C19orf10 promotes malignant behaviors of human bladder carcinoma cells via regulating the PI3K/AKT and Wnt/β-catenin pathways

Abstract

Background: Chromosome 19 open reading frame 10 (C19orf10) is a myocardial repair mediator overexpressed in hepatocellular carcinoma. However, its function and clinical value in bladder cancer (BC) have not been reported. This study aimed to investigate the role of C19orf10 in BC progression and explore underlying mechanisms. Methods: C19orf10 expression in BC tissues and human BC cell lines was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The correlation between the C19orf10 protein levels determined by immunohistochemical staining and the clinicopathological characteristics of 192 BC patients was evaluated. BC cell lines SW780, J82 and UMUC-3 were transfected with small interfering RNA (siRNA) targeting C19orf10 or plasmids overexpressing C19orf10. Cell proliferation, migration and invasion were measured by Cell Counting Kit-8, Colony formation, EdU incorporation and Transwell assays. The effect of small hairpin RNA (shRNA)-mediated stable C19orf10 knockdown on tumor formation was assessed in a xenograft mouse model. The expressions of epithelial-mesenchymal transition (EMT) markers, PI3K/AKT and Wnt/β-catenin signaling pathways-related molecules were determined by western blot assay. Results: C19orf10 was significantly upregulated in the BC tissues and a panel of human BC cell lines. High expression of C19orf10 was positively associated with malignant behaviors in BC. C19orf10 knockdown inhibited cell proliferation, migration, and invasion in SW780 and J82 cells, while C19orf10 overexpression in UMUC-3 cells resulted in opposite effects. In addition, C19orf10 silence in SW780 cells suppressed tumor growth in xenograft mice. Moreover, C19orf10 promotes the malignant behaviors and EMT of human bladder carcinoma cells via regulating the PI3K/AKT and Wnt/β-catenin pathways. Conclusion: C19orf10 is overexpressed in BC and functions as an oncogenic driver that promotes cell proliferation and metastasis, and induces EMT of BC cells via mechanisms involving activation of the PI3K/AKT and Wnt/β-catenin pathways. This study provides valuable insight on targeting C19orf10 for BC treatment.

Keywords: C19orf10; PI3K/AKT; Wnt/β-catenin; bladder cancer; migration and invasion; proliferation.

Figure 1

High expression of C19orf10 in bladder carcinoma tissues and bladder carcinoma cell lines. (A) Transcriptome sequencing was performed to analyze the C19orf10 mRNA levels in nine pairs of BC tissues and adjacent normal tissues. The expression level of C19orf10 in the adjacent normal tissue of each pair was set as 1, and the relative expression level in the BC tissue was calculated. (B) The expression levels of C19orf10 in BC tissues and normal bladder tissues from The Cancer Genome Atlas database were compared. (C) The C19orf10 mRNA levels in tissues from 42 pairs of BC and adjacent tissues were analyzed by qPCR. (D) The C19orf10 protein levels in five pairs of BC tissues and adjacent normal tissues were determined by western blot. (E) The C19orf10 mRNA levels in the normal bladder epithelial cell line SV-HUC-1 and six different BC cell lines were analyzed using qPCR. n = 3 for each group; **P < 0.01, ***P < 0.001, compared with the SV-HUC-1 group. (F) The C19orf10 protein levels in SV-HUC-1 cells and six different BC cell lines were determined by western blot analysis. Representative western blot images are shown.

Figure 2

Representative immunohistochemical images of C19orf10 expression in normal human bladder epithelial tissue and BC tissue from patients with different pathological grades. Scale bar, 100 µm.

Figure 3

Silencing of C19orf10 inhibited the migration and invasion of BC cells. (A-B) The mRNA (A) and protein (B) expression levels of C19orf10 were determined by qPCR and western blot assays, respectively. **P < 0.01, ***P < 0.001. (C-D) Transwell assays were used to assess the migration and invasion of SW780 cells (C) and J82 (D) cells at 48 h after transfection of negative control or C19orf10-specific siRNA oligos. Representative images of cell migration and invasion in the transwell experiments are shown (C-D), and the relative cell motility was quantitated. n = 3 for each group; ***P < 0.001, compared with the control group.

