| Colorectal cancer(CRC)is common malignant gastrointestinal tumor in the world.According to the global cancer statistics 2018,the morbidity and mortality of CRC ranked the 3rd and 2nd among all malignancies.Currently,the main treatments for colorectal cancer include surgery,chemotherapy and radiotherapy.However,long-term use of chemotherapy drugs in clinical treatment will lead to side effects and drug resistance.Multi-drug resistance(MDR)of colorectal cancer not only develop resistance to used chemotherapy drugs,but also develop cross-resistance to other unused drugs with different molecular structures,binding targets and multiple mechanisms,which has became a difficult problem in the treatment of colorectal cancer.Therefore,exploring the mechanism of multi-drug resistance in colorectal cancer and screening effective drug-resistant reversal agents are hot fields of tumor research.Small molecular compounds of plant-derived polyphenols have many advantages,such as a wide range of sources,low cost,low toxicity,high stability and large scale synthesis.In addition,many polyphenols with anti-tumor activity have the potential to develop into natural tumor drug resistance reversal agents.On the other hand,studies had shown that miRNAs were the center of the network regulating drug resistance,and they could affect multi-drug resistance in colon cancer by regulating numerous downstream target genes.However,many plant-derived polyphenols increase the sensitivity of colorectal cancer to drugs through regulating the expression of miRNAs and its downstream target genes.This indicates that miRNAs play a key role in mediating plant polyphenols to reverse tumor drug resistance.Our previous studies have found that millet inner shell as millet processing by-products,it contains rich polyphenol compounds.In addition,bound polyphenol of millet inner shell(BPIS)exhibit significantanti-colorectal cancer effect in vitro and in vivo.However,it is not clear whether BPIS has the activity of reversing multi-drug resistance in colorectal cancer,and its active components.Therefore,in this study,BPIS derived from millet bran was taken as the research object,and the active components and molecular mechanisms of BPIS reversing multi-drug resistance in colorectal cancer were elucidated from the perspective of plant polyphenol regulating miRNAs.These data provided the theoretical basis for it to become a reversal agent of multi-drug resistance in colorectal cancer.The research content and results mainly include the following aspects:(1)The main phenolic acid components with reverse drug resistance activity in the BPIS were determined.MTT assay showed that BPIS had a inhibitory effect in a concentration dependent manner on HCT-8/Fu cells,but had no significant effect on human normal colonic epithelial cells FHC.In our previous experiment,BPIS was determined to be composed of 12 phenolic acids by high performance liquid chromatography high resolution mass spectrometry.According to the molecular weight of phenolic acid,BPIS was divided into two parts after dialysis processes:molecular weight(MW)< 200 and MW > 200.MTT results showed that BPIS(MW < 200)had a significant inhibitory effect on the proliferation of HCT-8/Fu cells.On this basis,it was further found that only ferulic acid and p-coumaric acid could effectively inhibit the proliferation of HCT-8/Fu cells among the 6 phenolic acids of BPIS(MW < 200).According to the natural content ratio 1:1.13 of p-coumaric acid and ferulic acid in BPIS,the standards of p-coumaric acid and ferulic acid were mixed and prepared artificially.The results showed that artificially prepared ferulic acid and p-coumaric acid mixture had similar inhibitory effects.This indicated that the phenolic acid components in BPIS(MW >200)had synergistic effects on the reversal of drug resistance to ferulic acid and p-coumaric acid.(2)The multi-drug resistant reversal ability of BPIS in colorectalcancer HCT-8/Fu cells was analyzed.The results showed that HCT-8 was more sensitive to 5-fluorouracil(5-Fu),oxaliplatin(L-OHP)and vincristine(VCR)than HCT-8/Fu.The quantitative real-time PCR(RT-PCR)and westernblot results showed that three classical drug resistant proteins: the expression of multi-drug resistance protein 1(MRP1),P-glycoprotein(P-gp)and breast cancer resistance protein(BCRP)in multi-drug resistant HCT-8/Fu was significantly higher than HCT-8.The indicated that HCT-8/Fu had strong multi-drug resistance to5-Fu,L-OHP and VCR.By means of cell colony formation,nuclear staining and flow cytometry,the results showed that BPIS could reverse the multi-drug resistance of HCT-8/Fu cells by inhibiting the proliferation,inducing apoptosis,increasing the accumulation of chemotherapeutic drugs and inhibiting the expression of drug resistant proteins.(3)miR-149 plays a key role in the reversal of multi-drug resistance in colorectal cancer by BPIS.RT-PCR showed that the expression of miR-149 in HCT-8/Fu cells was significantly less than that of the sensitive strain HCT-8,and the expression level of miR-149 in HCT-8/Fu can be up-regulated by BPIS treatment.Then the cells were transfected with miRNA mimics and inhibitor to construct the cell model of knock-down or over-expression of miR-149.It was found that the expression level of miR-149 in cells was positively correlated with the chemosensitivity of cells to 5-Fu,L-OHP,and VCR.Therefore,it was confirmed that BPIS could reverse multi-drug resistance by increasing the expression level of miR-149 in HCT-8/Fu cells.(4)BPIS inhibited the methylation of miR-149 promoter region and up-regulated the expression level of miR-149.Methprimer software was used to analyze the 1000 bp sequence of the coding region of miR-149,and there is a 173 bp CPG island in this region.Methylation-Specific PCR(MSP)assay showed that the methylation of the promoter region of miR-149 in multi-drug resistant HCT-8/Fu was higher than that in colon epithelial cell line FHC and sensitive strain HCT-8,while BPIS,ferulicacid and p-coumaric acid could reduce the methylation of the promoter region of miR-149 in HCT-8/Fu.RT-PCR and westernblot experiments proved that BPIS,ferulic acid and p-coumaric acid could also inhibit the expression of DNMT3 a,DNMT3b and MECP2,the key genes regulating the degree of gene methylation,but the inhibition effect of BPIS was better than that of ferulic acid and p-coumaric acid.These results suggest that BPIS could up-regulate the expression of miR-149 through methylation regulation,which makes HCT-8/Fu more sensitive to drugs.In summary,BPIS has the ability to reverse drug resistance of HCT-8 /Fu cells with low toxicity and high efficiency.Ferulic acid and p-coumaric acid were found to be the main active components in BPIS(MW < 200);BPIS could reverse multi-drug resistance in HCT-8/Fu by inhibiting cells proliferation,activating apoptosis,increasing the accumulation of chemotherapy drugs in cells and down-regulating drug resistance genes.Furthermore,it was found that BPIS inhibited the methylation of miR-149 promoter region by down-regulating the expression of methylase,thereby activating miR-149 expression and increasing the sensitivity of HCT-8/Fu cells to 5-Fu,L-OHP and VCR.Therefore,this study provides substantial theoretical experimental data for the possible development and application of BPIS as a effective multi-drug resistance reversal agent for colorectal cancer. |
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