E2F1调控的长链非编码RNA LINC00668调节细胞周期促进胃癌增殖机制的初步研究

Recent studies indicated that lncRNAs involve in regulating tumor cells proliferation, apoptosis, invasion and metastasis. By utilizing publicly available lncRNAs expression profiling data of gastric cancer and integrating analyses, we screened out LINC00668, whose expression is significantly increased in gastric cancer. Moreover, E2F1 could induce the expression of LINC00668. Further experiments confirmed LINC00668 could significantly affect gastric cancer cell proliferation, cell cycle and affect CKIs and CDK6 expression. RIP assay proved that LINC00668 could bind to PRC2 in the nucleus, and sequence analysis revealed LINC00668 may be able to “sponge" miR-449a expression and regulation of CDK6 in the cytoplasm. Accordingly, the hypothesis is proposed: overexpression of E2F1 induces the expression of LINC00668. In the nucleus, LINC00668 could epigenetically silence expression of CKIs. In the cytoplasm, LINC00668 could serve as a “ceRNA”, competitive adsorption of miR-449a, releasing CDK6 expression, thus speeding up gastric cancer cell cycle progression, causing malignant proliferation of gastric cancer cells.We will documente this hypothesis by clinical samples, nude mice, ChIP, RIP, Luciferase reporter and RNA-pull down et.al. Moreover, our work will further the understanding about the molecular mechanisms of development of gastric cancer and provide a new basis for gastric cancer clinical individual therapy and molecular targeted therapy.

LncRNAs普遍参与肿瘤细胞增殖、凋亡、侵袭转移的调控过程。生物信息学分析发现在胃癌中lncRNA LINC00668明显高表达，并在组织中得到验证。同时发现E2F1能够诱导LINC00668表达。细胞实验证实LINC00668能够明显影响胃癌细胞的增殖和周期功能，并影响CKIs和CDK6表达。RIP实验证明LINC00668在细胞核中能够和PRC2绑定，序列分析发现LINC00668在细胞质中可能能够“吸附”miR-449a而调控CDK6表达。据此提出以下假设：在胃癌中，高表达的E2F1诱导LINC00668高表达，在细胞核表观抑制CKIs转录，在细胞质中发挥ceRNA活性，竞争性吸附miR-449a，释放CDK6表达，从而加速胃癌细胞周期进程，引起胃癌细胞恶性增殖。本课题组将扩大临床样本量，并用相关技术证实上述假设，丰富胃癌发生、发展的分子机制研究，为临床胃癌诊断、治疗提供理论依据。

胃癌是消化道常见的恶性肿瘤，严重威胁人类健康和生命。随着医疗技术的发展，胃癌的治疗效果和预后虽有提高，但整体的五年生存率仍然很低。近年来，lncRNAs被证实在胃癌的发生发展中发挥着重要的调控作用。我们通过对GEO数据库进行分析（GSE53137），发现LINC00668是在胃癌组织中上调倍数最高的lncRNA。随后在胃癌组织及癌旁组织中验证LINC00668的表达，发现LINC00668在癌组织显著上调。LINC00668的表达与肿瘤的浸润深度，TNM分期密切相关，且高表达的 LINC00668提示胃癌病人的不良预后。生物信息学分析发现LINC00668启动子区含有潜在的保守的E2F1结合位点，并通过干扰/过表达，荧光素酶、ChIP等实验证明了E2F1可结合于LINC00668的启动子区，调控LINC00668的表达。接下来，在胃癌细胞中干扰或过表达LINC00668，可影响胃癌细胞的增殖、克隆形成能力及细胞周期。体内动物实验表明干扰LINC00668后，平均瘤体体积，重量减少。核质分离实验表明LINC00668主要表达于细胞核。同时发现，在胃癌细胞中干扰LINC00668后能够引起CKIs (p15，p16，p21，p27，p57)表达明显升高。RIP实验发现LINC00668与PRC2相对特异性结合，且LINC00668与EZH2/SUZ12的在胃癌组织中的表达呈正相关。ChIP实验证实干扰LINC00668后，EZH2，H3K27me3与p15, p16, p27 and p57的启动子区结合降低。本课题证实了E2F1调控的LINC00668可通过表观沉默CKIs调节胃癌细胞的增殖。该研究可能为临床上胃癌治疗提供重要的靶点。