高压氧对mHESM的成熟调控在胶质瘤干细胞化疗增敏中的作用及机制研究

Hypoxic microenvironment is a main factor that maintains the chemotherapy resistance of glioma stem cells (GSCs). The preliminary studies show that the HBO combined with chemotherapy can prolong the survival time of transplantation tumors of GSCs in animal and play a role in the chemo-sensitization, but its mechanism has not been known clearly. According to some literatures, mHESM (miRNA regulated by hypoxia-dependent EGFR-suppressed maturation) refers to a miRNA that has the ability to promote tumor because of the maturation arrest in the hypoxic environment. It is supposed that HBO could play a role in the chemo-sensitization in this project. In order to verify such a hypothesis, multidrug-resistant cell lines of GSCs selected and determined previously, in combination with experimental platform of HBO, are applied to verify the main change types of mHESM and its phosphorylate change of mature splicing forms AGO2-Dicer with the intervention of HBO through conducting experimental screening in both vivo and vitro, with a view to determine the effect of mHESMs on HBO chemo-sensitization and potential mechanism through expression and inhibition assay. The study results will reveal the potential mechanism of regulation levels after the transcription of the effect of HBO on the chemo-sensitization , so as to provide a new idea for the targeted treatment strategy of GSCs.

缺氧微环境是维持胶质瘤干细胞（GSCs）化疗抵抗的主因，我们前期研究提示高压氧（HBO）联合化疗可延长胶质瘤干细胞移植瘤动物生存期,发挥化疗增敏作用，但机制不清，根据文献，mHESM(miRNAs regulated by hypoxia-dependent EGFR-suppressed maturation)为一类在缺氧环境下成熟受阻从而促进肿瘤进展的miRNAs。本项目推测, HBO通过影响mHESM成熟过程发挥化疗增敏作用。为验证假说,拟采用前期筛选鉴定的GSCs多药耐药细胞珠结合HBO实验平台,通过体内、外实验筛选并鉴定GSCs在HBO干预下主要mHESM改变种类及其对应成熟相关蛋白AGO2的磷酸化变化，通过过表达和抑制实验明确重点mHESMs在HBO化疗增敏中的作用及可能机制。研究结果将揭示HBO化疗增敏作用在转录后调控水平的可能机制，为GSCs靶向治疗策略提供新思路。

胶质瘤是现今最恶性的颅内肿瘤，平均生存期不到一年，并且展现出严重的化疗抵抗。本研究致力探索一种与传统化疗药物联用的方法，以增加化疗的敏感性。研究发现高压氧结合TMZ和ACNU较之单纯化疗组，其生存期缩短。在离体实验中，通过cck-8实验及BrdU标定细胞周期实验发现高压氧干预后U87胶质瘤细胞系的细胞增殖能力增强，细胞侵袭能力增强，高压氧结合化疗药物TMZ，ACNU及CIS较之单纯化疗组，细胞增殖能力及细胞侵袭能力增加。综合以上结果示高压氧会钝化化疗疗效。MRI检测U87荷瘤鼠颅内肿瘤，分析肿瘤体积示高压氧干预后肿瘤体积的增长加快。芯片分析高压氧及常氧对照组颅内原位瘤标本，经芯片筛选出的变化趋势显著的miR-A miR-B miR-D miR-E miR-F；经Qpcr筛选，不同氧干预条件下变化显著的为miR-C，miR-G。实验结果显示miR-B显著增高，miR-A/D/E/F显著降低。敲减miR-A后，细胞增殖能力下降，凋亡比例增加，细胞侵袭能力下降，过表达miR-A后恢复。过表达miR-A后，细胞凋亡比例显著增加，增殖能力下降，细胞侵袭能力下降，敲减后恢复正常；过表达miR-C后，U87/U251细胞系早期凋亡显著减少，反之，凋亡增加，较之对照组有显著差异。在cck8检测转染miR27 agomir/antagomir对细胞增殖的影响，结果显示：miR-27促进细胞增殖。