WDR76 regulates 5-fluorouracil sensitivity in colon cancer via HRAS

Abstract

Background: WD repeat domain 76 (WDR76) has been reported in multiple tumors, while without relation to chemotherapy resistance. 5-fluorouracil (5-FU) is widely adopted in treating colon cancer. However, the resistance of WDR76 and 5-FU in colon cancer remains unclear.

Methods: Limma package in R software was employed to analyze the differentially expressed genes. Western blot or quantitative real-time PCR (qRT-PCR) were run to assessed the gene expression. The cytotoxic effect was determined according to cell viability assay, colony formation assay in vitro. Cell apoptosis was assayed using flow cytometry. GSEA analysis was performed to identify pathways related to the target gene. Xenografted mice model was employed to evaluate the tumor growth.

Results: Bioinformatic analysis revealed the higher expression of WDR76 in 5-FU sensitive colon cancer cells compared to resistant colon cancer cells, accompanied by the decreased mRNA expression of WDR76 in 5-FU resistant colon cancer cells. The overexpressed WDR76 resulted in the apoptosis and the downregulated colony numbers in 5-FU resistant colon cancer cells, leading to the elevated sensitivity of 5-FU. Meanwhile, knockdown of WDR76 enhances the resistance of 5-FU in colon cancer both in vitro and vivo, which was reversed by a specific inhibitor of HRAS, Kobe006. An important molecular mechanism of 5-FU resistance lies the degradation of HRAS induced by WDR76.

Conclusion: Our findings demonstrated a role of WDR76 as a promising target for reversing the resistance of colon cancer to 5-FU.

Keywords: 5-fluorouracil (5-FU); Colon cancer; HRAS; WDR76.

Fig. 1

WDR76 is screened and verified in 5-FU resistant colon cancer. A Screening flowchart. B, C A volcano plot illustrating DEGs between DMSO and 5-FU treated HCT116 cells in GSE157300 or GSE158021. Values are presented as the log10 of tag counts. Pie chart revealed 1279 genes were upregulated and 1330 genes downregulated in GSE157300 and 297 genes were upregulated and 1251 genes downregulated in GSE158021. The hierarchical clustering of the RNA-seq analysis results shows all genes that were significantly differently expressed. D Venn analysis of the DEGs. The middle part represented the intersection of the results. E GO analysis based on the overlapped 120 DEGs. F Expressions of ACER2, CDKN1A, BTG2, MCM10, WDR76, BBC3 in HCT116-S and HCT116-R cell lines were performed by quantitative real-time PCR. All values displayed are mean ± SD and have been duplicated 3 times with similar results. All the experiments are repeated at least three times; ****P < 0.0001

Fig. 2

WDR76 regulates the sensitivity of resistant colon cancer cells to 5-FU. A Cell viability assays of sensitive HCT116 cells to 5-FU (HCT116-S) and resistant HCT116 cells to 5-FU (HCT116-R) transfected with empty vector or Flag-WDR76 and treated with 5-FU at gradient concentrations. B, C Colony formation and cell apoptosis of cells transfected as (A) and treated with 20 μm 5-FU. (D) Cell viability assays of HCT116-R transfected with control siRNA or si-WDR76 or si-WDR76 plus WDR76 re-expression (WDR76-R) and treated with 5-FU at gradient concentrations. E, F Colony formation and cell apoptosis of cells transfected as (D) and treated with 20 μm 5-FU. Illustrative images show colonies in plates. Histograms show colony number. Cell apoptosis were evaluated by flow cytometry and the apoptotic cell percentage was statistically analyzed. All values displayed are mean ± SD and have been duplicated 3 times with similar results. All the experiments are repeated at least three times; **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

WDR76 upregulates the sensitivity of resistant colon cancer cells to 5-FU by promoting the degradation of HRAS. A GSEA analysis was used to analyze the pathways correlated with WDR76. B, C Westernblot was applied to analyze the expressions of HRAS related proteins in resistant HCT116 cells to 5-FU (HCT116-R) transfected into Flag or Flag-WDR76 and control or si-WDR76 or si-WDR76 plus WDR76 re-expression (WDR76-R). D Cell viability assays of HCT116-R cells transfected with control or si-WDR76 and treated with DMSO or Kobe0065. E, F Colony formation and cell apoptosis of cells transfected as (D) and treated with 20 μm 5-FU. Illustrative images show colonies in plates. Histograms show colony number. Cell apoptosis were evaluated by flow cytometry and the apoptotic cell percentage was statistically analyzed. All values displayed are mean ± SD and have been duplicated 3 times with similar results. All the experiments are repeated at least three times; **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

WDR76 knockdown decreases 5-FU sensitivity of colon cancer cells through HRAS in vivo. A–C Resistant HCT116 cells to 5-FU stably expressing control shRNA or WDR76 shRNA were subcutaneously injected into the left flanks of 6-week female nude mice. Each group was injected with 5-FU (10 mg/kg) or normal saline once a week after 7 days. All mice were treated with 5-FU every 3 days after injected by cancer cells for 7 days. The volume of the tumors was measured at the interval of three days. D Westernblot of the representative tumor tissues to detect the expression of HRAS related proteins. E Schematic of the regulatory network in this study