Pseudogene OCT4-pg5 upregulates OCT4B expression to promote bladder cancer progression by competing with miR-145-5p

Abstract

Bladder cancer (BC) is one of the most common malignant neoplasms worldwide. Competing endogenous RNA (ceRNA) networks may identify potential biomarkers associated with the progression and prognosis of BC. The OCT4-pg5/miR-145-5p/OCT4B ceRNA network was found to be related to the progression and prognosis of BC. OCT4-pg5 expression was significantly higher in BC cell lines than in normal bladder cells, with OCT4-pg5 expression correlating with OCT4B expression and advanced tumor grade. Overexpression of OCT4-pg5 and OCT4B promoted the proliferation and invasion of BC cells, whereas miR-145-5p suppressed these activities. The 3' untranslated region (3'UTR) of OCT4-pg5 competed for miR-145-5p, thereby increasing OCT4B expression. In addition, OCT4-pg5 promoted epithelial-mesenchymal transition (EMT) by activating the Wnt/β-catenin pathway and upregulating the expression of matrix metalloproteinases (MMPs) 2 and 9 as well as the transcription factors zinc finger E-box binding homeobox (ZEB) 1 and 2. Elevated expression of OCT4-pg5 and OCT4B reduced the sensitivity of BC cells to cisplatin by reducing apoptosis and increasing the proportion of cells in G1. The OCT4-pg5/miR-145-5p/OCT4B axis promotes the progression of BC by inducing EMT via the Wnt/β-catenin pathway and enhances cisplatin resistance. This axis may represent a therapeutic target in patients with BC.

Keywords: OCT4-pg5; OCT4B; bladder cancer; competing endogenous RNA; miR-145-5p.

Figure 1.

OCT4-pg5 is upregulated and associated with poor prognosis in bladder cancer (BC). (a) expression of OCT4-pg5 in the TCGA BLCA cohort. (b) identification of differentially expressed molecules. A total of 133 DElncRNAs, 175 DEmiRNAs, and 250 DEmRNAs were identified. Red indicates RNAs upregulated in BCs and blue indicates RNAs downregulated in BCs. (c) the triple regulatory network in BCs. Red indicates miRnas, blue indicates mRnas, and green indicates lncRnas. (d) three hub RNAs in the network with a score of > 2. (e) functional enrichment analysis (GO and KEGG) of the DERNAs in the network. (f) Kaplan-Meier analysis of overall survival in BLCA patients in the TCGA with high and low expression of the three hub RNAs. Comparisons by log-rank tests. (g) levels of OCT4-pg5 expression in BC tissues (n = 140) and adjacent non-cancerous tissues (n = 34). (h) OCT4-pg5 mRNA expression levels in patients with different histological grades of BC, including papillary urothelial neoplasms of low malignant potential (PUN-LMP), low grade (LG), and high grade (HG) BCs. (i) relationship between OCT4-pg5 and OCT4B mRNA expression levels in BC tissues. (j)OCT4-pg5 mRNA expression in several bladder cell lines. ***p < 0.001, *p < 0.05.

Figure 2.

OCT4-pg5 and OCT4B function as oncogenes in BC cells in vitro. (a,b) CCK-8 assays showing numbers of viable (a) T24 and (b) 5637 cells over time following transfection with the indicated transcripts. (c,d) Colony formation assays estimating the proliferation rates of (c) T24 and (d) 5637 cells. (e,f) transwell assays determining the invasion capacities of (e) T24 and (f) 5637 cells. ***p < 0.001, *p < 0.01, ***p < 0.05.

Figure 3.

OCT4-pg5 3’UTR regulates the expression of OCT4B by sequestering miR-145-5p. (a) RNAhybrid analysis of the binding affinity and possible secondary structure. (b) luciferase activities of OCT4B reporters in T24 and 5637 BC cells. Cells were transfected with luciferase reports, along with miR-145-5p mimics, wild-type OCT4-pg5 3’UTR, or mutant OCT4-pg5 3’UTR. (c-e) relative levels of expression of OCT4B, miR-145-5p, and OCT4-pg5 mRnas in T24 and 5637 cells, as measured by qRT-PCR. (f) relative levels of expression of OCT4B protein in T24 and 5637 cells, as measured by Western blotting, after the indicated transfections. Densitometric measurements are shown to the right of each representative blot. (g) immunofluorescence analysis of OCT4B expression in T24 and 5637 cells transfected with the indicated vectors. Red: anti-OCT4B. Blue: DAPI nuclear staining. ***p < 0.001, *p < 0.01, ***p < 0.05.

Figure 4.

OCT4-pg5 promotes metastasis by inducing EMT through the Wnt/β-catenin pathway. (a-d) relative levels of expression of N-cadherin, vimentin, and E-cadherin mRnas in T24 and 5637 cells after transfection, as measured by RT-PCR. (e) relative levels of expression of E-cadherin, vimentin, and β-catenin proteins in T24 and 5637 cells after transfection, as measured by Western blotting (normalized to GAPDH). (f) relative levels of expression of MMP2, MMP9, ZEB1, and ZEB2 proteins in T24 and 5637 cells after transfection as measured by Western blotting (normalized to GAPDH). (g-i) immunofluorescence staining of T24 and 5637 cells with antibodies to E-cadherin, N-cadherin, and β-catenin in T24 cells and 5637 cells. Red: anti-E-cadherin, anti-N-cadherin, and anti-β-catenin. Blue: DAPI nuclear staining. ***p < 0.001, *p < 0.01, ***p < 0.05.

Figure 5.

Crosstalk between OCT4-pg5 and OCT4B enhances the resistance of T24 cells to cisplatin. (a,b) levels of expression of (a) OCT4-pg5 and (b) OCT4B mRnas in T24 cells as measured by qRT-PCR. (c) Western blot analysis of OCT4B protein level in T24 cells. GAPDH was used as the control. (d) effects of OCT4-pg5 and OCT4B on the apoptosis of T24 cells as measured by flow cytometry. UL: necrotic cells, UR: terminal apoptotic cells, LR: early apoptotic cells, LL: normal cells. Cells in UR and LR were counted and analyzed. (e) effects of OCT4-pg5 and OCT4B expression on the cell cycle stage distribution of T24 cells, as measured by flow cytometry. ***p < 0.001, *p < 0.01, ***p < 0.05.

Figure 6.

OCT4-pg5 promotes the tumorigenicity and enhances the resistance of BC cells to cisplatin in vivo. (a) tumor growth in vivo following OCT4-pg5 silencing in T24 cells. (b) tumor growth in vivo following OCT4-pg5 overexpressed in 5637 cells. (c) bioluminescence images from the control and OCT4-pg5 overexpressed groups. ***p < 0.01, *p < 0.05.