Dysreg?lated Expression Profiles And Function An Alysis Of MicroRNAs In Breast Cancer FFPE Tissues

Objectives:To screen for abnormal microRNAs (miRNAs) expression profile related breast cancer, analyze the relationship between differentially expressed miRNAs and clinicopathological features such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor2(Her-2), Ki-67index, proliferating cell nuclear antigen (PCNA) and mutant P53protein, evaluate the potential value of differential miRNAs as diagnosis and prognosis markers for breast cancer, and elucidate the roles of miR-15a and miR-141involved in the pathogenesis of breast cancer.Methods:MiRNA expression profile, using miRNAs expression chips, was executed in9formalin fixed paraffin embedded (FFPE) breast tissues, including3tumor tissues with luminal-like subtype and3corresponding para-carcinoma tissues, as well as3tumor tissues with Basal-like subtype. The results of microarray expression profile were validated in106cancer tissues,23para-carcinoma tissues and77breast benign tissues by quantitative real-time PCR technique. Genes targeted by candidate miRNAs were predicted by three softwares including Targetscan, PITA and miRNAorg. MDA-MB-231cells were transfected with candidate miRNAs mimics and normal control using Lipofectamine2000. Apoptosis and cell cycle assays were analysed by flow cytometry. Cell viability was measured by CCK-8assay, cell invasiveness was measured by transwell assay and cell migration was measured by wound healing assay; Expression of genes targeted by candidate miRNAs was detected by qPCR and western blotting. Dual luciferase report assay was used to validate the miRNAs target genes.Results:(1) For breast cancer tissues with luminal-like subtype, compared with para-carcinoma tissues, the stringent threshold for up-and down-regulated genes was set to be a fold change?2.0and p value?0.05, and there were37mature miRNAs up-regulated and8mature miRNAs down-regulated. For tumor tissues with luminal-like subtype, compared with basal-like subtype, the stringent threshold for up-and down-regulated genes was set as a fold change?1.5and p value?0.05, and there were4mature miRNAs up-regulated and4mature miRNAs down-regulated. (2) The results of microarray validated by qRT-PCR showed that miR-15a, miR-141and miR-205were downregulated in tumor tissues compared with benigh tissues (p<0.05), and miR-141was lower in tumor tissues with basal-like subtype tissues compared with luminal-like tissues (p<0.05); MiR-130b was upregulated in tumor tissues compared with benigh tissues (p<0.05).(3) Expression of miR-15a and miR-141were negative related with Ki67index and PCNA status in breast cancer tissues (p<0.05). Furthermore, miR-15a was negative related with mutant P53status in breast cancer tissues (p<0.05); MiR-205was related with patient's age, expression of miR-205was lower in older patients (?50years old) than in younger patients (<50years old)(p<0.05).(4) miR-141promoted MDA-MB-231cells apoptosis and G1cell cycle arrest, inhibited G1/S transition and cell proliferation, reduced cell invasiveness and migration. MiR-15a promoted cell apoptosis and inhibited cell proliferation.(5) The mRNA and protein expression levels for ANP32E were significantly decreased in MDA-MB-231cells transfected with miR-141mimics compared with cells transfected with mimics normal control (p<0.05). Similarly, the mRNA and protein expression levels for SNCG were significantly decreased in MDA-MB-231cells transfected with miR-15a mimics compared with cells transfected with mimics normal control (p<0.05).(6) The results of dual luciferase report assay showed that the relative luciferase activity decreased when psiCHECK2-ANP32E3'UTR was co-transfected with miR-141mimics, but not with mimics negative control. Similarly, the relative luciferase activity decreased when psiCHECK2-SNCG3'UTR was co-transfected with miR-15a mimics, but not with mimics negative control.Conclusion:miR-130b was considered as oncogene, while miR-141and miR-15a may function as tumor suppressor by targeting to3'-UTR of ANP32E and SNCG in process of development and progression of breast cancer. The expression level of miR-141was related with cell proliferation and invasiveness. Low expression level of miR-141indicated bad prognosis for breast cancer.