The Mechanism Of MiRna-320a Promotes 5-Fu Resistance In Human Pancreatic Cancer Cells

Background and objective: As the high mortality and high incidence rate of pancreatic cancer,most studies in pancreatic cancer focus on resectable ones,but few patients could benefit from surgery resection.Therefore chemotherapy become one of the important treatment and auxiliary treatment approaches for patients with pancreatic cancer.The drug-resistant nature of pancreatic cancer cells results in poor therapeutic effect.To predict the therapeutic effect of the chemotherapy drugs to specific patient and to reverse the resistances of pancreatic cancer cells are critical for chemotherapy of pancreatic cancer.MicroRNAs(miRNAs)have been reported to play important roles in the genesis of drug-resistance of various cancer types,also in human pancreatic cancer cells.There are also many advantages of miRNAs in diagnosis and therapy of disease.However,there were few reports about the mechanism of mi RNAs in regulating drug-resistant of pancreatic cancer cells.In this study,we created 5-FU-resistant pancreatic cancer cell lines and identified many mi RNAs,and found that miR-320 a was significantly up-regulated in the resistant cells(PATU8988/5-FU).Overexpression of miR-320 a in pancreatic cancer cells strongly promoted cell survival after treated with 5-FU and advanced cell proliferation,migration,invasion and EMT progression of pancreatic cancer cell lines PATU8988 and PANC-1.The primary investigation of miR-320 a expression in pancreatic specimens suggested that high expression of miR-320 a was correlated with worse outcome in PDAC patients treated with 5-FU.In addition,our study will research the mechanism of miR-320 a,and found the potential target of miR-320 a in modulating drug resistance of pancreatic cancer cells,which might be developed as a new prognostic marker for chemotherapy of pancreatic cancer to predict the effectiveness of the chemotherapy drugs for particular patients,and guiding the choice of chemotherapy drugs.Objective:1.To investigated the importment role of mi R-320 a in 5-FU resistance of pancreatic cancer.2.To explore the mechanism of mi R-320 a involved in 5-FU resistance in pancreatic cancer.3.To provide a new potential therapeutic target for the treatment of pancreatic cancer.4.To improve the status of chemotherapy resistance in pancreatic cancer.Methods: 1.Establishment of 5-FU-resistant cell lines and identification of differentially expressed miRNAs by miRNA expression microarray and RT-PCR analysis.2.The drug sensitivity was assessed by calculating IC50 values with cell counting and CCK8 assay.The migration and invasion capacity of pancreatic cancer cells was investigated by wound healing and transwell assay.3.Target prediction programs and the function information analysis of the target genes were used to screen the candidate genes.Double fluorescent assay and Western Blot were used to confirm the expression of the corresponding target genes.4.Survival curves were generated by Kaplan-Meier analysis and tested for significance using the Mantel-Cox log rank test using TCGA pancreatic adenocarcinoma(PAAD)miRNA expression by RNA seq.Results: 1.We firstly created 5-FU-resistant pancreatic cancer cell lines,and investigated the expression profile of miRNAs in PATU8988/5-FU and PATU8988 cells through microarray,we find that miR-320 a was significantly up-regulated in the PATU8988/5-FU cells.TCGA/GEO PAAD miRNA expression by RNA seq shows that miR-320 a was highly expressed in pancreatic cancer patients,which significantly affected the long-term survival and disease-free survival rate of pancreatic cancer patients.2.Overexpression of miR-320 a in pancreatic cancer cell lines(PATU8988,PANC-1)significantly enhanced the drug resistance of 5-FU/Gemcitabine in pancreatic cancer cells,and also advanced cell proliferation,migration,invasion and EMT progression of pancreatic cancer cell lines PATU8988 and PANC-1.3.Target prediction programs revealed that PDCD4 was the direct target of miR-320 a in pancreatic cancer cell lines,and overexpression of miR-320 a could decrease the expression of PDCD4.4.Improving the expression level of PDCD4 could rescue the increased proliferation rate and drug resistance induced by miR-320 a in pancreatic cancer cells.Conclusions: 1.MiR-320 a was significantly up-regulated in pancreatic cancer tissues compared with their paired normal tissues,and was negatively correlated with survival rate after chemotherapy in pancreatic cancer patients.2.Over-expression of miR-320 a induces resistance to 5-FU/gemcitabine in pancreatic cancer cells.3.Over-expression of miR-320 a promotes cell proliferation,migration,invasion and EMT progression of pancreatic cancer cell lines PATU8988 and PANC-1.4.MiR-320 a promotes the 5-FU/gemcitabine resistance of pancreatic cancer by targeting PDCD4.