The Role Of MiRNAs In Drug Resistance And Prognosis Of Breast Cancer Formalin-fixed Paraffin-embedded Tissues

PurposeChemo-resistance is the leading cause of failure in cancer therapy,however,much remains to be understood about the intrinsic mechanisms.At present,chemo-resistance mediated by miRNAs in breast cancer have been already validated by previous studies in vitro,while little is known concerning their expression in vivo.The aim of this study was to investigate the role of miRNAs linked to drug resistance in breast cancer formalin-fixed paraffin-embedded tissues and the association of prognosis with miRNAs,thus providing effective targets in chemotherapy.Methods1.MiRNAs microarray was used to screen miRNAs associated with the drug resistance and the results were validated by real-time fluorescent quantitative PCR(RT-qPCR).2.A retrospective search was conducted through the computerized database at the Department of Pathology in Nanjing Drum Tower Hospital and Jiangsu Province Cancer Hospital,for diagnosed breast cancer cases from January 2010 to February 2015.After comprehensive skimming,we selected 55 breast cancer cases with therapeutic response assessments of neoadjuvant chemotherapy.And 81 formalin-fixed paraffin-embedded(FFPE)breast cancer tissues in line with the standards of the study were collected.The expression levels of miRNAs in 26 paired FFPE and in the effective group with the ineffective group after neoadjuvant chemotherapies were compared.3.MiR-4443 mimics and inhibitors were transfected into drug sensitive and resistant cells respectively.The effects of miR-4443 on drug resistance was detected by MTT and flow cytometry.4.The specific target genes of miR-4443 were predicted by TargetScan software,and the expression of TIMP2 mRNA was detected by RT-qPCR.5.We use TCGA database to draw the Kaplan Meier survival curve to verify the correlation between the expression level of TIMP2 and the survival time of patients.Results1.According to the results of miRNAs chip combined with previous studies,a total of 28 miRNAs were selected for RT-qPCR verification in cells.The results were consistent with the microarray results(except for miR-671).2.After comparing the expression levels of miRNAs in the paired FFPE tissues,10 miRNAs displayed significantly higher expression in the specimens of post-neoadjuvant-chemotherapy than those pre-neoadjuvant-chemotherapy.Also,the detection in post-neoadjuvant-chemotherapy specimens showed that the expression levels of 8 miRNAs in the ineffective group was obviously higher than those in the effective group.3.The expression of miR-4443 in MDA-MB-231/Epi cells was 2.83 times higher than that of MDA-MB-231/S cells.Compared with the negative controls,the expression of miR-4443 in the MDA-MB-231/S cells transfected with miR-4443 mimics increased significantly.After the transfection,IC50 of cells increased significantly,with the Epirubicin induced apoptosis decreasing.In contrast,the expression of miR-4443 was downregulated in the MDA-MB-231/Epi cells transfected with miR-4443 inhibitors.The IC50 of the transfected cells was significantly decreased and the cell apoptosis increased.4.TargetScan software was used to identify the 3'UTR binding sites of TIMP2 complementary to miR-4443.Analytic results showed that,the expression level of TIMP2 mRNA in MDA-MB-231/S cells transfected with mimics significantly decreased,while the expression level of TIMP2 mRNA in MDA-MB-231/Epi cells transfected with inhibitors increased,when compared with the negative control cells.5.The Kaplan Meier survival curve drawn by TCGA database demonstrated that the patients with low expression of TIMP2 tended to have poor prognosis.Conclusions1.MiRNAs are involved in the regulation of drug resistance in breast cancer cells.2.MiR-29a,miR-34a,miR-140,miR-222,miR-423,miR-574,miR-744,miR-3178,miR-4443,miR-6780b are relative to breast cancer chemo-resistances.MiR-29a,miR-140,miR-222,miR-574,miR-744,miR-4443,miR-6780b,miR-7107 are negatively correlated to the efficacy of breast cancer chemotherapies.3.The expression of miR-4443 in breast cancer resistant cells was distinctly higher than that in sensitive cells.MiR-4443 was positively correlated with the resistance of breast cancer cells to doxorubicin after transfection with miR-4443 mimics or inhibitors.4.Bioinformatic analysis showed that TIMP2 was a specific target gene of miR-4443,along with its expression restrained by miR-4443.5.Low expression of TIMP2 was associated with poor prognosis of breast cancer patients.