The Research Of The Mechanism Of Low-dose DNA Demethylating Agent Decitabine Improving The Sensitivity Of Chemoresistant Gastrointestinal Cancers

ObjectiveChemotherapy is one of the mainstays of gastrointestinal cancer therapy,but chemoresistance has become the largest obstacle to the success of cancer chemotherapies.The abnormal expression of genes including miRNAs is closely related to chemoresistance.Recent studies confirmed that the abnormal epigenomics of cancer cells is the critical regulator of the malignant phenotypes of cancers including drug resistance and low-dose demethylating agents could improve the sensitivity of cancer cells to chemotherapy and overcome drug resistance.However,the mechanism is still not clear.This study is divided into three parts.First,we aim to explore the mechanism of DNA methylation-mediated repression of miR-181a/135a/302c expression and 5-fluorouracil(5-FU)resistance in microsatellite-unstable(MSI)colorectal cancer.The second is to confirm the efficacy of low-dose decitabine(DAC)as chemoresistance reversal agent of gastrointestinal cancers’in vitro and the final part is to identify the key genes of DAC overcoming chemoresistance.MethodsThe expression of three miRNAs(miR-181a,miR-135a and miR-302c)were validated by qPCR analysis in CRC samples and cell lines.The mechanism of miRNAs mediating MSI CRC 5-FU resistance was evaluated by proliferation and apoptosis assays,luciferase reporter system and western blotting.The methylation status of miR-181a/135a/302c was investigate by MSP analysis.The resistant gastrointestinal cancer cell lines were established.The re-sensitized effect of DAC were investigated in vitro.RNA-seq and qPCR were used to identify genes differentially expressed among the parent cell lines,drug-resistant cell lines and drug-resistant cell lines treated with DAC.ResultsThe expression levels of miR-181a,miR-135a and miR-302c were significantly repressed in MSI CRC.The underexpression of miR-181a/135a/302c mediated 5-FU resistance of MSI CRCs.PLAG1 is the functional target of miR-181a/135a/302c.Promoters hypermethylation of miR-181a/135a/302c were detected in MSI CRC.Compared with the parental cells,the drug sensitivity of drug-resistant cells was lower,the proliferation rate was slower with increased cell doubling time,the percentage of cells in G0/G1 phase was markedly increased and the cell apoptosis rate was decreased.After DAC treatment,the drug-resistant cells were re-sensitized to chemotherapy drugs,the percentage of cells in G0/G1 phase was decreased and the cell apoptosis rate was increased in comparison with those in untreated drug-resistant cells.The expression of genes PEG10,KIF14,PLA2G4A and SGK3 were up-regulated in drug-resistant cells and down-regulated in drug-resistant cells treated with DAC.ConclusionPromoters hypermethylation mediated repression of miR-181a/135a/302c expression and 5-FU resistance in MSI CRC.Hypermethylation status can be reversed and expression levels of miR-181a/135a/302c can be restored by demethylating agent.Low-dose DAC can improve the sensitivity of chemoresistant gastrointestinal cancers.The genes PEG10,KIF14,PLA2G4A and SGK3 can be used as predictive biomarkers of chemotherapy resistance and therapeutic targets.