Occludin facilitates tumour angiogenesis in bladder cancer by regulating IL8/STAT3 through STAT4

Abstract

Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT-qPCR analysis and Western blotting results showed that OCLN increased interleukin-8 (IL8) and p-signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual-luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.

Keywords: IL8; OCLN; bladder cancer; tumour angiogenesis.

FIGURE 1

Occludin (OCLN) expression is associated with the prognosis of patients with bladder cancer (BLCA). (A) Kaplan–Meier plot of TCGA‐BLCA clinical datasets showed the overall survival rate of patients with OCLN^High (red line) and OCLN^Low (black line) expression (raw p = .021). (B) The TCGA‐BLCA clinical datasets showed the relationship between OCLN expression and BLCA neoplasm staging, and the number of stage I patients was too low to be counted. (C) IHC staining for OCLN expression in BLCA tissues and adjacent normal tissues. Scale bar = 100 µm. (D) The relationship between OCLN expression and BLCA neoplasm stages was determined in 120 clinical patients with BLCA. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01; ***p < .001

FIGURE 2

Occludin (OCLN) promotes tumour angiogenesis in vitro and in vivo. (a and b) The knockdown efficiency was confirmed by performing (A) Western blotting and (B) RT‐qPCR assays using 5637 and T24 cells. (C), Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN‐silenced bladder cancer (BLCA) cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (d and e) The OCLN plasmid was transfected into 5637 and T24 cells; the efficiency of overexpression was analysed using (D) Western blotting and (E) RT‐qPCR. (F) Tube formation by EA.hy926 cells incubated with CM derived from 5637 and T24 OCLN overexpressing cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (H) Matrigel plugs containing 5637 and T24 OCLN stable knockdown cells were removed from BALB/c nude mice. (I) CD31 staining in the indicated cells embedded in Matrigel plugs after growth in BALB/c nude mice. Scale bar = 100 µm. (J) The density of microvessels in Matrigel plugs from BALB/c nude mice injected with the indicated cells. (K) IHC staining showing CD31 levels in clinical patients with high‐/low‐grade BLCA. Scale bar = 100 µm. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01

FIGURE 3

Occludin (OCLN) mediates bladder cancer (BLCA) angiogenesis by regulating IL8 expression. (A) Venn diagram showing the differentially expressed genes (DEGs) in the two T24 OCLN knockdown groups compared with the control groups (fold change ≥1, FDR < 0.1, p < .05). (B) Heatmap of the RNA sequencing analysis showing the relative levels of proangiogenic factors. Columns represent probe sets, and rows represent samples receiving the indicated treatments. (C) The relative mRNA levels of proangiogenic factors were detected in control and OCLN shRNA transfected 5637 and T24 cells. (D) The relative IL8 levels in control and OCLN shRNA‐transfected 5637 and T24 cells were measured using an ELISA (pg/ml). (E) The relative IL8 mRNA levels were detected in 5637 and T24 cells following transfection with the vector or OCLN plasmid. (F) Tube formation by EA.hy926 cells cultured with CM derived from 5637 and T24 OCLN‐silenced BLCA cells. IL8 was added, and the cells were stained with Calcein AM and then imaged with a fluorescence microscope. Scale bar = 100 µm. (G) The segment lengths were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01 < p < .05; **.001 < p < .01; ***p < .001

FIGURE 4

IL8/ STAT3 is involved in the process of Occludin (OCLN)‐mediated angiogenesis in bladder cancer (BLCA). (A) p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells. (B) 5637 and T24 cells were transfected with the OCLN plasmid and treated with or without the STAT3 inhibitor Stattic, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed by performing staining with Calcein AM and imagining using a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. D, p‐STAT3 and STAT3 protein levels were detected in OCLN knockdown 5637 and T24 cells after IL8 supplementation. (E) p‐STAT3 and STAT3 protein levels were detected in OCLN‐overexpressing 5637 and T24 cells after blocking IL8. (F) 5637 and T24 cells were transfected with the OCLN plasmid or cultured with the IL8‐neutralizing antibody; tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. G, The segment lengths in these images were analysed, and the meshes were quantified using ImageJ software. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01

FIGURE 5

STAT4 is involved in the mechanism by which Occludin (OCLN) regulates IL8/STAT3 signalling to mediate bladder cancer angiogenesis. (A) Relative IL8 mRNA levels were detected in control and STAT4‐overexpressing 5637 and T24 cells using RT‐qPCR. (B) The STAT4 plasmid or the indicated vector was transfected into 5637 and T24 cells, and tube formation by EA.hy926 cells incubated with CM derived from the indicated cells was assessed using staining with Calcein AM and imaging with a fluorescence microscope. Scale bar = 100 µm. (C) The segment lengths were analysed, and the meshes were quantified using ImageJ software. (D) The relative STAT4 mRNA levels were detected in control and OCLN shRNA‐transfected 5637 and T24 cells. (E) Cells were transfected with the indicated siRNAs or plasmids for 24 h. The relative knockdown and overexpression efficiency was determined using Western blotting and RT‐qPCR. (F) 293T cells were cotransfected with the indicated plasmids for 24 h, and the relative activity of the IL8 promoter was evaluated by performing a dual luciferase reporter assay. The results are shown as the mean ± SD. *.01< p < .05; **.001< p < .01