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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (3): 13-20    DOI: 10.13523/j.cb.20190303
研究报告     
干扰PKM2对人白血病细胞增殖和凋亡的影响及潜在机制 *
汪路,杨丽媛,唐雨婷,陶瑶,雷力,敬一佩,蒋雪坷,张伶()
重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆市重点实验室 重庆 400016
Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism
Lu WANG,Li-yuan YANG,Yu-ting TANG,Yao TAO,Li LEI,Yi-pei JING,Xue-ke JIANG,Ling ZHANG()
College of Laboratory Medicine, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education,Chongqing Medical University, Chongqing 400016, China
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摘要：

目的: 探讨糖酵解酶丙酮酸激酶M2型(PKM2)对人白血病细胞体外增殖和凋亡的影响及潜在机制。方法: 将靶向PKM2的慢病毒载体转染人K562细胞株(shPKM2组),同时设立空载体转染组为对照(Vector组)。采用qRT-PCR和Western blot技术分别检测Vector组和shPKM2组PKM2 mRNA和蛋白的表达以及自噬标志物的变化;CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期和凋亡情况;Western blot检测凋亡相关蛋白Bax、Bcl-2表达水平。结果: 在稳定干扰PKM2后,PKM2的mRNA(t=11.58,P=0.000 3)和蛋白水平(t=11.88,P=0.000 3)均明显降低。与Vector组比较,shPKM2组细胞体外增殖能力显著降低(F=118.87,P<0.000 1)。同时,干扰PKM2可使K562细胞周期阻滞在G1期,细胞凋亡率显著增加(t=37.23,P<0.000 1);使促凋亡蛋白Bax表达增加(t=15.36,P=0.000 1)、抗凋亡蛋白Bcl-2表达降低(t=9.965,P=0.000 6)。此外,干扰PKM2可减弱K562细胞自噬水平,表现为LC3II降低(tLC3II=10.32,PLC3II=0.000 5),而p62水平增加(tp62=14.59,Pp62=0.000 1)。还发现自噬诱导剂能逆转shPKM2引起的白血病细胞体外增殖能力减弱(F=96.32,P<0.000 1)。结论: 以上结果表明干扰PKM2可抑制人白血病K562细胞的体外增殖并促进其凋亡,其机制可能与PKM2介导的自噬活性降低有关,提示PKM2可能作为白血病诊疗的一个潜在靶点。
关键词： PKM2;  白血病;  增殖;  凋亡;  自噬    
Abstract:

Objective: Effects of PKM2 knockdown on proliferation and apoptosis of human leukemia cells and its potential mechanism were investigated in our experiment. Methods: Lentivirus-based short hairpin RNA (shRNA) vector targeting PKM2 was transfected into K562 cells (shPKM2 group), and the vector-treated cells were named as the Vector group. mRNA and protein levels of PKM2 in K562 cells were determined by qRT-PCR and Western blot techniques, respectively. Cell proliferation activity was evaluated by CCK-8 assay in vitro. Cell cycle and apoptosis rate were analyzed by flow cytometry, the expression levels of apoptosis-related proteins Bax and Bcl-2 were measured by Western blot and the autophagy activity was measured by qRT-PCR and Western blot, respectively. Results: mRNA (t=11.58, P=0.000 3) and protein (t=11.88, P=0.000 3) levels of PKM2 were significantly decreased after PKM2 knockdown in K562 cells. In comparison to the Vector group, cell proliferation was inhibited by PKM2 deleption in shPKM2 group (F=118.87, P<0.000 1). Furthermore, G1-phase cell cycle was arrested, and the cell apoptosis rate was increased after PKM2 knockdown (t=37.23, P<0.000 1). Meanwhile, upregulated pro-apoptotic Bax protein levels (t=15.3, P=0.000 1) and downregulated anti-apoptotic Bcl-2 protein levels (t=9.965, P=0.000 6) were observed in shPKM2 group compared with the Vector group. In addition, decreased expression of PKM2 significantly downregulated LC3II levels (tLC3II=10.32, PLC3II=0.000 5) and elevated p62 levels (tp62=14.59, Pp62=0.000 1) in K562 cells. Finally, autophagy activator rapamycin rescued the inhibitory cell proliferation due to PKM2 knockdown (F=96.32, P<0.000 1). Conclusion: Above-mentioned results indicate that PKM2 knockdown can inhibit cell proliferation and promote cell apoptosis, at least partially through the cell autophagy, and PKM2 might be a potential target in the treatment of leukemia.
Key words: PKM2    Leukemia    Proliferation    Apoptosis    Autophagy
收稿日期: 2018-10-21 出版日期: 2019-04-12
ZTFLH:  Q291  
基金资助: * 国家自然科学基金面上项目(81873973);重庆市渝中区科技计划(20170411);重庆市研究生科研创新资助项目(CYS17155)
通讯作者: 张伶     E-mail: lingzhang@cqmu.edu.cn
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汪路
杨丽媛
唐雨婷
陶瑶
雷力
敬一佩
蒋雪坷
张伶

引用本文:

汪路,杨丽媛,唐雨婷,陶瑶,雷力,敬一佩,蒋雪坷,张伶. 干扰PKM2对人白血病细胞增殖和凋亡的影响及潜在机制 *[J]. 中国生物工程杂志, 2019, 39(3): 13-20.

Lu WANG,Li-yuan YANG,Yu-ting TANG,Yao TAO,Li LEI,Yi-pei JING,Xue-ke JIANG,Ling ZHANG. Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism. China Biotechnology, 2019, 39(3): 13-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190303    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I3/13

基因Genes 序列Sequences(5' - 3')
PKM2 F: 5'-GCCTGCTGTGTCGGAGAAG-3'
R: 5'-CAGATGCCTTGCGGATGAATG-3'
LC3 F: 5'-GACCGCTGTAAGGAGGTGC-3'
R: 5'-CTTGACCAACTCGCTCATGTTA-3'
p62 F: 5'-GGGGACTTGGTTGCCTTTT-3'
R: 5'-CAGCCATCGCAGATCACATT-3'
β-actin F: 5'-TAGTTGCGTTACACCCTTTCTTG-3'
R: 5'-TGCTGTCACCTTCACCGTTC-3'
表1  定量PCR所用引物序列
图1  慢病毒干扰PKM2对K562细胞PKM2 mRNA和蛋白表达水平的影响
图2  干扰PKM2对K562细胞体外增殖的影响
图3  流式细胞术检测干扰PKM2对K562细胞周期的影响
Group G1(%) G2(%) S(%)
Vector 28.14±1.65 3.31±0.30 68.56±1.95
shPKM2 44.59±0.15* 17.15±0.76* 38.26±0.62*
表2  干扰PKM2对K562细胞周期分布的影响
图4  干扰PKM2对K562细胞凋亡的影响
图5  干扰PKM2对K562细胞自噬活性的影响
图6  干扰PKM2联合自噬诱导剂对K562细胞增殖的影响
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