TGF-β1 Regulates The Expression Of Chemokine Receptor CXCR4 In Human Breast Cancer Cell Line MCF-7

The most important biological characteristics of malignant tumour cells are the ability of invasion and migration, which are main reasons lead to death. Previous work has already revealed both transforming growth factor (TGF)-βsignaling system and chemokine-chemokine receptor are involve in tumor metastases. However, it is unclear that there is an relation between TGF-βsignaling system and chemokine-chemokine receptor during the process of tumor migration. In this research, TGF-β1-regulated CXCR4 expression was investigated in human breast cancer cell line MCF-7s.ⅠEffcet of human recombination TGF-β1 (rhTGF-β1) on the expression of CXCR4 and CCR7 in MCF-7sTo investigate whether TGF-β1 affects CXCR4 expression in MCF-7 cells, we cultured cells with 1, 10, 100 ng/ml human recombination TGF-β1 (rhTGF-β1) for 24 hours,then detected:1. the expression of CXCR4 in MCF-7s1)at the mRNA level:The experiment MCF-7s had a higher expression of CXCR4 transcripts in compared with those unstimulated cells (semiquantitative RT-PCR). The expression of CXCR4 transcripts of MCF-7s were further checked by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR) usingβ-actin mRNA as the control for cDNA input. The quantitative RT-PCR tests confirmed that the levels of CXCR4 transcripts were higher 6-, 6-, and 10-fold in the MCF-7s treated with 1, 10, 100 ng/ml rhTGF-β1, respectively, than in those unstimulated cells.2) at the protein level:MCF-7s were treated with rhTGF-β1 for 48 hours, and the expression of CXCR4 was analyzed by flow cytometry(FCM). TGF-β1induced or up-regulated the surface expression of CXCR4 and in a dose-dependent manner in MCF-7s.3)chemotaxis assay:The functional activity of CXCR4 receptor on the surface of MCF-7s to the respective ligand (SDF-1α) was assessed by applying recombinant human SDF-1αin a cell migration assay. MCF-7s treated with rhTGF-β1 had exhibited an efficient response to SDF-1αcompared with untreated cells.2. Effcet on the expression of CCR7 in MCF-7s treated with rhTGF-β1 It had no effect on the expression of CCR7 on MCF-7s treated with rhTGF-β1.ⅡEffcet of a soluble fusion protein(Fc:TβRII) on the expression of CXCR4 and CCR7 in MCF-7s1. Construction of an eukaryotic expression vector pIRES2-EGFP-TβRII-hIg To construct an eukaryotic expression plasmid of human soluble TGFβRII:human IgG1 Fc (Fc: TβRII) fusion gene, a portion of the extracellular domain of the human TGF-βtype II receptor was amplified by PCR from H2-3FF. Then the receptor fragments were cloned into the expression vector pcDNA3.1-hIg. Furthermore, human tβRII:Fc sequences were subcloned into the expression vector pIRES2-EGFP and the construct was termed pIRES2-EGFP-TβRII-hIg. Finally, authenticity was confirmed by sequencing.2. Expression of Fc:TβRII in MCF-7sTo examine the expression of Fc:TβRII, MCF-7 cells was transfected with pIRES2-EGFP-TβRII-hIg, and then the recombinant protein Fc:TβRII was expressed in the transfected cells and secreted into the medium confirmed by RT-PCR, western blotting and ELISA.Forty-eight hours later, total cellular RNA and the cell lysate samples were collected, and subsequently mRNA transcription were analyzed by reverse transcription- PCR. As expected, we can observe the～1200bp bands in pIRES2-EGFP-TβRII-hIg transfected cells, but no band was detected in parental and transfected cells with the empty vector. This demonstrated mRNA transcription of the Fc:TβRII encoding genes in transfected cells. Furthermore, the expression of Fc:TβRII was evaluated by western blotting using anti-hIgG polyclonal antibody, and the results suggested that the Fc:TβRII protein, characterized as expected by an apparent molecular weight of 41,000 Da, was present in pIRES2-EGFP-TβRII-hIg transfected cells. However in parental cells and empty vector transfected cells no Fc:TβRII protein can be seen. Finally, a soluble typeII TGF-βreceptor secreted by experiment MCF-7s was confirmed by ELISA, and the concentration of soluble Fc:TβRII in culture supernatant was ranged from 20 ng/ml to 80 ng/ml in the first 3 days.Together with RT-PCR , western blotting and ELISA experiments results indicated that fusion protein Fc:TβRII was secreted expression in pIRES2-EGFP-TβRII-hIg transfected MCF-7s.3. Effcet of Fc:TβRII on the expression of CXCR4 and CCR7 in MCF-7s To examine the effect of Fc:TβRII on CXCR4 expression, MCF-7s were transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg for 48 hours,then tested1. the expression of CXCR4 in MCF-7s1)at the mRNA level:cDNA samples from MCF-7s were then analyzed for CXCR4 mRNA expression by qRT-PCR. Cells secreted Fc:TβRII showed a significantly lower level of CXCR4 expression as compared with pIRES2-EGFP transfected cells. In detail, MCF-7s transfected with pIRES2-EGFP-TβRII-hIg resulted in a -2.54±0.34-fold decrease compared with control cells.2) at the protein level:We did FCM analysis for the change of cell surface CXCR4 expression in MCF-7s transfected with pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg. These secreted Fc:TβRII cells lost expression of CXCR4 after 48 hours transfection.3) the expression of CCR7 in MCF-7s:There is no significant difference in the expression of CCR7 between the two kinds of transfected cells could be detected.4) chemotaxis assay:We also evaluate the migration responses of MCF-7s transfected either pIRES2-EGFP or pIRES2-EGFP-TβRII-hIg . The data showed that MCF-7 cells transfected with pIRES2-EGFP-TβRII-hIg decreased numbers of migratory cells in response to SDF-1αcompared with control cells.ⅢConstruction of luciferase reporter and and dual luciferase assay 1.Promotor cloning and luciferase reporter constructionCXCR4 promoter sequences were amplified by PCR from human genome extracted from lympholeukocyte of human peripheral blood. A series of CXCR4 promoter fragments with different 5'ends (nt -231, -194, -179, -168, -160, -75, -63) and a common 3'end (+65), relative to the transcription start site, respectively, was ligated into the pGL3 promoterless plasmid (pGL3-basic) and validated them by DNA sequencing.2.5' Sequences controlling human CXCR4 gene transcription.To identify regions with transcriptional activity of CXCR4, cells were transfected with pGL3-basic vector that contains the luciferase gene after the successive 5' deletions of CXCR4 promoter and the pRL-TK vector encoding Renilla luciferase as a control for transfection efficiency. After 24 hours, luciferase assays were performed using the dual luciferase assay system.The functional expression studies indicated that the region upstream of the human cxcr4 minimal promoter contains a complex array of both positive and negative transcription control elements. Studies are in progress to identify the functional elements in this region and to determine if they could control cxcr4 gene expression by TGF-β1.Conclusion.Our studies revealed that MCF-7s treated with rhTGF-β1 could up-regulate the surface expression of CXCR4, while fusion protein Fc:TβRII secreted by MCF-7s transfected pIRES2-EGFP-TβRII-hIg could reduce the expression of CXCR4.Furthermore, we constructed a series of 5' truncations of CXCR4 promoter driving the expression of a luciferase gene, then identified the regions between -231bp and transcriptional start site with transcriptional activity of CXCR4...