5-FU blocks shuttling of HuR mediated by PKCδ in gastric cancer cells

Abstract

Background: 5-fluorouracil (5-FU) is a common chemotherapy drug for gastric cancer. Human antigen R (HuR) is an RNA-binding protein that is also known as ELAV like RNA binding protein 1 (ELAVL1) regulates gene expression by binding to target genes 3' UTR region and is highly expressed in tumor tissues.However, the regulatory mechanisms of HuR in 5-FU mediated chemotherapy in stomach cancer are not well understood. In this study, we aimed to investigate 5-FU regulated PKCδ expression and translocation of HuR in SGC cell lines.Methods: Using Cell viability assay to obtained IC50 doses when SGC cells were treated with 5-FU for 2 and 3 days. Western blot was used to detect the total protein content of HuR and PKCδ after treating with 20 mM 5-FU. Furthermore, after adding 20 mM 5-FU, knocking down PKCδ, HuR and using inhibitor Staurosporine respectively, the protein content of HuR in the cytoplasm were detected by Western blot and Immunofluorescence. MTT assay was used to verify the changes in cell proliferation after knockdown of HuR.Results: Using Cell viability assay, we obtained IC50 doses of 77 and 20 mM, respectively, when SGC cells were treated with 5-FU for 2 and 3 days. The total protein content of HuR and PKCδ in SGC cells treated with 20 mM 5-FU did not change. However, Western blot and Immunofluorescence detected that the protein content of HuR in the cytoplasm decreased with 20 mM 5-FU treatment. Staurosporine inhibits the nucleation of HuR and is an inhibitor of PKC. Consistently, the expression of HuR in cytoplasm declined while knockdown of PKCδ. And the proliferation of SGC cells decreased after knocking down the HuR.Conclusions: Our results showed that 5-FU blocked shuttling of HuR from the nucleus to the cytoplasm in SGC cells through PKCδ phosphorylation.

miR-325-3p Reduces Proliferation and Promotes Apoptosis of Gastric Cancer Cells by Inhibiting Human Antigen R

“…Western blot analyses were performed as described in a previously published paper [ 28 ]. Total proteins were extracted from SGC-7901 and HGC-27 cells 48 hours after transfection by the radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (PIC), and the protein concentration was measured using the bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China).…”