长非编码RNA LINC01197调节RNA m6A甲基化修饰促进胃癌细胞侵袭转移的机制研究

Metastasis is one of the major causes contributing to progression of gastric cancer. LncRNAs play important roles in tumor metastasis. Our high-throughput sequencing data showed high expression of LINC01197 was positively correlated with advanced stage and poor prognosis. Knockdown of LINC01197 expression could inhibit invasion and metastasis in gastric cancer cells. Besides, KLF5 could activate LINC01197 transcription by binding to the promoter region of LINC01197 in part. LINC01197 could bind to RNA m6A demethylases ALKBH5 which was identified by MeRIP and RIP. After knockdown of LINC01197 or ALKBH5, the expression of VEGF was upregualted and the m6a methylation levels in VEGF mRNA were also increased. So our hypothesis is that KLF5-induced LINC01197 enhances invasion and metastasis by binding to ALKBH5 to epigenetically increase the expression of VEGF in gastric cancer. The aim of our study is to demonstrate that LINC01197 promotes gastric cancer cell invasion and metastis by RNA m6A methylation modulating in both in vitro and in vivo. Moreover, it will provide us a novel theoretical basis for clinical treatment.

转移是胃癌治疗失败主要原因之一。lncRNA与肿瘤转移密切相关。高通量测序表明LINC01197高表达与胃癌患者临床分期晚、预后差相关；干扰LINC01197的表达后抑制胃癌细胞侵袭转移；KLF5可以与LINC01197启动子区结合，激活其转录；MeRIP、RIP实验证实LINC01197可与RNA m6A去甲基酶ALKBH5结合，敲低LINC01197或ALKBH5均可以下调VEGF的表达和增加VEGF mRNA中m6A甲基化水平。据此提出假设：KLF5激活LINC01197的转录，LINC01197通过ALKBH5的RNA m6A去甲基化修饰，上调VEGF的表达，从而促进胃癌细胞侵袭转移。本课题组拟从细胞模型、动物模型、临床样本三个层面，利用RNA pulldown、MeRIP等技术证实LINC01197调节RNA m6A甲基化促进胃癌细胞侵袭转移的分子机制，为临床治疗提供新依据。

m6A甲基化的调节与胃肠道肿瘤密切相关，其中去甲基化酶ALKBH5在胃癌中的研究还不够深入。我们发现ALKBH5在胃癌组织中成高表达，且与不良预后相关。通过体内外实验验证ALKBH5可以影响胃癌细胞的增殖和侵袭转移能力。对胃癌细胞进行甲基化RNA免疫共沉淀结合高通量测序以及后续实验发现ALKBH5可与长非编码RNA LINC00659相结合，通过募集ALKBH5发挥“脚手架”作用。同时，LINC00659在胃癌中高表达，且与不良预后相关。体内外研究证明了LINC00659能够促进胃癌细胞的增殖和侵袭转移能力。甲基化RNA免疫共沉淀结合高通量测序及转录组高通量测序，发现ALKBH5可对JAK1进行去m6A甲基化修饰，LINC00659可促进JAK1与ALKBH5的结合能力，使JAK1 m6A水平下降，竞争性抑制YTHDF2对JAK1的识别结合，抑制YTHDF2对JAK1的衰亡作用。进一步研究发现上调JAK1后可激活JAK1/STAT3通路，从而促进胃癌细胞的增殖和侵袭能力。综上所述，我们发现LINC00659通过募集ALKBH5发挥“脚手架”作用对JAK1 进行去m6A甲基化修饰，竞争性抑制YTHDF2对JAK1的m6A识别与结合，从而拮抗YTHDF2对JAK1的衰亡作用，上调JAK1的表达，激活JAK1/STAT3通路，促进胃癌的发生发展。