Prognostic Significance Of MiR-320a And Its Correlation With Rab14 In Human Breast Cancer

IntroductionBreast cancer is one of the most common malignant tumors among women in the world and threatens to women’s health seriously. Although improved early detection and effective treatment can help to prolong the survival times of breast cancer patients,many patients still die due to invasion and metastasis. Identification of sensitive and specific biomarkers for prognosis in breast carcinoma, therefore, is important for both early clinical detection and effective prophylactic therapy.miRNAs, 18-22 nucleotides long, are noncoding single stranded RNAs and possesses an important fuction in regulation of gene expression. The mature microRNA can bind to the 3’ untranslated regions(3’ UTR) of the target mRNAs and can regulate gene expression either by inhibiting the translation of target mRNAs or by promoting transcript degradation. microRNA can participate a series of biological processes including cell proliferation, dififerentiation, apoptosis and metabolism as well as tumor development, progression and metastasis and will become the potential treatment target in the future. Because of involving in multiple genes iregulation, it is able to get useful information about prevention, diagnosis, treatment and prognosis about tumor development and progression.miR-320 a is located on chromosome 8, and prior studies have shown that miR-320 a has abnonnal expression levels in several cancers and participates in the formation, progression, and metastasis of cancer, such as colon cancer, pancreatic cancer. The exact role miR-320 a plays in breast carcinoma proliferation, migration and invasion/metastasis is, however, still largely unclear. Specifically,there has been no report on the elation and mechanisms underlying miR-320 a expression and the prognostic significance of breast carcinoma in either Chinese or elsewhere.In this study, we focused on the expression pattern of miR-320 a in large amount cases of breast tissues. We used real-time PCR to detect miR-320 a expression in fresh,frozen invasive breast cancer and adjacent non-cancerous breast tissues. Then we used in situ hybridization to explore miR-320a expression in paraffin-embedded breast tissues and the relationship between miR-320 a and prognosis Then, we used computer software and a series of cell experiments to predict the possible target genes of miR-320a and got Rab14 as our candidate target gene. We further analyzed Rab14 expression in breast cancer tissues and the relationship with miR-320 a. Our findings possess not only obviously theoretical significance for understanding the biological characteristics of tumors, but also clinical and social effects.Part I Expression of miR-320 a in breast cancer and its correlation with clinicopathological valuables and prognostic analysis Objective To assess miR-320 a expression in fresh,frozen and paraffin-embedded breast cancer tissues and explore the relationship between invasive breast cancer and clinicopathological characters, including survival analysis.Methods We first determined the miR-320a expression levels in 19 pair firesh,frozen invasive breast cancer tissues and adjacent non-cancerous breast tissues using quantitative real-time PCR. Then we determined the miR-320 a expression levels in15 in situ breast carcinoma and 130 invasive breast cancer tissues using in situ hybridization(ISH). Statistical analysis software was used to analyze the Klationship between invasive breast cancer and clinic characters, including survival analysis.Results miR-320a was markedly down-regulated in invasive breast cancers compared to adjacent non-cancerous breast tissues, as determined by real-time PCR(P< 0.001). As determined using in situ hybridization,60 of 130(46%) showed high expression levels of miR-320a(staining index score > 4). miP-320a staining was observed in the nuclei and cytoplasm and was strongly related to tumor size(P =0.046), clinical stage(P < 0.001), lymph node metastasis(P < 0.001) and distant metastasis(P= 0.001)。Moreover,patients with low miR-320 a expression levels tended to have shorter overall survival times(P< 0.001). Univariate and multivariate analysis showed that miR-320 a was an independent prognostic biomarker for invasive breast cancer (P = 0.047, HR = 0.221, 95% CI: 0.050-0.979). Morover, clinical stage is also a prognostic biomarker for invasive breast cancer.Condusions miR-320 a was markedly down-regulated in invasive breast cancer. We did observe significant correlation of miR-320 a wkh dinieal stages,-lymph node metastasis, tumor sizes and distant metastasis. Patients with miR-320 a positive expression had longer survival times than with negative expression. miR-320 a.is a superior independent biomarker for diagnosis and prognosis comparing with currently widely-used diagnostic index in breast carcinoma.Part II The correlation of miR-320 a and its f;arget protein Rab14 in breast cancerObjective To clarify the possible mechanisms of miR-320 a in breast cancer, that is prediction and validation the downstream target genes of miR-320 a.Methods We predicted the potential targets by bioinfonnatics tools and further determined the target genes through dual-luciferase reporter(DLRTM) assay. Then we evaluated the mRNA and protein level of those targets by qRT-PCR and western blot after transfection of pre-miR-320 a. We fiirther investigate the target genes in clinical samples by western blotting and immunohistochemistry(IHC).Results According to bioinformatics and Kaplan-Meier Plotter analysis, we chow Rab14 as our potential target gene of miR-320 a. Based on DLR?assay, miR-320 a could significantly repress the luciferase activities of the 3’-UTR of Rabl4 mRNA.miR-320 a reduced the protein level of Rabl4 after transfected pre-miR-320 a. For Rab 14 protein expression, 12 of 19 invasive breast cancer tissues showed higher level than non-cancerous tissues by western Blot; Rab 14 immunoreactivity was readily detected in the cytoplasm. 77 of 130(59.2%) invasive breast cancer tissues showed high expression levels of Rab14. We 出d observe significant correlations of Rabl4 with tumor size (P = 0.034), lymph node metastasis (P < 0.001), distant metastasis {P=0.007),clinical stage(P=0.001) and histological grading(f = 0.035). Pearson,s correlation coefficient between miR-320a and Rab14 was- 0.50, P < 0.001.Kaplan-Meier survival analysis of 130 cases revealed a correlation between higher Rab 14 expression levels and shorter overall survival times(P=0.001)Conclusions miR-320 a may play its role though the downstream target gene Rab14.