| 摘要: |
| 目的 筛选敲减哺乳动物雷帕霉素靶蛋白（mTOR）基因后胶质瘤细胞表达上调的基因及信号通路，探讨联合阻断mTOR信号通路及其旁路激活通路抑制胶质瘤细胞生长的有效性。方法 选取5种人胶质瘤细胞系（U87、U251、U373、T98、LN229），采用蛋白质印迹法验证mTOR蛋白表达。构建靶向mTOR基因的短发夹RNA稳定转染的U87细胞，采用高通量测序筛选敲减mTOR基因细胞中表达上调最显著的基因及信号通路。利用细胞药物敏感试验筛选抑制率最高的通路抑制剂，用CCK-8实验进行细胞活性分析。结果 筛选出mTOR蛋白相对高表达的U87细胞建立mTOR基因敲减胶质瘤细胞模型。高通量测序共筛选出24 528个新转录本和1 906个差异表达基因，选取的log2|差异倍数|排在前12位的表达上调基因分别位于9条旁路激活通路。药物敏感试验结果显示信号转导因子和转录激活因子3（STAT3）通路抑制剂的抑制率最高，体外实验证实STAT3通路抑制剂可增强mTOR基因敲减对U87细胞增殖的抑制效果（P<0.05）。结论 敲减人胶质瘤细胞mTOR基因能激活旁路信号通路，联合应用旁路激活通路抑制剂可增强肿瘤抑制效果。 |
| 关键词: 哺乳动物雷帕霉素靶蛋白 神经胶质瘤 信号转导因子和转录激活因子3 高通量测序 |
| DOI：10.16781/j.0258-879x.2021.06.0596 |
| 投稿时间：2020-12-02修订日期：2021-01-07 |
| 基金项目:国家自然科学基金（81671206），上海市科学技术委员会国际科技合作项目（09410705100，14430721300），军队“十二五” 重点课题（BWS12J025）. |
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| Mechanism of combined blockade of mammalian target of rapamycin signaling pathway and its bypass activation pathway inhibiting glioma cell growth |
| HAN Shuo,LI Yi-ming,ZHANG Dan-feng,HAN Kai-wei,HUANG Zhen-yu,HOU Li-jun* |
(Department of Neurosurgery, Changzheng Hospital, Naval Medical University (Second Military Medical University), Shanghai 200003, China
*Corresponding author) |
| Abstract: |
| Objective To screen up-regulated genes and signaling pathways in glioma cells after knockdown of mammalian target of rapamycin (mTOR) gene, and to explore the efficacy of simultaneously blocking mTOR signaling pathway and its bypass activation pathways in inhibiting the growth of glioma cells. Methods Five human glioma cell lines U87, U251, U373, T98, and LN229 were selected to verify the protein expression of mTOR by Western blotting. U87 cells stably transfected with short hairpin RNA targeting mTOR gene were constructed, and the most significantly up-regulated genes and signaling pathways were screened in glioma cells after knockdown of mTOR gene by high-throughput sequencing. The pathway inhibitor with the highest inhibition rate was screened by cell drug sensitivity test. The cell activity was analyzed by cell counting kit 8. Results High mTOR expression cell line U87 was screened out, and the mTOR gene knockdown glioma cell model was successfully constructed. A total of 24 528 new transcripts and 1 906 differentially expressed genes were screened out by high-throughput sequencing. The top 12 up-regulated genes with high log2|fold change|value were located in 9 bypass activation pathways. The inhibitor of signal transducers and activators of transcription 3 (STAT3) pathway with the highest inhibitory activity was screened out by drug sensitivity test. In vitro experiments showed that the STAT3 pathway inhibitor could increase the inhibition effect of mTOR gene knockdown on the proliferation of U87 cells (P<0.05). Conclusion Knockdown of mTOR gene in human glioma cells can activate the bypass signaling pathways. The inhibition effect can be effectively enhanced by combined use of inhibitors of bypass activation pathways. |
| Key words: mammalian target of rapamycin glioma signal transducers and activators of transcription 3 high throughput sequencing |
