Fig 2: miR-6072 was identified to bind to both UBD and UBDP1. (A) Fluorescence in situ hybridization and immunofluorescence assay (magnification, ×400) determined the subcellular localization of UBDP1 and UBD in the cytoplasm. (B and C) Sequencing analysis detected potential binding sites of miR-6072 and miR-6818-3p in UBD mRNA and UBDP1. (D) The expression of UBD protein in different conditions of miR-6072 or miR-6818-3p expression pattern was determined by western blotting. (E and F) WT or MT UBDP1 or UBD vectors were transfected into the 293T cell lines with or without synthetic miR-6072 mimics, and luciferase assays demonstrated significantly decreased luciferase activities in both WT UBDP1 and WT UBD groups with miR-6072 mimics. n=3. ***P<0.001. miR, microRNA; WT, wild-type; MT, mutant; NC, negative control.

Fig 3: UBDP1 and UBD aggravate the malignancy of glioma cells. (A and B) Cell Counting Kit-8 assay revealed that overexpression of either UBDP1 or UBD could confer an enhanced proliferation ability on glioma cells, whereas knocking down UBDP1 or UBD inhibited cell proliferation. (C and D) Transwell assay (magnification, ×100) displayed an increased number of migratory glioma cells in the UBDP1 or UBD overexpressing group compared with control groups but decreased glioma cells in the UBDP1 or UBD knocked down group. (E and F) Matrigel assay (magnification, ×100) demonstrated stronger invasion of U87 and U251 with overexpression of UBDP1 or UBD but significantly less invasion ability of U87 and U251 with inhibition of UBDP1 or UBD when compared with that of NC. n=3. *P<0.05, **P<0.01, and ***P<0.001. KD, knockdown; NC, negative control; OE, overexpression.

Fig 4: UBD and UBDP1 could competitively bind to miR-6072 in glioma. (A) Western blot analysis detected the expression of UBD protein in different conditions of miR-6072 expression pattern and found miR-6072 inhibitor significantly upregulating UBD expression. (B) The expression of UBD protein was increased in glioma cells with UBDP1 overexpression but was decreased in the condition of UBDP1 inhibition. (C) Elevated expression of UBD protein in glioma cells with UBDP1 could be restrained when cells were co-transfected with miR-6072 mimics. (D) Cell Counting Kit-8 assay demonstrated that enhanced proliferation ability of glioma cells induced by UBDP1 overexpression could be restored by miR-6072 mimics. (E and F) Transwell and Matrigel assays (magnification, ×40) assessed the migration and invasion abilities of glioma cells in different conditions and revealed the inhibitory effects of miR-6072 mimics in glioma cells with UBDP1 overexpression. n=3. *P<0.05, **P<0.01 and ***P<0.001. miR, microRNA; KD, knockdown; NC, negative control; OE, overexpression.

Fig 5: Pseudogene UBDP1 is upregulated and correlated with poor outcomes in GBM. (A) MA plot revealed dysregulated pseudogenes in GBM (G, glioblastoma; N, normal brain). (B) Heatmap demonstrated the differently expressed pseudogenes between GBM and normal brain. (C) Reverse transcription-quantitative PCR of 30 GBM samples and 15 normal brain tissues confirmed both the expression of UBDP1 and UBD upregulated in GBM compared with normal brain. (D) Pearson correlation analysis determined a strong relationship between UBDP1 and UBD expression of the 30 GBM samples. (E and F) Kaplan-Meier curve showed either high UBDP1 or high UBD expression associated with a short overall survival time of patients with GBM. n=3. ***P<0.001. GBM, glioblastoma.