化疗压力下EGFR基因环化增强促进胶质瘤耐药及上皮间质转化的机制研究

Under the pressure of TMZ chemotherapy in glioma, the circulation of EGFR gene is enhanced, and chemotherapy resistance and EMT also rises up. According to early experiments, combined with bioinformatics analysis, we hypothesize that the RNA binding protein IGF2BP1 binds with pre mRNA of EGFR and induces the circulation to generate. The high expression of Hsa-circ_0080229 can lead to drug resistance and EMT. we furthermore hypothesize that Hsa-circ_0080229 can bind with RNA binding protein HuR, induce the output from nuclear. RNA binding protein HuR can bind with beta-catenin mRNA and accelerate the expression. We further speculated that Hsa-circ_0080229 can act as molecular sponge to adsorb and inhibit miR1827 then remove the targeted inhibition of functional protein. the former 2 pathway can lead to drug resistance and EMT of glioma. RNA IP,RNA pull down, immunofluorescence co-localization, luciferase reporter gene, cell proliferation, apoptosis, invasion and other experiments will be developed to verify this conjecture. And clinical specimen will be exploited to test moreover.

在替莫唑胺化疗压力下，EGFR基因环化增强，胶质瘤出现化疗抵抗以及上皮间质转化。 根据前期研究，我们推测蛋白IGF2BP1结合EGFR的pre-mRNA，诱导其环化生成Hsa-circ_0080229，后者表达的增强，导致胶质瘤的耐药，同时促进胶质瘤的上皮间质转化。进一步推测该circRNA结合HuR蛋白，诱导该蛋白出核，与胞浆中的beta-catenin mRNA结合，促进其表达。同时推测该circRNA能够作为分子海绵，吸附抑制miR1827，解除miR对靶蛋白的抑制。以上两种途径共同导致beta-catenin表达升高，促进胶质瘤的耐药和上皮间质转化。我们拟通过RNA IP，RNA pull down，免疫荧光共定位，荧光素酶报告基因，细胞增殖、凋亡、侵袭等实验来研究这一课题，同时在临床标本中加以验证。

胶质瘤是颅内最常见的原发恶性肿瘤。研究发现，许多非编码RNA在胶质瘤调控中发挥重要作用。据报道，环状RNA可以通过若干种途径参与到胶质瘤的分子调控当中。本课题聚焦于EGFR基因的一个环状RNA hsa_circ_0080229（circ-0080229），探讨circ-0080229调控胶质瘤增殖、迁移与侵袭的能力，并探索circ-0080229调控胶质瘤的分子机制。结果显示，circ-0080229在胶质瘤中显著高表达，并且其表达量与胶质瘤的病理级别呈显著正相关。我们用荧光素酶报告基因实验证实circ-0080229能靶向结合miR-1827，而miR-1827能够靶向结合下游MDM2 mRNA的3’非编码端(3’UTR)。结果显示，下调circ-0080229后能够抑制U87和U251胶质瘤细胞株的增殖、迁移、侵袭能力，而再转染miR-1827 inhibitor可以部分逆转其变化。同时，裸鼠皮下成瘤实验提示：下调circ-0080229的表达可以抑制在体胶质瘤的增殖进展。最后，qPCR及western blot实验提示，下调circ-0080229能通过促进miR-1827的表达来抑制MDM2的表达。我们通过标记RNA结合蛋白纯化实验拉取了circ-0080229所能结合的RNA结合蛋白，同时利用TCGA数据库对它们进行差异表达分析及生存分析，结果提示：ANXA2在胶质母细胞瘤（GBM）中相对较低级别胶质瘤显著高表达，且是胶质瘤患者预后不良的显著指标。在体外细胞实验中，下调ANXA2后能够显著抑制U251胶质瘤细胞的增殖、迁移及侵袭能力。结论：circ-0080229可以通过靶向miR-1827/ MDM2通路来促进胶质瘤的增殖、迁移与侵袭能力；同时circ-0080229能与多个RNA结合蛋白结合，并且可能通过特异性结合ANXA2协同促进胶质瘤的增殖、迁移与侵袭。