MiR-30a Inhibits Epithelial-mesenchymal Transition And Metastasis In Triple-negative Breast Cancer By Targeting ROR1

ObjectivesBreast cancer is the most frequently diagnosed cancer in women and one of the leading causes of cancer death for women.Triple-negative breast cancer(TNBC)is a highly aggressive breast cancer subtype that lacks effective targeted therapies.The features include the lack of expression of estrogen receptor(ER),progesterone receptor(PR)and an absence of human epidermal growth factor receptor 2(Her2)amplification demonstrate aggressive clinical behavior and poor outcome.Therefore,it is important to find a new molecular targeted therapy.MicroRNAs(miRNAs)are a large set of small endogenous non-coding RNAs of 20-22 nucleotides,which are involved in gene expression by targeting the 3’-UTR(untranslated regions)of mRNA,regulating various physiological and pathological processes.In the previous study,the expression of miR-30a was down-regulated in breast cancer.However,there were few reports on TNBC that is prone to invasion,metastasis and poor prognosis.To evaluate the expression and function of miR-30a in TNBC,there will be the following three aspects to explore.Methods1.Detection of miR-30a expression in TNBC tissues and cell lines by real-time PCR;analysis of the relationship between miR-30a expression and TNBC by clinicopathological data;use of bioinformatics public database,the relationship between miR-30a and prognosis of breast cancer was analyzed.2.Stable miR-30a-precursor expressing and negative control(miR-SCR)cell lines by lentiviral transfections were constructed.The effects of miR-30a expression on cell migration and invasion were analyzed by wound-healing assay and invasion assay.The expression levels of Epithelial-mesenchymal transition-related markers were detected by Western blot and immunofluorescence assays to investigate the effect of miR-30a on Epithelial-mesenchymal transition;The effect of miR-30a on tumor gro.^wth of TNBC was established by subcutaneous xenograft model.The model of lung metastasis was established by tail vein injection and the effect of miR-30a on lung metastases was analyzed.3.According to the prediction of miR-30a target genes by miR-base,Targetscandatabase and relevant reports in the literature,miR-30a regulate the expression of target gene by dual luciferase reporter assay.Results1-The expression of miR-30a was significantly down-regulated in TNBC cell linesand tumor tissues,and the down-regulation of miR-30a was associated with high histological grade and more lymph node metastasis(P<0.05).The relationship between the prognosis of miR-30a and the expression of miR-30a in breast cancer patients was analyzed by on-line Kaplan-Meier method.the low expression of miR-30a was associated with poor overall survival(P<0.05).2.The overexprssion of miR-30a suppressed epithelial-mesenchymaltransition(EMT)in TNBC cell,as demonstrated by the overexpression of miR-30a which increased the expression of epithelial marker E-cadherin but decreased the expression of mesenchymal markers N-cadherin and vimentin.The overexpression of miR-30a significantly suppressed TNBC cell invasion and migration,as well as inhibited tumor growth and metastasis in vivo.The volume of transplanted tumors of BT549 overexpressing miR-30a in nude mice was significantly lower(P<0.05)than that in the control group.The number of lung metastasis nodules was significantly lower than that of the control group.3.That transfection of miR-30a mimics significantly decreased the ROR1-3’-UTR wild-type but not the ROR1-3’-UTR mutant.luciferase levels in 293T cells.Thetransfection of anti-miR-30a increased the ROR1-3’-UTR wild-type luciferase activity.Similar results were found in TNBC cells.Overexpression of miR-30a significantly inhibited ROR1-3’-UTR wild-type luciferase activity.Furthermore,The overexpression of miR-30a significantly decreased the expression levels of ROR1.The overexpression of ROR1 partially restored cell invasion suppression by miR-30a.Conculsions1.The expression of miR-30a is significantly decreased in TNBC cell lines andtissues.miR-30a surpressed the migration and invasion,Epithelial-mesenchymal transition,the tumour growth and lung metastasis in TNBC.2.ROR1 is a direct target gene of miR-30a.miR-30a through targeting ROR1 toinhibit the growth and metastasis in TNBC.