MicroRNA-124 inhibits cancer cell growth （IF:5.621） MicroRNA-124 inhibits cancer cell growth through PTB1/PKM1/PKM2 feedback cascade in colorectal cancer 汇报人：侯仁浩 2015.11.26

背景知识 MiR-124：结直肠癌（Colorectal cancer，CRC） PTB1属于hnRNP家族，正常组织中低表达，癌组织中高表达 PKM1， PKM2—丙酮酸激酶（PKM）的亚型 PKM1特异表达于正常分化组织，促进氧化磷酸化 PKM2特异表达于胚胎，癌细胞，促进糖酵解 PKM2高表达有助于维持癌细胞生长 Warburg effect—癌细胞的生长需要大量的代谢变化， 癌细胞的代谢表型特征为糖酵解，而非有氧代谢，这个现象被称为Warburg effect。 IMI CONFIDENTIAL

本研究旨在阐明CRC中，MiR-124、PTB1、PKM1及PKM2的作用和相互关系 背景知识 在哺乳动物中，Warburg effect可通过调控PKM，从而选择性的少生成PKM1及多生成PKM2来实现。 本研究旨在阐明CRC中，MiR-124、PTB1、PKM1及PKM2的作用和相互关系 IMI CONFIDENTIAL

背景知识 实验方法：MiRNA-mimic、inhibitor转染和siRNA(siR-PTB1,PKM2) 转染、 荧光素酶报告实验、电镜、免疫荧光、乳酸测定与肿瘤移植 右磁共振波谱（ESR）:检测自由基 Hoechst33342染色：用于核染色，检测凋亡 实验材料： 组 织：来源于结直肠癌（CRC） 33位患者、结肠腺瘤（CRA）22位患者 细胞系：DLD-1和WiDr细胞系（活细胞数量通过台盼蓝染色确定） 抑制剂：Pan-caspase 抑制剂 : Z-VAD-fmk 自噬抑制剂: 3-MA 自由基清除剂: N-乙酰-L-半胱氨酸（NAC） Hoechst 33342是一种可以穿透细胞膜的蓝色荧光染料，对细胞的毒性较低。 染色常用于细胞凋亡检测，染色后用荧光显微镜观察 IMI CONFIDENTIAL

1. MiR-124 was down-regulated in clinical samples from CRA and CRC 正常的人组织 IMI CONFIDENTIAL

转染miR-124-mimic 下调结肠癌细胞系的活细胞率 2. MiR-124 induced apoptosis and/or autophagic survival in human colon cancer cells PI3K/Akt signaling 转染miR-124-mimic 下调结肠癌细胞系的活细胞率 IMI CONFIDENTIAL

N: 碎裂的核 ；V:小泡 ；M: 线粒体的自噬。 2. MiR-124 induced apoptosis and/or autophagic survival in human colon cancer cells N: 碎裂的核 ；V:小泡 ；M: 线粒体的自噬。 IMI CONFIDENTIAL

细胞自噬抑制剂 3-MA 和凋亡抑制剂Z-VAD逆转miR-124的效果 2. MiR-124 induced apoptosis and/or autophagic survival in human colon cancer cells 细胞自噬抑制剂 3-MA 和凋亡抑制剂Z-VAD逆转miR-124的效果 IMI CONFIDENTIAL

3 PTB1 is a direct target of miR-124 IMI CONFIDENTIAL

3 PTB1 is a direct target of miR-124 Anti-124逆转了miR-124的促凋亡效果，恢复了PTB1表达 IMI CONFIDENTIAL

小结1 MiR-124可以诱导结直肠癌细胞自噬和凋亡，此作用通过靶向抑制PTB1。 IMI CONFIDENTIAL

4 miR-124 repressed the expression of PKM2 and increased the expression of PKM1 PTB1能够抑制PKM的亚型PKM1的表达，诱导PKM转向表达PKM2。[1，2] IMI CONFIDENTIAL 1. C.J. David, et al. Nature 463 (2010) 364–368. 2. C.V. Clower, et al. Proc. Natl. Acad. Sci.U.S.A. 107 (2010) 1894–1899.

4 miR-124 repressed the expression of PKM2 and increased the expression of PKM1 Blue : DAPI IMI CONFIDENTIAL

5. Gene silencing of PTB1 induced apoptosis and/or autophagy IMI CONFIDENTIAL

5. Gene silencing of induced apoptosis and/or autophagy IMI CONFIDENTIAL

5. Gene silencing of PTB1 induced apoptosis and/or autophagy Hoechst33342对细胞核进行染色 IMI CONFIDENTIAL

5. Gene silencing of PTB1 increased the PKM1/PKM2 ratio 乳酸产生被极显著抑制 IMI CONFIDENTIAL

6. MiR-124, PTB1, PKM1, and PKM2 were involved in the feedback cascade IMI CONFIDENTIAL

6. MiR-124, PTB1, PKM1, and PKM2 were involved in the feedback cascade IMI CONFIDENTIAL

6. MiR-124, PTB1, PKM1, and PKM2 were involved in the feedback cascade IMI CONFIDENTIAL

小结2 Feedback cascade miR-124 PTB1 PKM2 此消彼长 PKM1 IMI CONFIDENTIAL

活性氧自由基主要激活TCA（三羧酸循环），诱导细胞凋亡和自噬 7.Oxidative stress was partly involved in growth inhibition and cell death induced by miR-124 活性氧自由基主要激活TCA（三羧酸循环），诱导细胞凋亡和自噬 IMI CONFIDENTIAL

7.Oxidative stress was partly involved in growth inhibition and cell death induced by miR-124 IMI CONFIDENTIAL

8. Anti-tumor effect of miR-124 or siR-PTB1 on DLD-1 cell xenografted tumor in nude mice IMI CONFIDENTIAL

8. Anti-tumor effect of miR-124 or siR-PTB1 on DLD-1 cell xenografted tumor in nude mice IMI CONFIDENTIAL

9. Down-regulation of miR-124 increased the ratio of PKM2/PKM1 in clinical CRA and CRC samples D: down ND: not down IMI CONFIDENTIAL

总结 IMI CONFIDENTIAL

谢 谢！ IMI CONFIDENTIAL