Analysis Of Differential Expression Profiles Of MiRNA And MRNA And Study On Drug Resistance In Colon Cancer

PART 1 BIOINFORMATICS ANALYSIS OF GENES ASSOCIATED WITH DRUG RESISTANCE IN COLON CANCERObjective Based on gene expression profiles and bioinformatics,to integratly analyze the roles of miRNAs and their target genes in the development of drug resistance in colon cancer cells.Methods miRNA and mRNA expression profiles of colon cancer cells resistant and sensitive to chemotherapeutics were searched and downloaded from GEO database.Differentially expressed miRNAs and mRNAs were screened by BRB-Array Tools.Target genes of the differential miRNAs were identified using miRWalk database in combination with negative correlation analysis of miRNA and mRNA expression.Then,these target genes were analyzed applying GO and KEGG databases,and miRNA-target gene network was established to visually display the regulating relations between miRNA and genes.Finally,the diversity of target genes as well as synergistic effect of miRNAs was studied.Meanwhile,critical miRNAs and target genes in the network were identified based on the principle of graph theory.Results 1 miRNA expression profile and 2 mRNA expression profiles were screened,including GSE28547,GSE9412 and GSE11440 datasets.According to the selection criteria(FDR<0.05,|log₂FC|?2),9 miRNAs and 395 genes with differentially expression were screened.Through integrated analysis of miRNA-mRNA expression profiles,96 targets of differential miRNAs were confirmed.Go analysis indicated that these target genes were closely associated with molecular functions including bile acid binding,actin binding and nucleotidyltransferase activity;mainly located in cell surface,plasma membrane,extracellular matrix;and further mainly involved in the biological processes including regulation of cell migration,proliferation,apoptosis,macromolecule metabolic process and signal transduction.KEGG pathway analysis enriched the metabolism of xenobiotics by cytochrome P450(CYP450),phosphatidylinositol signaling system and calcium signaling pathway,whereas there is no statistical significance(P>0.05).Based on 9 miRNAs and 96 target genes,miRNA-target gene network composed of 138 miRNA-target gene pairs was established.Combining analysis of target genes diversity and miRNAs functional synergy as well as graph theory,miR-149-5p,miR-552-3p and miR-338-3p were indicated to be potentially key miRNAs while CLN8 and GALNT10 to be potentially key target genes of miRNAs regulating drug resistance in colon cancer.Furthermore,it indicated that miRNAs were likely to affect drug resistance in colon cancer through collaboratively regulate the expression of common target genes.Conclusions miRNA and mRNA expression profiles associated with drug resistance to methotrexate in colon cancer were acquired on the basis of GEO database.With the help of integrated analysis,the roles of miRNAs and mRNAs as well as their regulatory relationships in the development of drug resistance in colon cancer were explained,the diversity of target genes and synergistic effect of miRNAs were confirmed,and further miR-149-5p,miR-552-3p,miR-338-3p,CLN8 and GALNT10 were indicated to probably have pivotal roles in the development of drug resistance in colon cancer,which provided firm foundation for future research.PART 2 MIR-93-5P MODULATE MULTIDRUG RESISTANCE IN HUMAN COLORECTAL CANCER CELLS THROUGH CDKN1A/MARI/P-GP SIGNAL AXISObjective To compare the expression levels of miR-93-5p between drug-resistant HCT-8/VCR cells and parent HCT-8 cells.To explore the potential role and mechanism of miR-93-5p in drug resistance in colorectal cancer.So as to further provide theoretical basis for clinical treatment of colorectal cancer.Methods The expressions of miR-93-5p and MDR1/P-gp were detected by polymerase chain reaction(PCR)or western blot.HCT-8 and HCT-8/VCR cells were transfected with miR-93-5p inhibitor,mimic or NC through Lipofectamine 2000.Then the expression levels of MDR1 mRNA and P-gp were assessed using PCR and western blot respectively,the activity of P-gp was analyzed applying Rhodamine 123(Rh-123)efflux experiment,and drug sensitivity was analyzed by CCK-8 assay.With the help of Pic Tar,Target Scan and miRanda,the target gene of miR-93-5p were then predicted.Furthermore,the expression levels of CDKN1 A mRNA and protein were measured using PCR and western blot respectively,and cell cycle was analyzed by FACS.Finally,the results were analyzed with SPSS 17.0 software(SPSS Inc.,Chicago,IL,USA).The difference between means was analyzed using two-tailed Student's t-test when only two groups was compared,or one-way ANOVA when more than two groups was compared.Statistical significance was considered to be existed when P<0.05.Results The relative expressions of miR-93-5p and MDR1/P-gp were much higher in HCT-8/VCR cells than that in the parental HCT-8 cells(P<0.05).Transfection of HCT-8/VCR cells with the inhibitors of miR-93-5p significantly decreased the expression of miR-93-5p and MDR1/P-gp(P<0.05),increased the fluorescence intensity of intracellular Rh-123(P<0.05),enhanced the sensitivity of HCT-8/VCR cells to vincristine(P<0.05),increased CDKN1 A expression(P<0.05),increased the percentage of cells at G1 phase(P<0.05),decreased the percentage of cells at G2/M and S phase(P<0.05).Meanwhile,expressions of MDR1/P-gp increased(P<0.05),the fluorescence intensity of intracellular Rh-123 decreased(P<0.05),CDKN1 A expression decreased(P<0.05),the percentage of cells at G1 phase decreased(P<0.01),the percentage of cells at G2/M and S phase increased(P<0.05)in HCT-8/VCR cells management with the mimics of miR-93-5p,while expression of miR-93-5p increased(P<0.01)and sensitivity to vincristine decreased(P<0.05)in HCT-8 cells treatment with the mimics of miR-93-5p.Conclusions In drug-resistant colorectal cancer cells,the up-regulation of miR-93-5p may be involved in the development of drug resistance,regulating the expression of MDR1/P-gp by targeting CDKN1 A,thereby increasing drug efflux and promoting cell cycle process as well as inhibiting the sensitivity of cancer cells to chemotherapeutics,which provide theoretical basis for research on multidrug resistance in cancers.