The Mechanism And Clinical Implication Of MiR-373 In Breast Cancer Metastasis

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The mechanism and clinical significance of down-regulation of TRPS1 by mi R-373 in promoting breast cancer metastasisBackground:Breast cancer is the most frequent malignancy occurring in women and the leading cause of cancer mortality in women.Although the significantly improving of early diagnoses and treatment in cancer patients,the high rate of distant metastasis and tumor recurrence after curative resection often results in a rapidly fatal clinical outcome for this disease.Notably,understanding the mechanisms for breast cancer metastasis,and identifying novel prognostic markers are critical.Breast cancer metastasis is a complex process comprising multiple sequential steps and the regulation of multiple factors.Many factors including oncogenes,tumor suppressors,metastasis-related genes and non-coding RNAs are involved in this complex process.Abnormal mi R-373 expression was shown to be closely associated with breast cancer metastasis.However,little is known about the mechanism by which mi R-373 promotes cancer metastasis.In our previous studies,we demonstrated that TRPS1 may act as a novel potential target of mi R-373 by SILAC quantitative proteomics.The study of TRPS1 mainly focused on its role in the development and differentiation.More importantly,the potential role and molecular mechanism of TRPS1 in cancer progression are not clear.In this study,we further to elucidate the biological functions,mechanism and clinical significance by which mi R-373 regulates its potential candidate,TRPS1,within breast cancer metastasis.Methods:1.Mi R-373 was over-expression or inhibited in cell lines,and TRPS1 protein level was determined by western blotting.2.The binding site of mi R-373 in the 3'UTR of TRPS1 was predicted by bioinformatics.Mi R-373 directly down-regulated TRPS1 level was determined by the Luciferase and Mutation assays.3.TRPS1 protein levels were detected in cancer cell lines with different metastasis properties.4.TRPS1 was silenced or over-expressed in cell lines,and the migration and invasion abilities and EMT markers were analyzed by a Transwell assay and western blotting,respectively.5.Lung metastatic nodules were determined in NOD-SCID mice after injection with cell lines in which TRPS1 expression was stably silenced.6.Cells were transfected with the TRPS1 wild-type and mutated-type plasmids and the TRPS1 protein level and migration and invasion were determined by western blotting and a Transwell assay,respectively.7.Cells were co-transfected with anti-TRPS1 si RNA and the mi R-373 inhibitor and the TRPS1 protein level,and migration and invasion were determined by western blotting and a Transwell assay,respectively.8.TRPS1-regulated FOXA1 were identified by SILAC quantitative proteomics.9.TRPS1 was silencing or over-expression in cell lines,the FOXA1 protein and m RNA levels were determined by western blotting and q PCR,respectively.10.Two binding sites of TRPS1 in the FOXA1 promoter were predicted by bioinformatics.TRPS1 directly bound to the FOXA1 promoter and induced FOXA1 transcription was determined by the Luciferase,Mutation and Ch IP assays.11.Cells were co-transfected with anti-TRPS1 si RNA together with FOXA1 plasmid,and the migration and invasion abilities and EMT markers were analyzed.12.Clinical correlations of TRPS1 with FOXA1 were analyzed in breast tumor samples.Clinical correlations between TRPS1 m RNA levels,pathological characteristics and survival were analyzed in breast tumor samples.Results:1.TRPS1 is targeted by mi R-373.Down-regulation of TRPS1 is induced by mi R-373 through binding to the TRPS1 3'UTR.2.TRPS1 are down-regulated in high metastasis cancer cell lines.Down-regulation of TRPS1 promotes EMT,migration and invasion in breast cancer.3.Down-regulation of TRPS1 stimulates breast cancer metastasis in vivo.4.Down-regulation of TRPS1 is critical for mi R-373-stimulated EMT,migration and invasion.5.TRPS1 