MeCP2调控lncRNA GPC5-AS1稳定GPC5 mRNA抑制胃癌细胞增殖的分子机制

Epigenetic regulation plays a key role in the process of tumorgenesis. A previous study by our group revealed an elevated expression of MeCP2 which could binds on the promoter region of a novel lncRNA GPC5-AS1 as well as decreases its expression in gastric cancer. Meanwhile, enforced GPC5-AS1 could accelerates the GPC5 expression on both mRNA and protein levels, and inhibits the cell proliferation. Here we hypothesize that the CpG islands of GPC5-AS1 was hypermethylation and its expression was epigenetic regulated by MeCP2, then play an anti-tumor effect through enhance GPC5 mRNA stability in gastric cancer. This study intends to (i) use BSP-PCR to detect the methylated sites and levels of CpG islands in GPC5-AS1 promoter region, and investigate the mechanisms of epigenetic regulation by MeCP2 with Chromatin Immunoprecipitation. (ii) analyze the regulation on GPC5 mRNA stability by GPC5-AS1 with RNA RIP and α-Amanitin, (iii) examine the expression of GPC5-AS1 and scrutinize its biological effects on gastric cancer cells by using CCK8, colony formation and flow cytometry in vitro, and (iv) investigate its influence on tumor formation in vivo through a xenograft model. The goal of this study is to illuminate the mechanism of dysregulations and functions of a novel lncRNA GPC5-AS1 in gastric cancer progression and offer a potential target or novel perspective for diagnosis and therapy in future.

表观遗传调控在肿瘤发生中起关键作用。本小组前期发现甲基化CpG结合蛋白MeCP2能结合lncRNA GPC5-AS1启动子区，影响其表达；上调GPC5-AS1可促进GPC5 mRNA与蛋白表达，抑制胃癌细胞增殖，提示GPC5-AS1是MeCP2的新靶点，可正向调控GPC5，发挥抑癌效应。本研究拟利用亚硫酸盐修饰测序法检测GPC5-AS1启动子区甲基化水平，ChIP解析MeCP2对其表达的表观调控机制；采用RNA RIP与α-Amanitin等方法分析GPC5-AS1对GPC5 mRNA稳定性的影响；明确胃癌中GPC5-AS1的表达与肿瘤发生相关性，通过CCK8、集落形成、流式细胞术等实验揭示其对胃癌细胞增殖的作用；慢病毒构建GPC5-AS1稳定表达细胞系，裸鼠皮下成瘤实验测定其在体内对肿瘤细胞成瘤能力的影响，为阐述GPC5-AS1在胃癌发生中的失调及作用机制与寻求潜在诊治靶点提供新的认识。

胃癌的发生发展是一种多因素参与、多基因改变的过程。近年研究表明表观遗传学改变也是胃癌发生发展的重要原因，对胃癌的诊断和治疗具有重要意义。MeCP2是表观调控的关键蛋白，课题前期通过免疫组化、Western Blot、qRT-PCR证实MeCP2在胃癌中表达显著上调并与G分期及肿瘤大小等临床病理特征相关。MeCP2通过与抑癌基因或原癌基因甲基化CpG位点结合调控其表达，进而影响肿瘤的发生发展。利用ChIP-seq技术联合高通量芯片对启动子区与MeCP2蛋白结合的潜在靶基因进行分析，发现有一富集丰度强且高度相关的新长非编码RNA：GPC5-AS1，其在胃癌中表达与功能均未见报道。应用染色质免疫共沉淀、报告基因系统、干扰RNA、TCGA大数据分析等方法证实MeCP2与GPC5-AS1在胃癌中表达呈负相关，MeCP2通过结合GPC5-AS1启动子区甲基化位点调控其表达，沉默MeCP2可促进GPC5-AS1表达。利用qRT-PCR证实GPC5-AS1在胃癌组织与细胞中表达均异常下调，构建GPC5-AS1表达载体，联合干扰shRNA，通过细胞功能学实验发现，过表达GPC5-AS1可抑制SGC-7901细胞增殖。流式细胞仪检测分析发现上调GPC5-AS1可诱导胃癌细胞凋亡，且胃癌细胞处于S期的比例明显减少。进一步的裸鼠成瘤实验证实了GPC5-AS1在体内对肿瘤细胞成瘤能力的抑制能力，明确了GPC5-AS1在胃癌发生发展中的抑癌定位。lncRNA可与微小RNA结合，作为其竞争性内源RNA发挥作用。GPC5-AS1与GPC5在胃癌中均表达下调，过表达GPC5-AS1能够诱导GPC5表达升高，抑制其RNA降解。生信分析发现miR-106家族成员既可与GPC5-AS1结合，也能靶向结合GPC5的3‘-UTR区。过表达GPC5-AS1后可观察到miR-93与miR-106a表达下调，且过表达miRNA能够逆转GPC5-AS1诱导的GPC5表达上调。同时发现miR-93与miR-106a在胃癌中均表达上调，其表达均与GPC5表达负相关。miR-93和miR-106a调控GPC5表达的靶向关系也得到了双荧光素酶报告基因的证实。本研究详尽的阐述一个新的lncRNA GPC5-AS1在胃癌中异常表达的表观修饰机制及其功能与作用方式，为寻求胃癌潜在治疗靶点与诊治手段提供新的思路。