MiR-143/145宿主基因CARMN低表达参与宫颈癌遗传易感及其机制研究

LncRNA CARMN is a host gene for miR-143/145, the most studied tumor suppressor miRNAs, which is low in most tumors, including cervical cancer (CC). The molecular mechanism underlying the underexpression of CARMN is instructive for elucidating the important biological role of miR-143/145. The literature reports that CARMN may be decreased expressed at the transcriptional and post-transcriptional levels. In the previous study, we identified the functional SNP rs12517403 in the promoter region of CARMN, which can influence gene transcription by affecting the binding of transcription factor; RNA pull-down combined with LS-MS/MS identified a batch of RNA-binding proteins, such as DDX3X, RBM8A, which are binding to CARMN and predicted to affect RNA stability of CARMN. Evidence shows that miR-143/145 inhibit and promote proliferation and apoptosis of CC cells, respectively. However, the exact signaling pathways involved have not yet been elucidated. The project intends to further explore the molecular mechanisms of rs12517403 regulating CARMN transcription; identify RBPs that can affect the stability of CARMN transcripts and elucidate their regulatory mechanisms; explore the pathways through which CARMN and its producing miR-143/145 regulate proliferation and apoptosis of CC cell; screen functional SNPs of CARMN, RBPs, and key molecules of signaling pathways, explore their association with susceptibility of CC and underlying molecular mechanisms, and determine their potential as biomarkers for CC susceptibility.

LncRNA CARMN是抑癌miRNAs miR-143/145的宿主基因，在大多数肿瘤包括宫颈癌中低表达。研究提示CARMN可在转录和转录后水平低表达。课题组前期研究筛选出CARMN启动子区可影响基因转录SNP rs12517403；RNA pull-down联合LS-MS/MS筛选出可能影响CARMN转录本稳定性的RNA结合蛋白（RBPs）如DDX3X、RBM8A。研究证实miR-143/145抑制宫颈癌细胞增殖、促进凋亡，但通过何种信号通路尚未阐明。本课题拟继续深入探讨rs12517403调控CARMN转录的分子机制；鉴定可影响CARMN转录本稳定性的RBPs并阐明其调控机制；探讨CARMN、miR-143/145通过何种信号通路调控细胞增殖和凋亡；寻找CARMN、RBPs、信号通路关键分子的功能SNPs，探讨其作为宫颈癌遗传易感的生物标志的可能性。

LncRNA CARMN在宫颈癌中显著下调，但其下调机制以及在宫颈癌中的生物功能尚不明确。本研究通过收集宫颈癌人群和健康对照人群宫颈组织进行全转录组测序分析发现CARMN是宫颈癌变过程中的关键基因，其在宫颈癌变过程中逐步下调。本研究中CARMN在宫颈癌中下调的机制通过转录水平和转录后水平分别探讨。在转录层面，与野生型T等位基因相比，SNP rs12517403 C等位基因可降低转录因子SP1与CARMN启动子区结合亲和力，并抑制CARMN转录活性。在转录后水平上，YBX1的减少可降低CARMN的稳定性，DHX9在此过程中充当了连接的支架。此外，DHX9的dsRBD I/II结构域和解旋酶核心结构域增强了与CARMN 561-754nt序列的结合亲和力。随后我们探究了CARMN在宫颈癌中的作用机制，结果发现，CARMN可显著增强胞浆中Keap1/Nrf2的相互作用，从而加速了泛素介导的Nrf2降解，减少了Nrf2的核异位，进而减少了抗氧化相关基因的表达，并最终引起宫颈癌细胞内活性氧（ROS）水平的升高。此外，升高的ROS可抑制Akt/mTOR信号通路，通过自噬通量阻断导致宫颈癌细胞自噬停滞并引起细胞周期停滞和细胞凋亡，最终降低宫颈癌细胞增殖和集落形成能力。CARMN还可通过与转录因子TFAP2α直接结合，抑制MAPK13的转录激活，进而抑制MAPK级联，最终也可发挥抗肿瘤作用。该研究的发现对于宫颈癌的临床诊治和预防具有一定的指导意义。