acts as a transcription activator.TRPS1 induces FOXA1 transcription through binding to the FOXA1 promoter.6.TRPS1 suppresses migration and invasion by up-regulating EMT negative modulator,FOXA1 expression.7.The expression of FOXA1 is significantly and positively correlated with TRPS1 expression in breast tumor samples,which is consistent with the results in our cell models.8.Breast cancer patients with TRPS1 low exhibit poor prognoses,suggesting that TRPS1 low is a potential biomarker for a poor prognosis in patients with breast cancer.Conclusions:In this study,for the first time,we report that TRPS1 functions as a transcription activator to induce a transcription of the negative EMT regulator,FOXA1.In addition,a novel molecular mechanism for mi R-373 in EMT and metastasis in breast cancer is elucidated.Down-regulation of TRPS1 induced by mi R-373 stimulates EMT and metastasis through repression of FOXA1.Part ? The function and mechanism of the mi R-373-TXNIP-HIF1?-TWIST-mi R-373 feedback loop in breast cancer metastasisBackground:Previously,we demonstrated that mi R-373 promoted breast cancer cell EMT and metastasis by directly inhibiting TXNIP.Based on our previous results,we carried out this study to identify how mi R-373 regulates TXNIP to stimulate cancer metastasis,and further to elucidate the mechanism and clinical implication of down-regulation of TXNIP by mi R-373 in promoting breast cancer metastasis.Methods:1.Lung metastatic nodules were determined in NOD-SCID mice after injection with cell lines with stable expressing mi R-373,mi R-373/TXNIP-3'UTR and mi R-373/TXNIP,respectively.2.Cells were transfected with mi R-373 precursor or anti-TXNIP si RNAs,following 1 ?M H2O2 treatment(non-toxicity)for 12 h,and then the migration and invasion were analyzed by a Transwell assay.3.Cells were transfected with mi R-373 inhibitor or TXNIP plasmid,following 2 m M NAC treatment(non-toxicity)for 24 h,and then the migration and invasion were analyzed by a Transwell assay.4.HIF1? was silenced or over-expressed in cell lines,and TWIST protein level was determined by western blotting.5.Cells were co-transfected with the TXNIP plasmids together with the mi R-373 precursor,and then western blotting and immunofluorescence assay were performed.6.HIF1?/TWIST was silenced or over-expressed in cell lines,and the mi R-373 level was analyzed by q PCR.7.Cells were co-transfected with HIF1? plasmid and anti-TWIST si RNAs,and then the mi R-373 level was analyzed by q PCR.8.Five binding sites of TWIST in the mi R-371-373 gene cluster were predicted by bioinformatics.TWIST directly bound to the mi R-371-373 promoter and induced mi R-373 transcription was determined by the Luciferase,Mutation and Ch IP assays.9.The mi R-373-TXNIP-HIF1?-TWIST-mi R-373 feedback loop was evaluated for its clinical relevance in breast tumor samples.Results:1.Mi R-373-induced metastasis occurs primarily through inhibiting TXNIP.2.Mi R-373 stimulates invasion and metastasis via TXNIP-dependent ROS reduction.3.The decrease of intracellular ROS levels induced by mi R-373 over-expression or TXNIP knockdown,increasing the HIF1? and TWIST level and induces their nuclear translocation.4.HIF1? and its downstream,TWIST,up-regulate mi R-373 expression.TWIST acts as a transcription activator.TWIST induces mi R-373 transcription through binding to the mi R-371-373 promoter.5.A novel mechanism by which mi R-373 promotes EMT and metastasis though the mi R-373-TXNIP-HIF1?-TWIST-mi R-373 feedback loop is elucidated in breast cancer.Clinically,activation of the feedback loop is closely associated with poor prognoses,implicating that this feedback loop is a potential biomarker for worse outcomes in patients with breast cancer.Conclusions:Based on our previous study,we further to demonstrate a novel mechanism by which mi R-373 promotes EMT and metastasis though the mi R-373-TXNIP-ROS-HIF1?-TWIST-mi R-373 feedback loop.Activation of the feedback loop may be an independent prognostic factor for patients with breast cancer.