长链非编码RNA-PTCHD3P1调控胰腺癌侵袭和转移的机制研究

Early invasion and metastasis of pancreatic ducatal adenocarcinoma(PDAC) are worldwide problem with respect to clinical treatment. Understanding the molecular mechanism underlying the process is urgent and necessary. Recently, many studies have shown that long non-coding RNA(lncRNA) play important roles in the regulation of invasion and metastasis of PDAC tumorigenesis. Corresponding to these findings, our preliminary work implied that lncRNA PTCHD3P1 was highly expressed in PDAC especially in those with metastasis, which indicated that PTCHD3P1 may associated with the migration and invasion of pancreatic cancer cell. Furthermore, bioinformatic analysis suggested that PTCHD3P1 may function as a ceRNA (competing endogenous RNA) to competitively combine to miRNA let-7a which regulated EMT (Epithelial-Mesenchymal Transition) by targeting to HMGA2. Since several studies have documented that let-7a-HMGA2-EMT axis plays a key role in invasion and metastasis of many cancers including PDAC, we propose that PTCHD3P1 could enhance the invasion and metastasis of pancreatic cancer cell by act as a ceRNA to facilitate HMGA2 expression through suppressing let-7a. Based on our prior works, we’ll try to elucidate the mechanism of PTCHD3P1 involved in the invasion and metastasis in pancreatic cancer cell, and illuminate the role of PTCHD3P1-let-7a-HMGA2-EMT axis in PDAC by both in vitro and in vivo studies.

胰腺癌的早期侵袭与转移的分子调控机制一直是该领域的难点与热点。长链非编码RNA（lncRNA）在胰腺癌等恶性肿瘤的侵袭、转移过程中起着重要调控作用。我们的前期研究发现lncRNA -PTCHD3P1在胰腺癌组织尤其是转移组织中表达显著增高，提示其与胰腺癌细胞的迁移、侵袭密切相关。进一步的分析提示PTCHD3P1可作为竞争性内源RNA（ceRNA）调控let-7a。而let-7a-HMGA2-EMT网络在包括胰腺癌等肿瘤的侵袭与转移中起着重要的作用。因此，我们推测PTCHD3P1可作为ceRNA调控let-7a并介导EMT，促进胰腺癌细胞的侵袭与转移过程。本研究拟在前期研究基础上，通过体内外实验进一步探讨在胰腺癌中PTCHD3P1、let-7a及其下游靶基因的表达变化规律，深入分析PTCHD3P1调控胰腺癌细胞侵袭与转移的功能，阐明PTCHD3P1调控胰腺癌侵袭与转移的分子机制。

本研究中我们利用生物信息学分析结合经典分子生物学技术等,在分子水平、细胞水平、实验动物水平及临床样本中探索异常表达的长链非编码RNA SVIL-AS1对胰腺癌生物学行为的影响及机制。研究组以60例胰腺癌患者的癌组织和癌旁组织为实验样本,采用qRT-PCR进行lncRNA SVIL-AS1表达验证。结果发现,lncRNA SVIL-AS1在癌组织中呈现低表达状态。结合患者的临床病理信息发现，lncRNASVIL-AS1的表达水平与胰腺癌患者的肿瘤大小、TNM分期及淋巴结转移呈明显负相关关系，提示lncRNA SVIL-AS1可能参与胰腺癌细胞増殖和侵袭转移有关。为探索lncRNA SVIL-AS1对细胞功能的影响,我们用慢病毒包装的过表达载体研究lncRNA SVIL-AS1的过表达效果,并对panc-1和Bxpc-3过表达系进行一系列细胞功能实验，观察其对胰腺癌细胞增殖、凋亡、迁移、侵袭等能力的影响。我们发现过表达lncRNA SVIL-AS1后，胰腺癌细胞的增殖、凋亡、迁移、侵袭能力未有明显变化。同时用裸鼠肿瘤异位种植实验验证lncRNA SVIL-AS1表达与肿瘤生长和转移的相关性，发现lncRNA SVIL-AS1可明显降低裸鼠体内肿瘤生长的速度，同时提高裸鼠的存活率。为了探明lncRNA SVIL-AS1造成这种现象的机制，我们对体外构建的PANC-1过表达系进行了二代测序，GSEA分析揭示SVIL-AS1过表达后发生重大变化的KEGG通路，注意到SVIL-AS1过表达后下降的途径多与免疫调节有关。于是研究组猜想lncRNA SVIL-AS1参与调控了肿瘤与肿瘤微环境的某种信号交流，导致了体内肿瘤减小而体外培养时癌细胞无明显变化。将过表达后下调的各个KEGG通路中的leading edge genes 取出，提取其中的差异表达基因，进行蛋白质相互作用网络分析，发现c-JUN、CCL2等处于网络的核心位置，提示他们可能是介导SVIL-AS1抗肿瘤作用的主要调控因子。CCL2的表达可以影响肿瘤免疫微环境中单核巨噬细胞的极化方向，于是研究组对THP-1（人单核细胞系）和胰腺细胞系进行共培养，发现lncRNA SVIL-AS1过表达时，可以使THP-1在分化时更多地由抑制肿瘤免疫的M2向促进肿瘤肿瘤免疫的M1极化，从而使肿瘤微环境更不利于胰腺癌的生长和转移。