DTX2 promotes glioma development via regulation of HLTF

Abstract

Background: Human Deltex 2 (DTX2) is a ubiquitin E3 ligase that functions as an oncogene and has been shown to participate in many human cancers. However, the role of DTX2 in glioma progression has remained obscure. In this study, we explore the mechanism underlying the function of DTX2 in glioma progression.

Methods: The associations between DTX2 expression and clinical characteristics of glioma were determined by bioinformatic analysis of data from The Cancer Genome Atlas and Human Protein Atlas. The expression of DTX2 in glioma tissues was detected using immunohistochemistry and western blotting. Lentivirus-mediated gene knockdown and overexpression were used to determine the effects of DTX2 and helicase-like transcription element (HLTF) on glioma cell proliferation and migration with CCK-8, cell colony formation, transwell, and wound healing assays; flow cytometry in vitro; and animal models in vivo. The interaction of the DTX2 and HLTF proteins was verified by immunoprecipitation assay and confocal microscopy.

Results: DTX2 was highly expressed in glioma samples, and this was correlated with worse overall survival. Silencing of DTX2 suppressed glioma cell viability, colony formation, and migration and induced cell apoptosis. In vitro ubiquitination assays confirmed that DTX2 could downregulate HLTF protein levels by increasing ubiquitination of the HLTF protein. We also observed that HLTF inhibited proliferation and migration of glioma cells. Subcutaneous xenografts with DTX2-overexpressing U87 cells showed significantly increased tumor volumes and weights.

Conclusions: We have identified DTX2/HLTF as a new axis in the development of glioma that could serve as a prognostic or therapeutic marker.

Keywords: DTX2; Glioma; HLTF; Ubiquitination.

Fig. 1

DTX2 is highly expressed and predicts poor prognosis in glioma samples. A Differences in mRNA levels of DTX2 in 33 cancer and paraneoplastic tissues from TCGA. B Differential expression of DTX2 between normal tissues from the Genotype-Tissue Expression (GTEx) database and cancer tissues from TCGA. C, D Differential expression of DTX2 in LGG (C) and glioblastoma (GBM) (D) was further analyzed in combination with normal brain tissue data from the GTEx database. Blue and red represent normal and tumor tissues, respectively. E DTX2 exhibited favorable prognostic value within the glioma cohort, particularly concerning OS, DSS, and PFI patterns. (Low: median =  < 50%; High: median > 50%). F AUC values of time-dependent ROC curves for the past decade. G Kaplan–Meier survival plots for glioma patients with high and low DTX2 expression from HPA. (Low: negative and weak staining; High: median and strong staining). H IHC staining to investigate the expression of DTX2 in clinical glioma tissues. I DTX2 protein expression in glioma tissues (n = 3) and controls (n = 3) as detected using western blotting

Fig. 2

DTX2 promotes cell proliferation. A Western blots demonstrating the knockdown efficiency of DTX2. B Role of DTX2 knockdown in glioma cell propagation as determined by CCK-8 assay (**P < 0.01, ***P < 0.001). C Numbers of colonies formed by U87 and U251 cells transfected with shDTX2#1 and shDTX2#2 (**P < 0.01, ***P < 0.001). D Western blots showing the overexpression efficiency of DTX2 following transfection with overexpression plasmids. E Role of DTX2 overexpression in glioma cell proliferation as determined by CCK-8 assay (**P < 0.01). F Numbers of colonies formed by U87 and U251 cells transfected with DTX2-overexpression plasmids (**P < 0.01, ***P < 0.001)

Fig. 3

DTX2 promotes cell migration and invasion of glioma cells. A, B The role of DTX2 knockdown in the migration (A) and invasion (B) of glioma cells as determined by transwell assay (***P < 0.001). C, D Role of DTX2 overexpression in the migration (C) and invasion (D) of glioma cells as determined by transwell assay (***P < 0.001). (E, F) Wound healing assay showing cell migration of glioma cells after DTX2 knockdown (E) and overexpression (F) (***P < 0.001)

Fig. 4

Interaction between DTX2 and HLTF. A HLTF protein was verified using immunoprecipitation. B Immunofluorescence signals showing colocalization of DTX2 and HLTF. C HLTF expression in glioma cells after DTX2 knockdown and overexpression as investigated by western blotting. D Role of DTX2 knockdown and overexpression in HLTF ubiquitination in glioma cells. E HLTF protein expression in glioma tissues (clinical samples)

Fig. 5

HLTF inhibits cell proliferation and migration of glioma cells. A The knockdown efficiency of HLTF in glioma cells was confirmed using western blotting. B The role of HLTF knock down in glioma cell proliferation was detected using the CCK-8 assay. (**P < 0.001). C Numbers of colonies formed by U87 and U251 cells transfected with shHLTF #1 and shHLTF #2. (***P < 0.001). D The effects of HLTF knock down in glioma cell migration were investigated using a Transwell assay. (***P < 0.001). E The efficiency of HLTF overexpression using HLTF overexpression plasmids in glioma cells was confirmed using western blotting. F The role of HLTF overexpression in glioma cell proliferation was detected using the CCK-8 assay. (***P < 0.001). G Numbers of colonies formed by U87 and U251 cells transfected with HLTF overexpression plasmids. (***P < 0.001). H The effects of HLTF overexpression in glioma cell migration were investigated using a Transwell assay. (H) The effects of HLTF in glioma cell migration were investigated using the wound healing assay

Fig. 6

DTX2/HLTF axis contributes to glioma cell proliferation and migration through HLTF. A The expression of DTX2 and HLTF was detected DTX2 knock down and double knock down of DTX2 and HLTF (shDTX2#1 + shHLTF#1) in U87 and U251 cells by western blot. B, C The role of DTX2 and HLTF knock down in glioma cell proliferation was detected in U87 and U251 cells using the CCK-8 assay. (***P < 0.001). D Numbers of colonies formed by U87 and U251 cells with DTX2 knock down and double knock down of DTX2 and HLTF (shDTX2#1 + shHLTF#1) in U87 and U251 cells (***P < 0.001). E The effects of DTX2 and HLTF knock down in glioma cell migration were investigated using a Transwell assay. (***P < 0.001)

Fig. 7

DTX2 promotes the development of glioma through HLTF in vivo. A Representative images of tumor size in DTX2 overexpression in the tumor xenograft model. B, C Tumor volume and tumor weight in mice xenografts in the Ctrl and DTX2 groups. (**P < 0.01). D The expression of DTX2 and HLTF in mice tumors in the Ctrl and DTX2 groups detected using western blot. E Representative images of tumor size in shCtrl, shDTX2 and shDTX2 + shHLTF xenograft models. F, G Tumor volume and tumor weight in shCtrl, shDTX2 and shDTX2 + shHLTF xenograft models. (**P < 0.01). H The expression of DTX2 and HLTF in mice tumors in shCtrl, shDTX2 and shDTX2 + shHLTF mice tumors detected using western blot