The MiR19a Induces Resistance To5-fluorouracil By Affcecing Tumor Protein53-induced Nuclear Prottein1in HCT8Colon Cancer Cells

Objectives:To investigate the effect of miR19a on tumor protein53-induced nuclear protein1(TP53INP1)-induced resistance to5-Fu in HCT8colon cancer cells.Methods：1.Collecting formalin-fixed, paraffin-embedded (FFPE) tissue samples fromcolon cancer patients, we screened miRNAs from formalin-fixed paraffin-embedded(FFPE) tissue samples of colorectal tumors and adjacent tissues by microarrays.2. A5-Fu resistance cell line from human colon HCT8cancer cells wasestablished by a dosage increase gradient method, the drug resistance ofHCT8/5-Fu cells was evaluated using MTT assay.3.The real time fluorescence quantitative PCR (qRT-PCR) was used to detectmiR19a expression in HCT8cells and5-Fu-resistant HCT8/5-Fu cells.4.Transfecting miR19a mimic（mi）and miR19a negative control（mi-NC）intoHCT8cells, Drug sensitivity to5-Fu after transfect ion was tested using MTTassay.5.Two commonly databases including Targets can and Pictar. miRNAs were usedto forecast the targets with the search terms of miR19a.TP53INP1was selected ascandidate targets, Identifying the TP53INP1as the target of miR19a by theLuciferase reporter system.6.Transfecting miR19a mimic（mi）and miR19a negative control（mi-NC）intoHCT8cells, the protein expression of TP53INP1after transfection were detected byWestern blotting.Results:1. The microarray chip results show that the expression of miR19a in colorectalcancer tissues wass ignificantly higher than that in adjacent tissues(P<0.05).2. MTT assay showed that the drug resistance index of HCT8/5-Fu cells relativeto the parental HCT8cells was18.2.3.QRT-PCR results showed that miR19a was up-regulated7.12-fold in the5-Fu resistant cells (HCT8/5-Fu), which was significantly higher than that in HCT8cells(P<0.05).4. MTT assay showed that compared with miR19a negative control, aftertransfection of miR19a mimics into HCT8cells, the half inhibition concentration(IC50) of5-Fu was significantly higher than that in negative control group (P <0.05).5.The Luciferase reporter system showed that miR19a reduced the luciferaseactivity containing the wild-type of TP53INP13’-UTR, showed that miR19a andTP53INP13’-UTR can specifically bind(P <0.01).6.The Western blot results showed that compared with miR19a negative control,after transfection of miR19a mimics into HCT8cells, miR19a downregulated theexpression of TP53INP1(P <0.05), TP53INP1was the target gene of miR-19a.Conclusion:1.expression of miR19a is associated with the resistance to5-Fu in human coloncancer HCT8cells.2. miR19a targeting the TP53INP1gene could negatively regulate the resistanceto5-Fu in human colon cancer HCT8cells.