Downregulation of HuR Inhibits the Progression of Esophageal Cancer through Interleukin-18

Abstract

Purpose: The purpose of this study was to investigate the effect of human antigen R (HuR) downregulation and the potential target genes of HuR on the progression of esophageal squamous cell carcinoma (ESCC).

Materials and methods: In this study, a proteomics assay was used to detect the expression of proteins after HuR downregulation, and a luciferase assay was used to detect the potential presence of a HuR binding site on the 3'-untranslated region (3'-UTR) of interleukin 18 (IL-18). In addition, colony formation assay, MTT, EdU incorporation assay, Western blot, flow cytometry, immunohistochemistry, transwell invasion assay, and wound healing assay were used.

Results: In the present study, we found that the expression of both HuR protein and mRNA levels were higher in tumor tissues than in the adjacent tissues. HuR downregulation significantly suppressed cell proliferation. In addition, the metastasis of esophageal cancer cells was inhibited, while the expression of E-cadherin was increased and the expression of matrix metalloproteinase (MMP) 2, MMP9, and vimentin was decreased after HuR knockdown. Moreover, silencing of HuR disturbed the cell cycle of ESCC cells mainly by inducing G1 arrest. Furthermore, proteomics analysis showed that downregulation of HuR in TE-1 cells resulted in 100 upregulated and 122 downregulated proteins, including IL-18 as a significantly upregulated protein. The expression of IL-18 was inversely regulated by HuR. IL-18 expression was decreased in ESCC tissues, and exogenous IL-18 significantly inhibited the proliferation and metastasis of ESCC cells. The 3'-UTR of IL-18 harbored a HuR binding site, as shown by an in vitro luciferase assay.

Conclusion: HuR plays an important role in the progression of esophageal carcinoma by targeting IL-18, which may be a potential therapeutic target for the treatment of ESCC.

Keywords: Cell proliferation; Esophageal squamous cell carcinoma; Human antigen R (HuR); Interleukin-18; Neoplasm metastasis.

Fig. 1.

Both human antigen R (HuR) protein and mRNA levels were significantly higher in tumor tissues than that in the adjacent tissues. (A) Representative images of the different staining patterns are presented. (B) RNA was extracted from four tumor tissues and matched normal tissues, and HuR mRNA expression was detected by polymerase chain reaction. ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 2.

Downregulation of human antigen R (HuR) inhibited cell proliferation. (A) Western blot was used to detect the expression of HuR in esophageal cancer cells after shRNA transfection. (B) The effect of HuR on the viability of the TE-1 and Eca-109 cells infected with the HuR knockdown virus. The cells were seeded in a 96-well plate and incubated for 48 hours before being analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. (C) The proliferating TE-1 and Eca-109 cells were labeled with 5-ethynyl-20-deoxyuridine (EdU). The Click-iT reaction revealed EdU staining (green). The nuclei were stained with Hoechst 33342 (blue). The images are representative of the results obtained. (D) Colony formation assays of TE-1 and Eca-109 cells after treatment. After 10 days, the cells were stained with crystal violet. Colonies consisting of more than 50 cells were counted. The data are presented as the mean±standard error of mean (n=3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001, compared to the negative control.

Fig. 3.

Downregulation of human antigen R (HuR) disturbed the cell cycle of esophageal squamous cell carcinoma cells. TE-1 and Eca-109 cells infected with the HuR knockdown virus were stained with propidium iodide in preparation for flow cytometry with the FACSCalibur system as described in Materials and Methods. (^**p < 0.01 and ^***p < 0.001).

Fig. 4.

Downregulation of human antigen R (HuR) inhibited cell invasion and metastasis in esophageal squamous cell carcinoma cells. (A) Migration of the cells was assessed using a transwell-based assay. The TE-1 and Eca-109 cells infected HuR knockdown virus were seeded in the upper chamber and incubated for 30 hours at 37°C. The cells in the upper chamber were carefully scraped off using a cotton swab, and those that had migrated to the basal side of the membrane were fixed in 4% paraformaldehyde and stained with Giemsa. The membranes were mounted and dried at 37°C for 30 minutes. (B) Wound healing was observed 48 hours after the cells treated. The open wound area after incubation was normalized to the initial wound area. The data are presented as the mean±standard error of mean normalized to the control cells (^**p < 0.01 and ^***p < 0.001). (C) TE-1 and Eca-109 cells infected with the HuR knockdown virus were subjected to Western blot analysis for β-actin, HuR, matrix metalloproteinase (MMP) 2, MMP9, E-cadherin, and vimentin.

Fig. 5.

Human antigen R (HuR) negatively regulates the expression of interleukin 18 (IL-18) in esophageal cancer cells. (A) Western blot was used to evaluate the expression of IL-18 in esophageal carcinoma cells in which HuR was downregulated. (B) The diagram indicates the relative positions of the transcription factor HuR on the IL-18 promoter. Truncated promoters were cloned upstream of the firefly luciferase reporter gene. (C) Luciferase reporters containing IL-18 promoters were transfected into TE-1 and Eca-109 cells. After 24-hour incubation, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). NS, not significant. ^**p < 0.01, ^****p < 0.0001.

Fig. 6.

Interleukin 18 (IL-18) inhibited proliferation and invasion of esophageal squamous cell carcinoma cells. (A) The effect of human antigen R (HuR) on the viability of TE-1 and Eca-109 cells seeded in 96-well plates and treated with IL-18 (5 ng/mL) for 48 hours were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (^*p < 0.05, ^**p < 0.01). (B) Colony formation assays of TE-1 and Eca-109 cells treated with IL-18 (5 ng/mL). After 10 days, the cells were stained with crystal violet. Colonies consisting of more than 50 cells were counted. The data are presented as the mean±standard error of mean (SEM) (n=3). ^*p < 0.05 compared to the negative control. (C) TE-1 and Eca-109 cells were treated with IL-18 for 48 hours, and stained with propidium iodide in preparation for flow cytometry with the FACSCalibur system as described in Materials and Methods. (D) Migration of cells was assessed using a transwell assay. The TE-1 and Eca-109 cells treated for 48 hours with IL-18 were seeded in the upper chambers and incubated for 30 hours at 37°C. Then, the cells in the upper chambers were carefully scraped off using a cotton swab, and the ones that had migrated to the basal side of the membrane were fixed in 4% paraformaldehyde and stained with Giemsa, and the membranes were mounted and dried at 37°C for 30 minutes (^**p < 0.01). (E) Wound healing was observed 48 hours after the cells were treated with IL-18, and the open wound area after incubation was normalized to the initial wound area. The data are presented as the mean±SEM normalized to the control cells (^****p < 0.0001). (F) Protein extracts from TE-1 and Eca-109 cells treated for 48 hours with IL-18 were subjected to Western blot analysis for HuR, IL-18, matrix metalloproteinase (MMP) 2, and MMP9.

Fig. 7.

Low expression of interleukin 18 (IL-18) in esophageal cancer tissues. (A) Representative images of the different staining patterns are presented. (B) Box plots depicting IL-18 staining scores as assessed by immunohistochemisty (IHC) in tumors and adjacent tissues in our series of 52 paired esophageal squamous cell carcinoma (ESCC) and tumor adjacent tissue samples. ^****p < 0.0001.