Mechanism On Suppression Of Breast Cancer Cell Metastasis Mediated By MiR-568

?Background?The development of cancer involves multiply levels and stages.Metastasis plays a critical role in its progress,and ultimately leads to high fatality rate.In light of complexity of metastasis itself,we should deeply explore its molecular mechanism from preclinical and clinical aspects.Subsequently,we obtain early alarming signals and make timely intervention to improve therapeutic effect and life quality as well as relieve social and personal burdens.Tumor metastasis is very intricate,which involves many metastasis-associated molecules.There is little effect on metastasis through single molecule-targeting intervention.Mi RNAs that target many molecules together possibly can resolve the above problem.Especially,much attention was paid to breast cancer(BC)metastasis-related miRNA,however,only a little progress was made and many problems remained unsettled.Are there novel miRNAs involved in BC metastasis except discovered BC-suppressed miRNA? If any,what metastasis-associated molecules are downregulated? What mechanisms do miRNA-targeting molecules employ to involve in BC metastasis? Are there the uniform data from preclinical and clinical research to support the phenotype reversed by novel miRNA? That many problems are presented and settled will significantly improve therapeutic effect and prognosis.?Aims?To screen and identify novel miRNAs involved in breast cancer metastasis and to explore its function in metastatic progress,with the aim of defining the uniform of preclinical research and clinical investigation and form a solid theoretical base for early alarm,molecular diagnosis and therapy of BC metastasis.?Methods?1.According to GEO profiles,we searched and analyzed expression levels of NFAT5 in breast cancer metastatis-associated microarray data.To obtain highly and lowly expressed NFAT5 cells,we test the abundance of NFAT5 in breast cancer cells utilizing q RT-PCR and WB assay.After MDA-MB-231 cells were transfected with si NFAT5 or infected with lentivirus p LKO.1-GFP-sh NFAT5,wound-healing assay,and tail vein injection metastasis assay,then migration and invasion assay were performed to investigate the metastatic ability of established cells.Tumor formation assay was employed for examining cell survival in vivo.2.According to microarray data and bioinformatics prediction,NFAT5 could be manipulated by miR-568.Dual Luciferase reporter gene assay was made to clarify the above speculation.The abundance of miR-568 in breast cancer cells and clinical samples were detected via q RT-PCR.We tested the NFAT5 expression levels and investigated the invasive properties of MDA-MB-231 cell transfected with miR-568 mimics as well as MCF-7 cell with antagomiR-568,respeltively.We screened and established MDA-MB-231 cell lines stably infected lentivirus PGCsil-miR-568,with the aim of investigating its tumor formation and lung metastasis potential.3.The expression level of S100A4 in cancer cells were examined by q RT-PCR and WB,and the speculation that NFAT5 binds to S100A4 promoter in breast cancer cell was confirmed by Ch IP assay.We tested the expression levels of S100A4 m RNA and protein in MDA-MB-231 transfected with different density of si NFAT5 mimics.After MDA-MB-231 and MCF-7 cells were transfected with miR-568 and antagomiR-568 mimics respeltivly,q RT-PCR was used to measure expression change of S100A4.q RT-PCR,WB and IF assay were employed to examine the expression of EMT-related molecules(mainly E-cadherin and vimentin)in MDA-MB-231 cells transfected with si NFAT5 or si S100A4.The levels of NFAT5,S100A4,E-cadherin and vimentin in breast cancer tissues were assessed by IHC assay,and correlations between them were analyzed.Followed by MDA-MB-231 cell were transfected with si NFAT5,si S100A4 or miR-568 mimics,q RT-PCR were performed to measure the expression levels of CD44,Rho A,MMP2,and MMP9.FC was made to assess cell cycle distribution.WB was utilized to evaluate ERK,Akt and their phosphorylation levels.4.The expression levels of VEGF-C in cancer cells and patients' blood were examined via q RT-PCR and ELISA assays,and correlations between VEGF-C and breast cancer metastasis were analyzed.We confirmed that NFAT5 could bind to the promoter of VEGF-C using Ch IP assay.We examined VEGF-C levels in MDA-MB-231 cells transfected with miR-568 or si NFAT5 mimics using q RTP-CR and ELISA assays.5.The expression levels of Hotair in breast cancer cells and patitein tissues were tested by q RT-PCR.We examined metastatic properties of cancer cells accompanied with upregulation or downregulation of Hotair.After Hotair,EZH2 and