Effects of PKM2 Knockdown on Proliferation and Apoptosis of Human Leukemia Cells and Its Potential Mechanism

doi:10.13523/j.cb.20190303

China Biotechnology

2019, Vol. 39

Issue (3): 13-20 DOI: 10.13523/j.cb.20190303

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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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 (t_LC3II=10.32, P_LC3II=0.000 5) and elevated p62 levels (t_p62=14.59, P_p62=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

Received: 21 October 2018 Published: 12 April 2019

ZTFLH:

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Corresponding Authors: Ling ZHANG E-mail: lingzhang@cqmu.edu.cn

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	Lu WANG
	Li-yuan YANG
	Yu-ting TANG
	Yao TAO
	Li LEI
	Yi-pei JING
	Xue-ke JIANG
	Ling ZHANG

Cite this article:

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.

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

Table 1 The qRT-PCR primer sequences for each gene

Fig. 1 Effect of PKM2 shRNA transfecting on the levels of PKM2 mRNA and protein in K562 cells (a) K562 cells were infected with shRNA lentivirus targeting PKM2 and its Vector control, qRT-PCR was performed to analyze PKM2 mRNA levels, normalized to β-actin (b) Western blot was performed to measure PKM2 protein levels, normalized to β-actin (c) The relative protein levels of PKM2 Vector means shRNA vector control group, shPKM2 means PKM2 knockdown lentivirus group. * P<0.05, compared with the Vector group

Fig.2 Effect of PKM2 knockdown on proliferation of K562 cells in vitro * P<0.05, compared with the Vector group

Fig.3 Effects of PKM2 knockdown on cell cycle in K562 cells by FCM

Table 2 Effects of PKM2 knockdown on cell cycle distribution

Fig.4 Effect of PKM2 knockdown on the apoptosis of K562 cells (a) The apoptosis of K562 cells by FCM (b) The apoptosis rate of K562 cells (c) Western blot was performed to measure Bax and Bcl-2 protein levels (d) The relative protein levels of Bax and Bcl-2 Vector means shRNA vector control group, shPKM2 means PKM2 knockdown lentivirus group. * P<0.05, compared with the Vector group

Fig.5 Effect of PKM2 knockdown on the autophagic activity in K562 cells (a) qRT-PCR was performed to analyze LC3、p62 mRNA levels (b) Western blot was performed to measure LC3、p62 protein levels (c) The relative protein levels of LC3II、p62. Vector means shRNA vector control group, shPKM2 means PKM2 knockdown lentivirus group. * P<0.05, compared with the Vector group

Fig.6 Effects of PKM2 knockdown combined with autophagy activator on the proliferation of K562 cells in vitro * P<0.05, compared with the Vector group; # P<0.05, compared with the shPKM2+Rapamycin group


[1]	Pfrepper C, Klink A, Behre G , et al. Risk factors for outcome in refractory acute myeloid leukemia patients treated with a combination of fludarabine, cytarabine, and amsacrine followed by a reduced-intensity conditioning and allogeneic stem cell transplantation. J Cancer Res Clin Oncol, 2016,142(1):317-324. doi: 10.1007/s00432-015-2050-y pmid: 26424692

[2]	Jang M, Kim S S, Lee J . Cancer cell metabolism: Implications for therapeutic targets. Exp Mol Med, 2013,45:e45. doi: 10.1038/emm.2013.85 pmid: 24091747

[3]	Dayton T L, Jacks T , Vander Heiden M G . PKM2, cancer metabolism, and the road ahead. EMBO Rep, 2016,17(12):1721-1730. doi: 10.15252/embr.201643300 pmid: 27856534

[4]	Yang W, Lu Z . Pyruvate kinase M2 at a glance. J Cell Sci, 2015,128(9):1655-1660. doi: 10.1242/jcs.166629 pmid: 25770102

[5]	Orsini M, Morceau F, Dicato M , et al. Autophagy as a pharmacological target in hematopoiesis and hematological disorders. Biochem Pharmacol, 2018,152:347-361. doi: 10.1016/j.bcp.2018.04.007 pmid: 29656115

[6]	Takahashi H, Inoue J, Sakaguchi K , et al. Autophagy is required for cell survival under L-asparaginase-induced metabolic stress in acute lymphoblastic leukemia cells. Oncogene, 2017,36(30):4267-4276. doi: 10.1038/onc.2017.59

[7]	Cheng T Y, Yang Y C, Wang H P , et al. Pyruvate kinase M2 promotes pancreatic ductal adenocarcinoma invasion and metastasis through phosphorylation and stabilization of PAK2 protein. Oncogene, 2018,37(13):1730-1742. doi: 10.1038/s41388-017-0086-y

[8]	Wang Y H, Israelsen W J, Lee D , et al. Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis. Cell, 2014,158(6):1309-1323. doi: 10.1016/j.cell.2014.07.048 pmid: 25215489

[9]	Panchabhai S, Schlam I, Sebastian S , et al. PKM2 and other key regulators of Warburg effect positively correlate with CD147 (EMMPRIN) gene expression and predict survival in multiple myeloma. Leukemia, 2017,31(4):991-994. doi: 10.1038/leu.2016.389 pmid: 28025580

[10]	Lin Y, Meng F, Lu Z , et al. Knockdown of PKM2 suppresses tumor progression in human cervical cancer by modulating epithelial-mesenchymal transition via Wnt/beta-catenin signaling. Cancer Manag Res, 2018,10:4191-4202. doi: 10.2147/CMAR

[11]	Goldberg M S, Sharp P A . Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression. J Exp Med, 2012,209(2):217-224. doi: 10.1084/jem.20111487 pmid: 22271574

[12]	Zheng B, Liu F, Zeng L , et al. Overexpression of pyruvate kinase type M2 (PKM2) promotes ovarian cancer cell growth and survival via regulation of cell cycle progression related with upregulated CCND1 and downregulated CDKN1A expression. Med Sci Monit, 2018,24:3103-3112. doi: 10.12659/MSM.907490 pmid: 29752805

[13]	Tooze S A, Dikic I . Autophagy captures the Nobel Prize. Cell, 2016,167(6):1433-1435. doi: 10.1016/j.cell.2016.11.023 pmid: 27912049

[14]	Polak R , Bierings M B, van der Leije C S , et al. Autophagy inhibition as a potential future targeted therapy for ETV6-RUNX1 driven B-cell precursor acute lymphoblastic leukemia. Haematologica, 2018, DOI: 10.3324/haematol.2018.193631. doi: 10.3324/haematol.2018.193631

[15]	Piya S, Andreeff M, Borthakur G . Targeting autophagy to overcome chemoresistance in acute myleogenous leukemia. Autophagy, 2017,13(1):214-215. doi: 10.1080/15548627.2016.1245263 pmid: 27797294

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