Hypoxia inducible factor-1 HIF-1 promotes myeloid-derived suppressor cell accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma

Transcriptome sequencing data HC

PKM2 expression in human HCC.

<p>(A) mRNA expression of PKL, PKM1, and PKM2 in HCC and NT tissues. Values = 2^ΔΔCT, ΔΔCT = (CT_PK – CT_HPRT) of HCC - (CT_PK– CT_HPRT) of NT. <i>P</i> values, Wilcoxin signed rank test (B) Waterfall plot shows that, at the mRNA level, PKM2 was up-regulated (HCC/NT2 folds) in 29/60 (48.33%) human HCC samples. (C) Representative pictures of IHC staining with antibody against PKM2 in HCC tissue microarray. PKM2 protein was drastically up-regulated in human HCCs as compared to the paired NT tissues. (D) Mann Whitney test showed that PKM2 over-expression was associated with multiple aggressive clinicopathological features in HCC including the presence of tumor microsatellites, presence of venous invasion, and absence of tumor encapsulation. (E) Over-expression of PKM2 in human HCC was associated with poor prognosis. HCC patients were categorized into two groups: PKM2 over-expression and PKM2 normal/under-expression. PKM2 was considered to be over-expressed when HCC/NT2 folds and was considered to be normal/under-expressed otherwise. HCC patients with PKM2 over-expression had a higher 1-year tumor recurrence rate after surgical resection than HCC patients without PKM2 over-expression, 46.667% Vs 25%. (F) Patients with PKM2 over-expression had lower 5-year overall survival rates after surgical resection. <i>P</i> values were calculated by Kaplan-Meir log rank test.</p

PKM2 promoted HCC growth <i>in</i><i>vitro</i> through regulating aerobic glycolysis and ROS levels.

<p>(A) Two stable PKM2 knockdown clones were generated in MHCC-97L and SMMC-7721 cells. Expression of PKM2, PKM1, and β actin were evaluated by Western Blots. (B) Knockdown of PKM2 by two independent sequences consistently reduced HCC cell proliferation rate by cell counting. (C) Knockdown of PKM2 reduced lactate accumulation in multiple HCC cell lines. (D) Colorimetric assay showed that knockdown of PKM2 reduced the glucose consumption rate of multiple HCC cell lines. (E) Glucose uptake in HCC cells was confirmed with 2-NBDG staining. (F) Knockdown of PKM2 increased ROS accumulation in multiple HCC cells. (G) Knockdown of PKM2 decreased NADPH level in SMMC-7721 cells. Values were normalized to NTC of the according cell lines. *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i><0.001 Student’s <i>t</i> test (n≧3).</p

PKM2 promoted HCC growth <i>in</i><i>vivo</i>.

<p>(A) Left: Subcutaneous tumors derived from SMMC-NTC, -shPKM2 cells. Middle: volumes (mm³) of SMMC-NTC and -shPKM2 tumors were measured and plotted against time. Right: mass (g) of SMMC-NTC, -shPKM2 tumors were measured at the end of the experiment. (**<i>P</i><0.01, Student’s t test) (B) Left: subcutaneous tumors derived from MHCC-97L-NTC and -shPKM2 cells. Middle: volumes (mm³) of MHCC-97L-NTC and -shPKM2 tumors were measured and plotted against time. Right: mass (g) of MHCC-97L-NTC and -shPKM2 tumors were measured at the end of the experiment. (C) Left: orthotopic tumors derived from MHCC-97L-NTC and -shPKM2 cells. Right: Tumor volume was measured at the end of the experiment. (D) Left: bioluminescent signals of the lung tissues in the mice orthotopically implanted with luciferase labeled-MHCC-97L-NTC and -shPKM2 cells. Right: mRNA expression of human hexokinase 2 (HK2) in lung tissues of mice orthotopically implanted with luciferase-labeled MHCC-97L-NTC and -shPKM2 cells. Values were normalized to mouse GAPDH. *<i>P</i><0.05, **<i>P</i><0.01,***<i>P</i><0.001, Student’s t test. Scale: 1 cm.</p

Re-expression of miR-122 suppressed HCC growth through modulating aerobic glycolysis.

<p>(A) miR-122 expression in MHCC-97L cells stably expressing miR-122 precursors. MiR-122 expression was normalized to U6 expression and to empty vector (EV) control. (B) Lactate accumulation was reduced in miR-122 over-expressing MHCC-97L cells. (C) Glucose uptake rate was reduced in miR-122 over-expressing MHCC-97L cells. (D) Glucose uptake in MHCC-97L-EV and –miR-122 cells was confirmed with 2-NBDG staining. (E) Glucose uptake in SMMC-EV and –miR-122 cells was confirmed with 2-NBDG staining. (F) Glucose uptake in PLC/PRF/5 cells transfected with LNA-Ctrl and LNA-miR-122. (G) Left: Orthotopic tumors derived from MHCC-97L-EV and -miR-122 subclones. Right: Tumor volume was measured at the end of the experiment. (H) Bioluminescence (left) and H&E staining (right) in lung tissues from mice implanted with MHCC-97L-EV and –miR-122 subclones. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001, Student’s t test or paired t test. Scale: 1 cm.</p