PTCH1-3'UTR 作为lncRNA MALAT1的ceRNA参与调控SLC39A6促进肺癌转移的机制

A gene's 3'UTR may act as ceRNA to regulate gene expression, and its function is independent of CDS region. PTCH1 is the key regulator of Hedgehog signal pathway, but it's role in NSCLC is till unclear. PTCH1 has multiple splicing forms, which all share the same 3'-UTR sequence, indicating the importance of PTCH1-3'UTR. MALAT1 is a lncRNA that promotes NSCLC metastasis, and miR-101 can bind MALAT1, down-regulation its expression. SLC39A6 also play a vital role in NSCLC by promoting metastasis. We identified SLC39A6 as a target of miR-101; PTCH1-3'UTR can promote invasion and migration in lung cancer cell H1299, as well as increasing the expression of MALAT1 and SLC39A6. We thus hypothesis that PTCH1-3'UTR may compete with MALAT1 by binding to a specific miRNA, induce the miRNA transporting to cytoplasm. The released MALAT1 thus induce another miRNA, miR-101, to enter into the nuclear, result in accumulation of SLC39A6 that may enhance tumor matastasis. The proposed project intend to employ cultured cells, animal model and clinical samples to elucidate the interplay of PTCH1-3'UTR, MALAT1, SLC39A6 by miRNA and the mechanism of miRNA shuttle between cytoplasm and nuclear. Our study may shed light on the underlying mechanisms of PTCH1-3'UTR in the progression of lung cancer.

基因的3’UTR可作为ceRNA调控基因表达，独立发挥与CDS区无关的生物学功能。MALAT1定位于核，可结合miR-101。我们前期发现，Hh信号通路调控因子PTCH1的3’UTR不影响增殖，但促进转移，提升转移促进因子MALAT1和SLC39A6的水平，具有独立于Hh通路的作用，并且SLC39A6是miR-101的靶基因。我们推论，PTCH1-3’UTR可与MALAT1竞争结合某未知miRNA，促使该miRNA出核，被释放的MALAT1再诱导细胞质miR-101入核，引起SLC39A6上调促进转移。项目拟在细胞、动物及病理等层次深入研究PTCH1-3’UTR-miRNA-MALAT1-miR101-SLC39A6调节环路对上皮间质转化和细胞转移的影响，阐明相关miRNA核质运输的机制，揭示PTCH1-3’UTR在肺癌进展中的作用，为肺癌干预提供理论依据。

PTCH1 是Hh信号通路的负调控因子，PTCH1 的7个不同的剪接体的3’UTR序列完全相同，提示其重要性。过表达PTCH1-3'UTR促进细胞迁移，侵袭和粘附，但对细胞增殖没有影响。通过与PTCH1共享相同的microRNA响应元件基因的加权基因网络共表达分析，结合PTCH1-3'UTR转染细胞后的微阵列数据的芯片分析，两者取交集后进行了一系列基因的筛选，锚定SLC39A6进行研究。通过RT-PCR验证了在H1299和A549中过表达PTCH1-3'UTR能够促进SLC39A6的表达，敲低PTCH1后可以抑制SLC39A6的表达。Q-PCR和荧光素酶实验表明，PTCH1和SLC39A6是miR-101的直接靶标。与用miR-NC转染的细胞相比，用miR-101转染的A549细胞中PTCH1和SLC39A6蛋白水平也显著降低。SLC39A6介导PTCH1-3'UTR对NSCLC细胞迁移和侵袭的影响，低水平miR-101和PTCH1以及高水平SLC39A6与NSCLC进展呈正相关，证实了PTCH1-3'UTR/miR-101/SCL39A6通路。.MALAT1也是miR-101的靶基因，MALAT1主要定位于细胞核内，我们猜测miR-101是否分别靶向MALAT1和SLC39A6来实现PTCH1-3'UTR的功能。AGO2和TRNC6A形成复合物后，共同介导miRNA的出核与入核。在H1299细胞中，敲低TRNC6A后，MALAT1有比较明显的下调，暗示某种miRNA的出核受到影响。敲低AGO2后，抽提总miRNA，发现miR-101在细胞核中的比例下降，在细胞质内的比例上升。敲低AGO2后，抽提总RNA，A549中PTCH1和MALAT1的表达量基本不变。在这些实验中，miR-101-/MALAT1中的miR-101出入核与PTCH1-3'UTR/SCL39A6的轴线的关系没有明确结论。根据生物信息学的分析结果，我们继续筛选了一批miRNA，并对miRNA-429进行研究。过表达miR-429后，总RNA水平上，MALAT1和PTCH1表达量均下调，A549细胞中细胞核里MALAT1下调，但对PTCH1-3'UTR/SCL39A6的轴线影响不显著。.Hh信号通路的靶基因FOX家族，我们进一步研究发现了circFOXO3/miR-29a-3p/SLC25A15通路。