and then influence the level of m6A modification and the expression of HIF-1α, finally plays an anti-tumor effect in colorectal carcinoma. We intend to elaborate the exact mechanism through experimentation in vitro and vivo, and also explore the effects of hypoxia on m6A modification in colorectal carcinoma. From the new direction about regulating the m6A modification, the aim of this project is to study the underline mechanism how microRNA works in colorectal carcinoma, and provides a new strategy for the targeted therapy of colorectal carcinoma.

近期研究热点——N6-甲基腺嘌呤（m6A）这一RNA表观遗传新修饰，在细胞生存、发育及肿瘤形成等过程中发挥重要调控作用，但目前m6A在结直肠癌中的作用和功能仍未有报道。我们前期发现METTL3（m6A甲基转移酶）在结直肠癌中表达上调，利用软件预测METTL3可能是miRNA-186-5p的作用靶点，并在预实验中得到了验证。缺氧微环境可促进肿瘤的发生发展，生物信息学分析显示METTL3与HIF-1α呈正相关，且HIF-1α非编码区及外显子区存在多个m6A修饰位点。据此我们提出假设：miRNA-186-5p通过靶向调控METTL3，改变m6A修饰水平、改变HIF-1α的表达，在结直肠癌中发挥抑癌作用。本研究拟从体外和体内两方面详细阐明上述机制，此外还探究缺氧对m6A修饰的影响。本课题从microRNA调控m6A修饰这一新角度深入认识结直肠癌的发病机制，为结直肠癌的靶向治疗提供新的策略。

在胰腺癌的治疗中，吉西他滨的耐药一直是一个重要的挑战。在过往的研究中，长链非编码RNA（lncRNA）被证实在胰腺癌的肿瘤发生中起到重要调控作用，但是在吉西他滨抵抗的胰腺癌中lncRNA起到什么作用还未明确。在本研究中，我们证实了在吉西他滨耐药的胰腺癌细胞中，HIF-1a的反义RNA1（HIF1A-AS1）显著升高。功能获得和功能丧失实验证实HIF1A-AS1在体外和体内都促进了胰腺癌细胞对吉西他滨的耐药性。我们进一步发现HIF1A-AS1上调HIF-1a表达，从而促进糖酵解以增强胰腺癌细胞的吉西他滨耐药性。在机制上，HIF1A-AS1促进丝氨酸/苏氨酸激酶Akt和Y盒结合蛋白1（YB1）之间的相互作用，从而促进YB1的磷酸化（pYB1）。同时，HIF1A-AS1将pYB1招募到HIF-1a mRNA，从而促进HIF-1a的翻译。此外，HIF-1a通过直接结合HIF1A-AS1启动子区域中的HIF-1a响应元件来促进HIF1A-AS1转录，形成正反馈。一直以来，HIF1A-AS1和HIF-1a在胰腺癌组织中均上调，并与总体生存率低相关。总之，我们的结果强调了HIF1A-AS1和HIF-1a的相互循环，这增强了胰腺癌的吉西他滨耐药性，并表明HIF1A-AS1可能作为胰腺癌吉西他滨耐药性的新治疗靶点。