OLFM2入核正向调控TGF-β自分泌环路促进GBM细胞获得干细胞特性的机制研究

The remaining stem cells existed in the resection margin of glioblastoma(GBM) are the origin of tumor recurrence, transforming growth factor-β(TGF-β) is one of the most important cytokines induced stemness of GBM cells. However, the mechanism of TGF-β highly express and autocrine in GBM still remains unclear. Previously, we have cultured several paired of primary GBM cells isolated from tumor core and peritumoral region. Through transcriptomics analyses and verification of cell experiments, we have found that the expression of TGF-β, TGF-β receptor and OLFM2 were up-regulated in peritomoral GBM cells, and these cells were easier to gain stem cell characteristics. Through further experiments we have confirmed that both TGF-β1 and TGF-β2 could induce the nucleus translocation of OLFM2, and OLFM2 could bond with SRF (serum responding factor) which is a transcription factor. Further, OLFM2 is predicted to bond with transport protein KPNA2 and SRF is predicted to bond with the promoter region of TGF-β1/2 promotor region after bioinformatic analyses. In this research, using advanced methods such as Crispr/Cas9 and two-photon imaging, in vivo and vitro, we will confirm that TGFβ/SMADs/KPNA2 axis promote the expression and nucleus translocation of OLFM2, a complex formed with transcription factor SRF and OLFM2 which act as a co-activate factor could bond with TGF-β1/2 promotor region and promote the expression and autocrine of TGF-β1/2 in GBM cells, the TGF-β1/2 autocrine loop result in the stem cell transformation of GBM cells and lead to the tumor recurrence finally. We will reveal the key role OLFM2 played in GBM stem cell transformation and tumor recurrence, and provide molecular oncotarget of GBM therapy.

术后残存于瘤腔边缘的胶质母细胞瘤（GBM）干细胞是导致复发的根源，TGFβ是诱导GBM细胞获得干性的重要因子，但GBM中高表达、自分泌TGFβ的机制仍待阐明。我们前期从GBM瘤心、瘤周分离了多对肿瘤细胞，转录组学及实验发现：GBM瘤周细胞高表达TGFβ、TGFβ受体、OLFM2，且更容易发生干细胞转变；进一步实验发现：TGFβ1/2可促进OLFM2入核，OLFM2可结合转录因子SRF；转运蛋白KPNA2预测可结合OLFM2，SRF预测可结合TGF1/2启动子区。本研究将运用双光子成像、Crispr/Cas9等技术，在体内、体外验证：TGFβ/SMADs/KPNA2促进OLFM2表达及入核，OLFM2入核后结合SRF并作用于TGFβ启动子区，促进GBM细胞高表达、自分泌TGFβ，并获得干细胞特性。本项目将揭示OLFM2在GBM细胞获得干性、导致复发中的关键作用，为GBM的治疗提供候选靶点。

胶质母细胞瘤（GBM）是颅内最为常见的原发恶性脑肿瘤，术后残存的肿瘤干细胞是导致肿瘤治疗抵抗、复发进展及治疗失败的根源，分子机制仍不明确。为研究GBM不同区域肿瘤干性差异以及介导治疗抵抗及复发的分子机制，本项目：（1）利用双光子成像策略，研究GBM肿瘤中心（T1增强）、水肿带（T2/FLAIR异常）以及相对正常区域（正常影像）的肿瘤干细胞、微环境差异，并鉴定OLFM2等关键分子指标的差异；（2）室管膜下区（SVZ）被认为是GBM肿瘤干细胞起源的地方，利用临床组织标本、动物造模等工具，研究GBM肿瘤与室管膜之间的相对关系，并鉴定相关分子指标；（3）研究关键转运蛋白DHC2与OLFM2蛋白之间关系，并鉴定DHC2蛋白介导GBM细胞治疗抵抗与复发的分子机制。研究结果发现：（1）GBM的不同区域存在显著的微环境差异，肿瘤周边区域干细胞特性更强；（2）室管膜对室管膜下区的GBM肿瘤干细胞、GBM肿瘤细胞存在非机械应力的阻隔作用，可能与高表达某些蛋白分子相关；（3）关键转运蛋白DHC2转运DNA损伤修复蛋白进入细胞核内是介导GBM细胞产生治疗抵抗的关键机制。本项目阐明了GBM中肿瘤不同区域干细胞特性、微环境的差异，同时也阐明了介导GBM治疗抵抗的关键分子机制。本项目可为GBM治疗提供候选靶点。