SLD1 were knockdown singly or combinedly,q RT-PCR was performed to evaluate miR-568 expression.?Results?1.According to GEO profiles,we seatched and analyzed breast cancer metastatisassociated microarray data,then we knew that NFAT5 expression was high in breast cancer cells of highly metastatic potential.Highly expressed(MDA-MB-231)and lowly expressed(MCF-7)NFAT5 breast cancer cells were obtained via screening.Knockdown of NFAT5 led to the decrease in migratory and invasive properties of MDA-MB-231 cells,and accordingly inhibited tumor formation and lung metastasis in vivo.According to in vitro and in vivo characteristics,we confirmed that NFAT5 prompted breast cancer metastasis.2.In line with microarray data and bioinformatics prediction,dual luciferase reporter gene assay showed that miR-568 inhibited the expression of NFAT5.q RT-PCR results demonstrated that miR-568 was negatively with NFAT5 expression in breast cancer cells and clinical tissues.The invasive and metastatic protential of breast cancer cell got downgraded or upgraded accompanied with upregulation or downregulation of miR-568,and the expression of NFAT5 showed the similar tendency.In vivo,miR-568 partly inhibited breast cancer growth,but significantly suppressed lung metastasis.Colletively,miR-568 inhibited breast cancer metastasis by downregulating NFAT5 expression.3.S100A4 expression showed the similar tendency with NFAT5 in breast cancer cells.Ch IP assay comfirmed that NFAT5 bound to the promoter of S100A4 and positively regulated its expression.q RT-PCR,WB and IF assay showed that NFAT5 regulated the expression of EMT-assciated molecules such as E-cadherin,through upregulation of S100A4.IHC assay demonstrated that the expression of NFAT5 and S100A4 was negatively with E-cadherin and positively with Vimentin,and the correlations were in line with TNM stages.After MDA-MB-231 cells were transfected with miR-568,si NFAT5 or si S100A4,cancer metastasis-related molecules CD44,Rho A,MMP2 and MMP9 were downregulated,and the cell cycle was placed in S phase arrest,and the phosphorylation of ERK and AKT were lowered,and anoikis effect was inhibited.Colletively,miR-568 indirectly suppressed S100A4 expression through downregulating NFAT5,and ultimately prompted breast cancer cell metastasis.4.q RT-PCR and ELISA assay showed that the VEGF-C expression levels were high in cancer cells with highly metastatic potential.Compared with normal and begnin disease patient,the leves of VEGF-C in blood samples from patients with lymph node metastasis or lung metastasis were statistically different.Similar to S100A4,VEGF-C expression was upregulated by NFAT5 in a transcription dependent manner.q RT-PCR and ELISA assay confirmed that VEGF-C expression and secretion was lowered in MDA-MB-231 cells transfected with miR-568 or si NFAT5 mimics.Collectively,miR-568 as a tumor suppressor indirectly inhibited VEGF-C expression and secretion by downregulating NFAT5.5.q RT-PCR assay was employed to examine Hotair expression in breast cancer cells and breast cancer tissues and their-matched noncancerous tissues,and the result clarified that Hotair expression was positively with cancer metastasis.Single or combinational knowdown of Hotair,EZH2 and SLD1 led to of miR-568.According to issues,linc RNA Hotair suppressed gene expression via epigenetic mechamism.Collectively,we speculated Hotair possibly epigeneticly silenced miR-568 expression and prompted breast cancer metastasis.?Conclusions?1.In vivo and in vitro assay confrmed that NFAT5 prompted breast cancer metastasis,which possibly acted as a potentially effect target.2.miR-568 was downregulated in breast cancer,and it inhibited breast cancer cell metastasis by lowering NFAT5 expression.3.NFAT5 indirectly induced EMT and cell survivial signal by positively regulating S100A4 expression.Either miR-568 overexpression or NFAT5 knockdown reversed the above phenotype.Predinical functional research data was well uniform to IHC results from clinical samples.Collectively,miR-568 indirectly suppressed S100A4 expression through downregulat-ing NFAT5,and ultimately prompted breast cancer cell metastasis.4.NFAT5 stimulated lymphangiogenesis by inducing VEGF-C expression and secretion.Either miR-568 overexpression or NFAT5 knockdown suppressed VEGF-C expression.Combining data of clinical blood test with the above results,we speculated that miR-568 indirectly lowered VEGF-C levels by suppressing NFAT5 expression.As a result,this effect inhibited lymphangiogenesis,and ultimately led to low frequency of lymph node metastasis.5.Linc RNA Hotair possibly epigeneticly silenced miR-568 expression to prompt breast cancer metastasis.