Table 1.
Clinicopathological features of 21 patients with glioma.
Fig 1.
Immunohistochemistry to detect the expression of HK2 in glioma tissues.
HK2 localized mainly to the cytoplasm. The expression of HK2 in different grades of glioma samples: (A). Non-neoplastic brain tissue; (B). Pilocytic astrocytomas (WHO I); (C). Diffuse astrocytomas (WHO II); (D). Anaplasia astrocytomas (WHO III); (E). Primary glioblastomas (WHO IV). Original magnification, 200×. (F). The immunoreactive score demonstrating a significant positive correlation between HK2 expression and glioma grade. Error bars represent SEM. **, P<0.01.
Fig 2.
Silencing HK2 expression of glioma cells.
(A). Successfully established HK2 silenced stable U87 and U251 cells (B). qRT-PCR analysis demonstrated the downregulation of HK2 expression in transfected U87 and U251 cells. (C). Western-blot analysis demonstrating the downregulation of HK2 protein in transfected U87 and U251 cells. Error bars represent SEM. ***, P<0.001.
Fig 3.
Silencing HK2 inhibits glioma cell invasion, migration, and proliferation in vitro and tumorigenesis in vivo.
(A). Growth curves after cell transfections were assessed by CCK-8 assay. The rate of cellular proliferation was significantly decreased in both the HK2-silenced cell lines (B-C). The wound healing assay showed a significant difference in cell migration between transfected U87/U251 cells and their corresponding control. (B). Representative images taken at different time points. (C). Quantification of cell motility by measuring the wound width. The amount of motility was expressed as a migration percentage, setting the width of the wound at 0 hour as 0%. The rate of cellular migration was significantly decreased in both the HK2-silenced cell lines. (D-E). Transwell assay of transfected glioma cells. (D). Representative fields of invaded cells on the membrane. (E). Quantitative analysis of the invasive cells from three independent experiments. The rate of cellular invasion was significantly decreased in both the HK2-silenced cell lines. (F). In-vivo tumorigenesis; Nude mice were subcutaneously injected with HK2 silenced stable U87 cells and their corresponding negative controls (seeding density = 3.0×106). (G). Determination of the tumor growth. Tumor volume was calculated every two days after injection, demonstrating a significant (p = 0.011) decrease in the tumor volume in HK2 silenced stable U87 cells. The mean volumes of xenograft tumors generated from HK2-silenced U87 cells were significantly smaller than those originating from its negative control cells. (H). IHC and H&E staining of xenograft tumor tissues. Magnification, 100×. Error bars represent SEM, *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 4.
HK2 levels are inversely correlated with miR-218 levels in glioma cells, but HK2 is not a direct target of miR-218; miR-218 may regulate HK2 through Bmi1.
(A). Establishment of stable LV-infected U87 and U251 cells overexpressing miR-218. (B). qRT-PCR analysis confirmed the increased miR-218 expression in glioma cell lines. (C). Overexpression of miR-218 down-regulated the mRNA level of HK2. (D). Overexpression of miR-218 down-regulated the protein level of HK2. (E). Luciferase reporter assay showed HK2 was not the direct target of miR-218. (F). Establishment of Bmi1 knocked-down stable U87 and U251 cells. (G). Knocking-down Bmi1 decreased the HK2 mRNA level. (H). Knocking-down Bmi1 also decreased the HK2 protein level.