LncRNA RPL37AP1通过调控HNF4A/CEBPA/RPSA轴促使贲门腺癌侵袭迁移的新机制

In our previous study, it was found that LncRNA RPL37AP1 was up-regulated in metastatic gastric cardiac adenocarcinoma (GCA) cancerous tissues compared with primary loci pairs. Depletion of LncRNA RPL37AP1 led to down-regulation of HNF4A, CEBPA, RPSA and up-regulation of miR-190a, resulting in decreased potential of metastasis and invasion of GCA cells. Especially, CEBPA and RPSA are targets of miR-190a, and CEBPA are the potential transcription factor of LncRNA RPL37AP1/ HNF4A/ RPSA gene. Hence, it was speculated that LncRNA RPL37AP1/ HNF4A/CEBPA/RPSA axis played an important role in the progress of metastasis and invasion of GCA. This study aims to examine the roles of LncRNA RPL37AP1/ HNF4A/miR-190a/ CEBPA/RPSA in the GCA metastasis and invasion using gain and loss experiments; to investigate the mechanisms by which LncRNA RPL37AP1 regualtes HNF4A, and miR-190a regulates CEBPA/RPSA; to investigate the roles of LncRNA RPL37AP1 on the epigenetics status of HNF4A; to investigate the chromatin binding sites of LncRNA RPL37AP1 using ChIRP and CHART methods; and to interrogate the transcription factors such as CEBPA of LncRNA RPL37AP1 using PAR-Clip method so as to provide new trategies preventing GCA metastasis and invasion.

本课题组前期在国际上率先发现：LncRNA RPL37AP1（L）在贲门腺癌转移组织中的表达相对原发灶组织显著上调，下调其表达可显著下调HNF4A（H）、CEBPA（C）及RPSA（R）表达并上调miR-190a表达，进而显著降低贲门腺癌细胞侵袭、迁移能力。尤其是，C、R均是miR-190a靶基因，且C还是L/H/R等的潜在转录因子。 因此推测: RPL37AP1/ HNF4A/CEBPA/RPSA轴在贲门腺癌侵袭迁移过程中发挥重要作用。本项目拟利用体内外Gain-及Loss-实验验证上述分子对贲门腺癌细胞侵袭迁移能力的影响；以荧光报告基因实验验证L对H、以及miR-190a对C/R的调控作用；以ChIP及n-MSP法检测L对H基因表观遗传学影响；以ChIRP和CHART方法验证L与染色体结合位点；并以PAR-Clip等技术确定与L结合的C等转录因子，以期为贲门腺癌侵袭迁移防治提供新策略。

1本研究自开展以来基本按照原制定计划实施，部分研究方法、内容等在原计划基础上略有调整。在本研究中，我们通过qRT-PCR、Northern等方法发现RPL37AP1在胃癌细胞系AGS、SK-GT2中相对于GES显著上调，通过免疫组织化学方法发现RPL37AP1在贲门腺癌组织中显著上调；敲除RPL37AP1进行细胞增殖检测表明：RPL37AP1表达水平下调后显著抑制SK-GT-2、OACP4C细胞增殖；RPL37AP1表达水平下调与对照组相比S期细胞比例显著下降（转染24h，48h；转染siRNA 50nM, 及100 nM），侵袭细胞数量也较对照组显著下降（转染时间48h及72h；转染siRNA 25nM, 及100 nM）；高内涵实验观察PRL37AP1干扰实验对胃癌细胞系增殖、凋亡等的影响，发现：100nM和50nM的RPL37AP13 siRNA分别转染SK-GT-2细胞，均表现为抑制增殖，促进细胞凋亡作用，与正常组比较，差异显著（P<0.0001，n=12）；而25nM低浓度的RPL37AP13-siRNA转染SK-GT-2细胞，对细胞增殖没有表现出明显的抑制作用，与正常组比较，差异不明显（P>0.05，n=12)；但促进了细胞凋亡的发生，与正常组比较，差异显著（P<0.0001，n=12）；全基因合成与载体构建；对pGL4-basic、pGL4-2458、pGL4-2459、pGL4-2460、pGL4-2461、pGL4-2462等质粒在人胃癌细胞AGS中进行双荧光报告实验，发现：pGL4-2458、pGL4-2459、pGL4-2460、pGL4-2461、pGL4-2462分别高于pGL4-basic组比值，并分别与pGL4-basic做比较，P<0.05有统计学差异。