Mechanisms of pyruvate kinase M2 isoform inhibits cell motility in hepatocellular carcinoma cells

Figure 1 Knockdown of pyruvate kinase M2 isoform suppressed cell proliferation in HepG2 and Huh-7 cells. A: HepG2 and Huh-7 cells were stably transfected with shRNA directed against pyruvate kinase M2 isoform (PKM2). The specific knockdown of PKM2 was monitored by immunoblot (bottom). Cells stably transfected with pcPUR + U6-siPKM2 are referred to as siPK, and those transfected with pcPUR + U6-siRenilla are referred to as pu6; B: PKM2 expression levels were analyzed by quantitative real-time PCR in stable HepG2 and Huh-7 cells. Error bars represent the mean ± SD of triplicate experiments (^aP < 0.05 between groups); C and D: Proliferation of the stably transfected cells. The cell number was determined with the CCK-8 assay, and the relative number of cells is shown.

Figure 2 Knockdown of pyruvate kinase M2 isoform promoted the migration and invasion of HepG2 and Huh-7 cells upon epidermal growth factor stimulation. A: A cross-shaped wound was created in the monolayer, and stably transfected HepG2 and Huh-7 cells were cultured for an additional 24 h with epidermal growth factor (EGF) (50 ng/mL). Representative images of the wounded region are shown; B: The results of the migration assay are also shown as graphs (^aP < 0.05); C: The invasion potential of the HepG2 and Huh-7 stable cells was assessed using the BD transwell chamber assay with 50 ng/mL EGF in the lower chamber for 36 h. The cells that migrated to the lower side of the filter were stained, photographed, and counted; D: The data are expressed as the mean ± SD from three independent experiments, ^aP < 0.05 between groups.

Figure 3 Differential gene expression between HepG2-pu6 and HepG2-siPK cells. A: The number of statistically significant differentially expressed transcripts identified from the F-test. Color plots show the expression levels of significantly differentially expressed transcripts (P < 0.05 between groups and false discovery rate 0.05) between HepG2-pu6 and HepG2-siPK cells. The upregulated genes are indicated in red, and the downregulated genes are indicated in green. Genes that did not show a statistically significant difference are indicated in blue; B: The graph shows gene expression in relation to cell migration and invasion. Fold differences were calculated based on the log2 ratio of siPK/pu6; C: A comparison of the RT-qPCR and RNA sequence expression analysis; D: The expression levels of genes that were determined to have statistically significant differences in expression were analyzed by immunoblot analysis in stable HepG2 and Huh-7 cells.

Figure 4 Depletion of pyruvate kinase M2 enhanced the activities of the epidermal growth factor/EGFR and transforming growth factor beta 1/TGFBR downstream signaling pathways. A: Stable HepG2 and Huh-7 cells were exposed to epidermal growth factor (EGF) (50 ng/mL) for different times. Western blots of the cell lysates are shown. The protein levels of phospho-EGFR (Tyr¹⁰⁶⁸), phospho-PLCγ1 (Tyr⁷⁸³), and phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) are shown as indicated; B: The expression of E-cadherin was knocked down in HepG2 and Huh-7 cells by transient transfection of siRNA, and after 48 h, these cells were stimulated with EGF (50 ng/mL). The protein levels of phospho-EGFR (Tyr¹⁰⁶⁸) and phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) are shown as indicated; C and D: MMP1 expression levels were analyzed by quantitative real-time PCR in HepG2 and Huh-7 stable cells. Error bars represent the mean ± SD of triplicate experiments (^aP < 0.05 between groups); E: Phospho-Smad2 (Ser^465/467) protein levels are shown as indicated in stable HepG2 and Huh-7 cells stimulated with TGFβ1 (20 ng/mL); F: The expression of TGFBRI was knocked down in HepG2 and Huh-7 cells by transient transfection of siRNA, and after 48 h, these cells were stimulated with transforming growth factor beta 1(TGFβ1) (20 ng/mL). The protein levels of phospho-Smad2 (Ser^465/467) are shown as indicated.