Study On Mechanism Of MiR-21-5p/Tiam1 In Breast Cancer Progression

Background:Breast cancer(BC),one of the most abundant malignant solid tumors,considered as the main cause of cancer-related death in women globally.The present studies have demonstrated that invasion and metastasis is a continuous process of multi-step and multi-factor participation,which is not only the main biological character of BC but also the main cause that affected the mortality of BC patients.With the development of molecular biology of BC,molecular targeted therapy has become research hotspot,which promotes the process of individualization treatment of BC.Since trastuzumab received approval for use in the treatment of solid tumors in 1998,more and more molecular targeted drugs have emerged,such as the CDK4/6 inhibitors,palbociclib,anti-estrogen,tamoxifen,mTOR inhibitor,everolimus,etc.Despite considerable progress has been achieved in the treatment effect of BC with the clinical application of these drugs,the prognosis of metastatic BC patients is still poor.Hence,in-depth study of the molecular mechanisms of BC,and found the more effective drugs and therapeutic targets for reducing BC mortality and improve the quality of life of patients has a very important significance.microRNA(miRNA)are a major class of endogenous,small noncoding RNA molecules approximately 18-25 nucleotides in length,play a key role in the regulation of protein expression through base pairing with the 3'-untranslated region(3'-UTR)of their target mRNAs.As the gene mutation is a common event in the onset of cancer and the changes of gene expression will influence the initiation and progression of tumor growth,it is well accepted that an increasing number of alterations in miRNA expression are associated with biological processes in diverse malignant tumors.T lymphoma invasion and metastasis 1(Tiam1)was first identified in 1994 in an in vitro gene research that confer an invasive phenotype of murine T-lymphoma cells.Human and mouse gene products present 95%homology,and Tiam1 gene is located on chromosome 21q22.I in human,whereas on chromosome 16 in mouse homolog.Recently,studies indicated that Tiam1 is essential in various aspects of tumor progression,such as cell proliferation,differentiation,angiogenesis,migration and invasion.Huang et al reported that Tiaml had a higher probability of being over-expressed in hepatocellular carcinoma(HCC)patients with metastasis.High Tiaml expression level was also considered as a prognostic factor for poor overall survival in HCC patients,which suggested an association between Tiaml over-expression and metastasis in HCC.Liu et al indicated that silencing Tiam1 lead to a decline on cancer cell growth,invasion,and migration in esophageal squamous cell carcinoma(ESCC),enhanced cell apoptosis and caused a G0/G1 cell cycle arrest.The previous study from our lab demonstrated that Tiam1 is over-expressed in BC and is correlated with poor survival outcomes,but its regulatory mechanisms in BC progression remains unclear.Thus,there is an urgent need to explore functional roles of Tiaml in BC,which providing a new molecular option for its diagnosis and prognosis.Objectives:This study was aimed to evaluate the impact of miR-21-5p/Tiaml on BC biological behaviors,and to explore the related molecular mechanisms.(1)To determine whether Tiaml expression is important in tumorigenesis and to evaluate its prognostic value in BC.(2)To elucidate the functional roles of Tiaml in cell proliferation,metastasis and glycolysis in BC in vitro and in vivo,and explore its possible regulatory mechanism.(3)To identify the potential miRNA candidates in Tiaml targeted,and further reveal the mechanisms of miR-21-5p/Tiaml axis in BC tumorigenesis and metastasis regulation.Materials and methods:(1)Clinical specimens and database analysis:To investigate the role of Tiaml in human BC,157 BC tissues and 37 normal breast tissues were examined by IHC.The relationship between Tiaml expression and the clinicopathological characteristics of BC was then analyzed.Kaplan Meier-plotter was used to evaluate the expression level of Tiaml in the prognosis association of BC patients.