Figure 4

Silencing of C19orf10 inhibited the proliferation and colony formation of BC cells. (A-D) SW780 and J82 cells were transfected with negative control or C19orf10-specific siRNA oligos, and cell proliferation and colony formation were assessed at 48 h after transfection. (A-B) The cell viability after silencing C19orf10 in SW780 (A) and J82 (B) cells was measured using the CCK-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001. (C)The cell proliferation rates of SW780 cells and J82 cells after silencing C19orf10 were assessed using a colony formation assay, and the number of cell colonies was normalized to that of the negative control group. n = 3 for each group; ***P < 0.001, compared with the control group. (D) Detection of cell proliferation by EdU incorporation assay. The relative proliferation rate was quantitated by calculating the percent of EdU-positive cells. *P < 0.05, ***P < 0.001.

Figure 5

Overexpression of C19orf10 promoted the proliferation, migration and invasion of BC cells. (A-B) The mRNA (A) and protein (B) expression levels of C19orf10 in UMUC-3 cells after transfection of negative control (NC) or C19orf10-expressing plasmids were determined by qPCR and western blot assays, respectively. ***P < 0.001. (C) Transwell assays were used to assess the migration and invasion of UMUC-3 cells at 48 h after transfection of NC or C19orf10-expressing plasmids. Representative images of cell migration and invasion in the transwell experiments are shown (C), and the relative cell motility was quantitated. n = 3 for each group; ***P < 0.001, compared with the control group. (D) CCK-8 assay was performed to evaluate the cell viability for different days (1-3 day). *P<0.05, **P<0.01. (E) Colony formation assay was used to assess cell proliferation (left panel) and quantification (right panel) of formation by counting total colony numbers. ***P < 0.001. (F) Detection of cell proliferation by EdU incorporation assay. The relative proliferation rate was quantitated by calculating the percent of EdU-positive cells. **P<0.01.

Figure 6

Deficiency of C19orf10 inhibited the malignant growth of subcutaneous cancer xenografts in vivo. (A) The protein levels of C19orf10 in stable SW780 cells with infection of control or C19orf10-shRNA-expressing lentivirus were determined by western blot assays. (B) The infection efficiency of lentivirus in the above cells was observed under a fluorescence microscope. (C) Growth curves show the tumor sizes in nude mice inoculated with the indicated SW780 cell lines (NC, sh-C19orf10#1, and sh-C19orf10#3 cells) during an observation time of four weeks. n=5 per group; *P < 0.05, **P < 0.01. (D-E) Mice were sacrificed at 4 weeks after tumor cells inoculation. The images of mice at end-point are shown (D), and the excised tumor tissues were images and weighed (E). *P < 0.05, **P < 0.01. (F) Representative images of immunohistochemical staining (IHC) analysis of PCNA and Ki67 protein levels in tumor tissues. Scale bars, 100 µm.

Figure 7

C19orf10 promotes malignant behaviors and EMT of human bladder carcinoma cells via regulating the PI3K/AKT and Wnt/β-catenin pathway. (A) Western blotting analysis of PI3K, p-AKT, total AKT, p21, and p27 expressions in control or C19orf10 siRNA-expressing SW780 and J82 cells. (B) Western blotting analysis of PI3K, p-AKT, AKT (pan), p21, and p27 expressions in control or C19orf10-overexpressed UMUC-3 cells. (C) Western blotting analysis of N-cadherin, Vimentin, Snail, Slug, β-catenin, and p-β-catenin expressions in control or C19orf10 siRNA-expressing SW780 and J82 cells. (D) Western blotting analysis of N-cadherin, Vimentin, Snail, Slug, β-catenin, and p-β-catenin in control or C19orf10-overexpressed UMUC-3 cells.