HOXD-AS1调控miR-20a介导的神经胶质瘤细胞替莫唑胺耐药的机制研究

Glioma is the most common malignant tumor in central nervous system. It has been well known that temozolomide-resistance (TMZ-resistance) in glioblastoma cells is a major obstacle in the treatment of glioblastoma. However, the molecular mechanism of glioblastoma chemoresistance is still unclear. Our preliminary results showed that miR-20a mediated TMZ-resistance in glioblastoma cells via negatively regulating LRIG1 expression. As one candidate of putative targets determined by bioinformatics analysis (Starbase 2.0), HOXD-AS1 stood out through detailed survey. However, it is poorly understood with the effect of HOXD-AS1 on the TMZ-resistance in glioblastoma cells. Based on the foregoing issues, we speculate that HOXD-AS1 down-regulating miR-20a expression may suppress the temozolomide-resistance in glioblastoma cells. In present study, clinical samples with glioblastoma, glioblastoma cells, and the subcutaneous xenografts in nude mice are utilized and the methods like real time PCR, Western blot, establishment of TMZ-resistant cells and RNA interference are performed. The significant role of HOXD-AS1 in TMZ-resistance of glioblastoma cells will be explored from the respects of tissues, cells, molecules and animals. We aim to explicit the molecular mechanism of HOXD-AS1 through regulating the expression of miR-20a to inhibit the TMZ-resistance of glioblastoma cells. This study will lay the foundations for bringing insight into the molecular mechanism of TMZ-resistance in glioblastoma cells from the new perspective of HOXD-AS1/miR-20a, and provide new ideas for the prevention of temozolomide-resistance development in glioblastoma.

胶质瘤是最常见的神经系统恶性肿瘤，替莫唑胺(TMZ)是治疗胶质瘤的一线药物。但胶质瘤细胞对TMZ耐药是治疗失败的主因，具体分子机制不清。我们前期研究显示miR-20a调控胶质瘤细胞TMZ耐药。生物信息学在线预测发现miR-20a与HOXD-AS1具有潜在结合位点，而HOXD-AS1在胶质瘤细胞TMZ耐药中的作用知之甚少。为此我们提出假说，HOXD-AS1可能通过抑制miR-20a的表达降低胶质瘤细胞TMZ耐药发生。为验证此假说，我们将通过胶质瘤临床样本、胶质瘤细胞系、裸鼠移植瘤模型，从组织、细胞及动物水平探讨HOXD-AS1在胶质瘤细胞TMZ耐药中的作用，明确HOXD-AS1通过调节miR-20a的表达降低胶质瘤细胞TMZ耐药的分子机制。本研究将从HOXD-AS1 / miR-20a这个新视点为揭示胶质瘤细胞TMZ耐药的分子机制奠定基础，为胶质瘤TMZ耐药的防治提供新思路。

胶质瘤是由脑和脊髓中的胶质细胞癌变引起的最常见的原发性颅脑恶性肿瘤，具有高发生率，高复发率，高死亡率和低治愈率的特点。研究表明26S蛋白酶体参与许多生物学过程，例如细胞周期进程、细胞凋亡、代谢调节和信号转导。PSMC2是26S蛋白酶体19S调控亚基的关键成员。PSMC2的部分基因组缺失在多种肿瘤中被发现，并且PSMC2与肿瘤细胞密切相关，这表明PSMC2可能是治疗肿瘤的潜在靶标。目前，PSMC2在多种癌症中呈现高表达且与肿瘤的进展密切相关。.本项研究发现，PSMC2在神经脑胶质瘤组织和正常组织中存在明显的表达差异，且在肿瘤组织中高表达。此外，通过U87和U251细胞功能实验，我们发现PSMC2的敲除可以明显抑制肿瘤细胞的增殖和克隆形成，促进细胞凋亡。体内实验同样证明，PSMC2的敲除可以显著抑制肿瘤的生长。通过表达谱芯片对shCtrl和shPSMC2两组细胞的差异基因进行IPA分析及实验验证发现，E2F1作为PSMC2的潜在下游目标，在mRNA和蛋白水平均出现显著下调。功能实验验证证明，PSMC2和E2F1的敲减可以显著抑制肿瘤细胞的增殖和迁移，促进细胞的周期阻滞。上述这些结果初步提示，PSMC2可以通过调控E2F1的表达进而促进脑胶质瘤的进程。