Vitamin D receptor suppresses proliferation and metastasis in renal cell carcinoma cell lines via regulating the expression of the epithelial Ca2+ channel TRPV5

Abstract

We previously demonstrated that transient receptor potential vanilloid subfamily 5 (TRPV5) expression was decreased in renal cell carcinoma (RCC) compared with that in normal kidney tissues, a finding that was correlated with vitamin D receptor (VDR) expression, but further investigations is warranted. The aim of this study was to elucidate whether VDR could regulate the expression of TRPV5 and affect proliferation and metastasis in RCC. In this study, we used lentivirus to conduct the model of VDR overexpression and knockdown caki-1 and 786-O RCC cell lines in vitro. The results demonstrated that VDR overexpression significantly inhibited RCC cells proliferation, migration and invasion, and promoted apoptosis by the MTT, transwell cell migration/invasion and flow cytometry assays, respectively. However, VDR knockdown in RCC cells had the opposite effect. The RNA-sequence assay, which was assessed in caki-1 cells after VDR overexpression and knockdown, also indicated that significantly differentially expressed genes were associated with cell apoptotic, differentiation, proliferation and migration. RT-PCR and western blot analysis showed that VDR knockdown increased TRPV5 expression and VDR overexpression decreased TRPV5 expression in caki-1 cells. Furthermore, knockdown of TRPV5 expression suppressed the VDR knockdown-induced change in the proliferation, migration and invasion in caki-1 cells. Taken together, these findings confirmed that VDR functions as a tumour suppressor in RCC cells and suppresses the proliferation, migration and invasion of RCC through regulating the expression of TRPV5.

Fig 1. VDR expression levels in the caki-1 and 786–0 RCC cell lines with different treatments.

VDR mRNA expression was measured by RT-PCR and was normalized to that of β-actin. VDR protein expression was analysed by WB, and β-actin was used as a loading control. *p <0.05, **p <0.01, ***p <0.001. (A) VDR expression levels in caki-1 cells were higher in 786-O cells. (B) & (C) Overexpression of VDR by infection with the VDR-expressing lentivirus (leVDR) and knockdown of VDR with shRNA lentivirus (shVDR) in caki-1 and 786-O cells.

Fig 2. VDR inhibits RCC proliferation, migration and invasion.

(A) The MTT assay showed that the proliferation of caki-1 and 786-O cells was significantly inhibited by VDR overexpression and promoted by knockdown of VDR compared with their respective control cells. Each data point represents the mean ± SD of absorbance values. Transwell migration (B) and invasion (C) assays (magnification, ×100) showed that the migration and invasion of 786-O cells were significantly decreased following infection with leVDR lentivirus and were increased following infection with shVDR lentivirus compared with each vector control. The graph indicates the mean ± SD of the number of cells from 5 random high-power fields (magnification, ×200). *p <0.05, **p <0.01, ***p <0.001.

Fig 3. VDR promotes RCC apoptosis.

Flow cytometry indicated that VDR overexpression increases caki-1 cell apoptosis, and VDR knockdown decreases caki-1 cell apoptosis. Graphs for each treatment group and the percentage of cell apoptosis are shown. X-axis: PL1-H Annexin V; Y-axis: PL2-H PI. The data were expressed as means ± SD of three independent experiments. *p <0.05, ***p <0.001.

Fig 4. VDR regulates TRPV5 expression.

(A) RT-PCR and (B) WB were performed to examine the expression of TRPV5 following VDR overexpression or knockdown in caki-1 cells. Changes in TRPV5 expression were increased following infection with shVDR lentivirus and were decreased following infection with leVDR lentivirus. *p <0.05, **p <0.01, ***p <0.001.

Fig 5. TRPV5 knockdown reverses the effect of VDR knockdown on caki-1 cell proliferation, migration and invasion.

(A) TRPV5 mRNA expression was detected by RT-PCR and was normalized to that of β-actin. TRPV5 protein expression was detected by WB using β-actin as a loading control. shV+T is shVDR+shTRPV5 co-treated. (B) Cell proliferation ability was analysed by the MTT assay in control, shVDR-treated, or shVDR+shTRPV5-treated caki-1 cells; (C-D) Cell migration and invasion ability were measured by the transwell assay. The graph indicates the mean ± SD of the number of cells from 5 random high-power fields (magnification, ×200). *p <0.05, **p <0.01.

Fig 6. Bioinformatics analysis of differently expressed genes after VDR overexpression and knockdown in caki-1 cells.

After VDR overexpression, GO analysis of biological process (A) indicated differentially expressed genes were associated with cell apoptotic, differentiation, proliferation and migration. KEGG pathway enrichment analysis (B) indicated differentially expressed genes were associated with the TNF signalling pathway, TGF-beta signalling pathway and carcinogenesis pathway. In the VDR knockdown cells, (C) KEGG pathway enrichment analysis indicated that significantly differentially expressed genes were associated with the TNF signalling pathway, apoptosis, Wnt signalling pathway and the carcinogenesis pathway.