| BRK1protein is a subunit of WAVE/SCAR complex that activates the Arp2/3complex. Arp2/3leads to nucleation and assembly of actin filaments, which mediate the formation of pseudopodia and ultimately drive tumor cell migration. In our previous study on non-small cell lung cancer (NSCLC), BRK1is more highly expressed in tumor tissues than in adjacent normal tissues, and the high level of BRK1is associated with lymph node metastasis, pathological grade and poor differentiation. So it is likely that BRK1promotes the tumor metastasis. In this study, we clarified the role of Spl in the transcriptional regulation of BRK1in cell lines A549, H1299and H520.We analyzed the sequence-84～-1nt (segment named S831) upstream of the ATG codon of BRK1gene by TFsearch software, at which the cis-acting elements might locate. We found a putative Spl-binding site at-73～64nt, and mutation of the proximal5bases led to a substantial decrease of Relative Luciferase Unit (RLU) in A549, HI299and H520. Mithramycin A, an Sp1inhibitor, could inhibited the transcriptional activity of S831, too. Knocking down of Spl could decrease the mRNA level of BRK1by at least30%, but couldn’t affect BRK1protein expression. However, overexpression of Spl had little effect on the expression of BRK1for some reason. At last, both an Electrophoretic Mobility Shift Assay (EMSA) and a Chromatin Immunoprecipitation (ChIP) assay demonstrated that Spl bound to the promoter area of BRK1gene.In summary, our data identified a functional and positive Spl regulatory element from-73to-64nt in the BRK1promoter area, which may likely explain the overexpression of BRK1gene in NSCLC. Structural maintenance of chromosome4(SMC4) is a member of chromosome ATPase superfamily, and combines with homologous protein SMC2and non-SMC regulatory subunits to form the condensin complexes. In eukaryotes, SMC4and SMC2function as the core of the condensin complexes that are essential for chromosome assembly and segregation during mitosis, previous study of gene expression profiles and immunohistochemistry on lung cancer showed a up-regulated expression of BRK1in tumor tissue than in adjacent normal tissue, which was associated with the clinical prognosis of adenocarcinoma patients. So it is indicated that the interaction between SMC4and other proteins may involved in metastasis of lung cancer. In this study, Co-immunoprecipitation (Co-IP) combined with Liquid Chromatography-Mass spectra/Mass spectra (LC-MS/MS) was used to identify the proteins interacting with SMC4in immortalized bronchial epithelial cell line (M cell) and A549.In present study, we found that SMC4had a different distribution in cytoplasm in comparison with that in nucleus. In Co-IP of SMC4, Coomassie brilliant blue stain showed4different protein bands in M cell, and2different bands in A549, compared to Normal IgG control. The analysis of these bands by LC-MS/MS identified a total of20nonredundant proteins that matched to the Mascot Database, including SMC4, SMC2, NCAPG, NCAPD2, MCM5, IQGAP1, MAP4, MAP7, OPA1, CTNND1, DDX1, DDX46, DIS3, HNRNPU, LRRFIP1, EEF2, PFKP, MTHFD and ACLY. The biological function of these proteins covered mitosis, cytoskeleton regulation, cell adhesion, RN A processing and so on. Gene expression profile showed that SMC4, NCAPD2, NCAPG and MCM5were overexpressed and IQGAP1was down-regulated in adenocarcinoma. In addition, the expression of SMC4was tightly correlated with SMC2, NCAPD2, NCAPG, respectively (R>0.65).Here, we preliminarily identified19proteins interacting with SMC4using Co-IP and LC-MS/MS, which need validation in future. |
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