Curcumin regulates sensitivity of pancreatic cancer to gemcitabine by inhibiting nuclear translocation of NF-κB
Objective To explore the mechanism of curcumin in enhancing gemcitabine sensitivity by regulating pancreatic cancer cells.Methods In order to verify the effect of curcumin on p53 and NF-κB p65 nuclear translocation in pancreatic cancer cell PANC1,the pancreatic cancer PANC1 cells were treated with 0,10,20,30 μmol/L curcumin to obtain the blank group,10 μmol/L curcumin group,20 μmol/L curcumin group and 30 μmol/L curcumin group.The intracellular distribution of NF-κB p65 was determined by immunofluo-rescence staining under fluorescence microscope.The expression level of p53 protein in cells was determined by Western blot.The expression level of p53 mRNA was determined by reverse transcription-real-time fluo-rescence quantitative PCR.The combined use of miRstar and JASPAR softwares predicted to seek microRNA (miRNA) potentially regulated by NF-κB p65 nuclear translocation.The double luciferase reporting assay was used to respectively verify the relationship between miRNA and p53.In order to verify whether curcumin in-fect the sensitivity of PANC1 cell through NF-κB p65/miR-266-5p/p53 axis,the gemcitabine group was ob-tained by 10 μmol/L gemcitabine treating cells,and the curcumin combined gemcitabine group was obtained by 20 μmol/L curcumin and 10 μmol/L gemcitabine treating cells.After the cells were treated with curcumin (20 μmol/L) and gemcitabine (10 μmol/L),oligonucleotides mimic NC and miR-26b-5p mimic were trans-fected into cells,and the mimic NC group and miR-26b-5p group were obtained.pcDNA3.1 no-load plasmid and pcDNA3.1-p53 overexpression plasmid were transfected into cells,respectively,and the pcDNA3.1 group and p53 group were obtained.The cell activity was detected by MTT assay.The cellular apoptosis level was determined by Annexin V/PI double staining.Results Compared with the blank group,the intracellular p53 mRNA and protein expression levels in the various curcumin treated groups (10,20,30 μmol/L curcumin groups) all were increased.Compared with the blank group,the expression level of NF-κB p65 in the nucleus of 20μmol/L curcumin group was decreased.The miRstar and JASPAR prediction software found 8 miRNA that may be regulated by NF-κB p65 nuclear translocation,in which 3 miRNA had potential to target p53 genes,especially miR-26b-5p effect was most significant.The double luciferase report experiment confirmed that miR-26b-5p did interact with p53.Compared with the mimic NC group,the expression levels of p53 mR-NA and protein in the mimic miR-26b-5p group were decreased.Compared with the gemcitabine group,the cell viability in the curcumin combined gemcitabine group was decreased and the apoptosis rate was increased.Compared with the mimic NC group,the cell activity in the mimic miR-26b-5p group was increased and the apoptosis level was decreased.Moreover compared with the pcDNA3.1 group,the cell activity in the pcDNA3.1-p53 group was decreased and the apoptosis level was increased.Conclusion Curcumin reduces the expression of miR-26b-5p gene by inhibiting the nuclear translocation of NF-κB p65 protein,and then increase the p53 gene and protein expression,and enhance the sensitivity of pancreatic cancer cells to gemcitabine by the NF-κB p65/miR-26b-5p/p53 axis.
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