MicroRNA455-3p Regulates Tumor Invasion And Migration In Triple Negative Breast Cancer

BackgroundBreast cancer is one of the most common malignant tumors and a major cause of cancer-related death for women worldwide. Contributing to heavier environmental pollution, aging population, the incidence has increased year by year. Breast cancer is a kind of heterogeneous disease, and the morphology, clinical manifestations and sensitivity to treatment are different. Triple negative breast cancer (TNBC) is a special type of breast cancer, and is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) expression. TNBC accounts for 15%-20% of all breast cancers approximately and mostly occurs in young women. It possesses a worse cell differentiation and more aggressive behavior than other subtypes, so it is easy to take an early recurrence and distant metastasis resulting in a much poorer prognosis. Now the main treatment therapies are surgery operation, chemotherapy and radiotherapy. However, due to short of conventional molecular targets such as ER and HER-2 which can be used for target therapy in other subtypes, it lacks of the more effective ways in TNBC. In recent years, the molecule known as a kind of endogenous small no-coding RNAs was found in prokaryotic organism and eucaryon, miRNAs contrain 18-22 nucleotides and are important gene regulatory factors which may paly critical roles in cell proliferation, progression, differentiation and apoptosis. MiRNAs can inhibit the transciption of target genes or induce the degradation of mRNA by combining with the 3’-UTR region, and play role of tumor inhibitor genes or oncogenes. Based on analysis of bioinformics,92% of human genes are regulated by miRNAs. Recent studies have also demonstrated that there is a close correlation between miRNAs and the development of breast cancer. High expression of miR-10b was found in metastatic breast cancers and promoted metastasis and infiltration, and MiR-145 could inhibit cell proliferation, infiltration and metastasis in breast cancers inversely. But the function of miRNAs in TNBC remains unknown.EI24, a p53 responsive pro-apoptotic factor, plays an important role in the suppression of cell growth and the activation of autophagy. Furthermore, reduced expression of EI24 is associated with the induction of EMT and tumor progression. Loss of EI24 contributes to etoposide resistance, suggesting that EI24 status could be used as a prognostic marker for chemotherapy responsiveness. Jung-Min’s study indicated that reduced expression of EI24 confers resistance to gefitinib through IGF-1R signaling in NSCLC cells. MiR-483-3p plays an oncogenic role in esophageal squamous cell carcinoma by targeting tumor suppressor EI24, so there is a significant correlation between miRNA and EI24. However, no study has been reported about EI24 in TNBC.AimTo detect the differential expression level of microRNAs in TNBC and confirm the microRNAs candidate. Further, we verify its function and target gene in TNBC.MethodsScreening out differentially expressed microRNAs as candidates by analyzing the Microarrays. qRT-PCR assay was applied to evaluate the expression of selected microRNA in 117 cases of TNBC tissues,42 cases of IDC tissues (ER++-+++, PR++-+++). miR-455-3p mimics, negative control, miR-455-3p inhibitor and inhibitor negative control were respectively transfected into TNBC cell lines MDA-MB-231 and MDA-MB-468, then cell proliferation and migration were assessed by cell viability assay and Transwell assay. Luciferase assay and Western blot were used to identify the underlying target gene of miR-455-3p.Results(1) We screened out 3 candidate microRNAs, and they were miR-455-3p、 miR-425-5p and miR-196a-5p. The expression level of miR-455-3p in 117 TNBC was 10-fold more than that in 42 IDC by qRT-PCR. Then we identified and verified that the expression levels of miR-455-3p were up-regulated markably in TNBC cell lines by qRT-PCR(P<0.05).(2) Comparing with the negative controls which have been transfected with miR-455-3p mimics, we found that there was a significant effect of miR-455-3p on promoting migration and invasion in cell lines MDA-MB-231 and MDA-MB-468 (P <0.05). Meanwhile, by transfection with miR-455-3p inhibitor in MDA-MB-231 and MDA-MB-468, the abilities of cell migration and invasion were suppressed significantly comparing with the negative control (P<0.05).(3) The results of MTS assay confirmed that there was a significant statistical difference between miR-455-3p group and negative control group. So miR-455-3p made effect on promoting growing and proliferation in cell lines MDA-MB-231 and MDA-MB-468.(4)Through co-transfection of pmirGLO-cad vector and miR-455-3p mimics in cell lines MDA-MB-231 and MDA-MB-468, the luciferase assay indicated that EI24 was the target gene of miR-455-3p.(5) Western blot showed that EI24 protein was down-regulated by transfecting miR-455-3p mimics in cell lines MDA-MB-231 and MDA-MB-468, which verified that EI24 was the target gene of miR-455-3p. These suggested that miR-455-3p could be a novel and potential therapeutic target of TNBC.ConclusionsOur findings indicate that miR-455-3p acts as an oncogene in TNBC tissues and cell lines, and it may increase the abilities of invasion and metastasis of TNBC through a mechanism involving in EI24.