Abstract
The objectives of the present study are to explore role of pyruvate kinase isoenzyme type M2 (PKM2) in progression of Kazakh’s esophageal squamous cell carcinoma (ESCC) in Xinjiang, China, and to clarify mechanism of PKM2 in malignant phenotype. PKM2 expression was examined using immunohistochemistry (IHC) in 101 matched pairs of ESCC and normal adjacent tissues (NATs) and using enzyme-linked immunosorbent assay (ELISA) in 35 serum samples of Kazakh’s ESCC and 8 serum samples of healthy subjects. To investigate mechanism, small interfering RNA (siRNA)-PKM2 was transfected into ESCC cells. Cell migration and invasion were evaluated by wound healing and Transwell assays. Apoptosis and cell cycle were analyzed by flow cytometry (FCM). PKM2 expression was significantly higher in ESCC tissues (77.2 %, 78/101) compared with matched NAT (P = 0.003) and also higher in serum samples of Kazakh’s ESCC patients (78.84 ng/mL) compared with healthy subjects (13.55 ng/mL) (P = 0.001). Patients with overexpression of PKM2 had a poor prognosis (P = 0.032). After knockdown of PKM2, cell proliferation, migration, and invasion were significantly reduced (P = 0.001), apoptosis increased (P = 0.001), and cell cycle was arrested at G1 phase. PKM2 overexpression was significantly correlated with the worse outcome of Kazakh’s ESCC. Furthermore, PKM2 was involved in progression of ESCC by promoting proliferation and suppressing apoptosis, accelerating invasion, and influencing cell cycle. PKM2 could be a potential biomarker for molecular classification of ESCC.
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Acknowledgments
This project was supported by the Natural Science Foundation of China (Nos. 81160303, 81260359, 81201891, U1303321) and support from Major Science and Technology Projects of the Xinjiang Uygur Autonomous Region (No. 201430123-1).
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Supplementary figure 1
Specificity of primary antibody against human PKM2 was evaluated using antigen preadsorption test method. A: primary antibody against human PKM2 was diluted at 1:100, followed by incubation with formalin-fixed paraffin-embeded ESCC section. B: primary antibody against human PKM2 was incubated with the synthetic peptide of PKM2 (from human PKM2 ELISA kit) at 50ug/mL, followed by incubation with formalin-fixed paraffin-embeded ESCC section. The magnification was 200×. (DOC 859 kb)
Supplementary figure 2
Observation of Eca109 cells transfected with siRNA-PKM2 by fluorescence microscopy (Magnification × 100). A: 24 h after transfection with siRNA-PKM2; B. 48 h after transfection with siRNA-PKM2; C. 72 h after transfection with siRNA-PKM2. (DOCX 810 kb)
Supplementary figure 3
Expression of PKM2 mRNA levels in Eca109 after siRNA-PKM2 transfection. Expression level of PKM2 mRNA in Eca109 was significantly down regulated after transfection with siRNA-PKM2 as compared with negative control and untreated cells. *P < 0.05, **P < 0.01 (DOCX 52 kb)
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Liu, Q., Liang, M., Liu, T. et al. M2 isoform of pyruvate kinase (PKM2) is upregulated in Kazakh’s ESCC and promotes proliferation and migration of ESCC cells. Tumor Biol. 37, 2665–2672 (2016). https://doi.org/10.1007/s13277-015-4073-z
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DOI: https://doi.org/10.1007/s13277-015-4073-z