(2)Experiments in vitro:Stable knockdown or over-expression of Tiaml was generated by transducing the sh-Tiaml or Tiam1 lentiviral expression construct in BC cells.To demonstrate the potential effects of Tiam1 on proliferation promotion in BC cells,we performed MTT,Colony and EdU assays.To determine whether Tiaml could promote BC cells invasion and migration in vitro,wound healing and transwell assays were performed.The tube formation ability of HUVECs was examined to explore whether Tiaml expression affect the angiogenesis in BC.Metabolic Kits were used to detect the effects of Tiaml modulation on cell metabolism products,glucose,lactate,and ATP.Western blot and IF were used to investigate the effect of Tiaml on EMT and metabolic markers in BC cells.qRT-PCR and luciferase reporter assay were used to determine the relationship between miR-21-5p and Tiaml.(3)Experiments in vivo:The xenograft model was utilized by BC cells injection with either stable Tiam1 knockdown or over-expression into nude mice subcutaneously to further investigate whether Tiaml promoted BC tumor growth in vivo.To ascertain the role of Tiaml in BC metastasis in vivo,we injected MDA-MB-231-shTiam1,MDA-MB-231-shCon,and MCF-7-Tiaml,MCF-7-vector cells into the tail vein of nude mice and detected the lung metastatic nodules.The tumor sections were stained to quantitatively evaluate expression of the EMT associated markers.E-cadherin and Vimentin.Results:(1)Tiaml was up-regulated in BC and predicted poor prognosis:The positive and strongly positive rate of Tiaml expression was significantly up-regulated in the BC tissues(77.7%,65.6%)compared with the normal breast tissues(29.7%,10.8%).Higher Tiam1 expression was found to be correlated with clinical stage,pathological grade,LN metastasis and Her2 status.Analysis from Kaplan Meier-plotter showed that BC patients with high expression of Tiaml displayed a poor overall survival(OS).(2)Tiaml promoted cell proliferation and tumorigenesis of BC:MTT,Colony and EdU assays showed that Tiaml knockdown or over-expression significantly associated with cell growth in corresponding BC cells,respectively.The xenograft tumor model further confirmed that Tiam1 promotes BC tumor growth in vivo.(3)Tiaml promoted BC metastasis via EMT in vitro and in vivo:Using wound healing and transwell assays,we found that knockdown of Tiaml significantly reduced the ability of migration and invasion of BC cells.The in vivo metastatic experiments further validated the role of Tiam1 in BC metastasis.Western blotting and IF assays showed that Tiaml knockdown increased epithelial markers expression,and suppressed mesenchymal markers levels in MDA-MB-231/Hs-578T cells.Tiaml over-expression displayed the opposite effect in MCF-7/MDA-MB-468 cells.(4)Tiaml promoted angiogenesis in BC:BC cells' ability to induce tube formation of HUVECs was significantly reduced in Tiaml knockdown cells,while enhanced in Tiam1 over-expressing cells.Western blot analysis revealed that knockdown of Tiaml suppressed,while over-expression of Tiaml enhanced the expression level of MMP2 and VEGF in BC cells.(5)Tiaml promoted BC cells growth and metastasis through Warburg effect:Tiaml knockdown markedly reduced the glucose uptake,lactate production and ATP level,whereas Tiam1 over-expression increased these results in BC cells.Consistent with these observations,the protein levels of metabolic markers were down-regulated by Tiaml silencing and up-regulated by Tiam1 over-expressing in BC cells.The glycolytic inhibitor 2-deoxy-D-glucose(2-DG)or 3-bromopyruvate(3-BrPA)greatly abrogated the effect of Tiaml on BC cell proliferation and metastasis.(6)Tiaml was a direct target of miR-21-5p:Using four target prediction programs,miR-21-5p was picked as one potential miRNA candidates that targeting Tiam1.And then luciferase reporter assays further demonstrated that miR-21-5p inhibits Tiaml production by directly targeting its 3'-UTR in BC cells.(7)miR-21-5p/Tiaml regulated Warburg effect and tumor metastasis in BC:To determine whether miR-21-5p might influence glycolysis and metastasis through the inhibition of Tiaml expression in BC cells,we co-transfected BC cells with miR-21-5p inhibitor and si-Tiaml.We found that miR-21-5p inhibition significantly enhanced glycolysis and metastasis,while Tiaml suppression obviously weakened these effects.Conclusions:(1)Tiam1 over-expression was correlated with clinical stage,pathological grade,LN metastasis,Her2 status and poor prognosis of BC patients.(2)The over-expression of Tiam1 promoted cell proliferation and metastasis in BC.(3)Mechanistic study showed that Tiam1 influence BC cells glycolysis by relevant metabolic enzymes affection,and therefore enhanced cell proliferation and metastasis.(4)miR-21-5p/Tiaml promoted BC cell growth and metastasis through Warburg effect,which revealed that the miR-21-5p/Tiaml plays an important role in the progression of BC.