ABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of long non-coding RNA growth arrest-specific transcript 5 (lncRNA-GAS5) in breast cancer progression and epithelial-mesenchymal transition (EMT) of the cancer cells.</p><p><b>METHODS</b>Real-time quantitative PCR (qRT-PCR) was used to detect the expression of lncRNA-GAS5 in 37 pairs of breast cancer and adjacent non-tumor tissues and in parental MCF-7 cells and paclitaxel-resistant MCF-7 (MCF-7/PR) cells, and the correlation of lncRNA-GAS5 expression with the clinical stage and lymph node metastasis of breast cancer was investigated. The expressions of the genes related with cell cycle and EMT at both the mRNA and protein levels were detected using qRT-PCR, Western blotting and immunohistochemistry. The changes in the biological behaviors and morphology of breast cancer cells with either lncRNA-GAS5 knockdown or overexpression were observed. Nude mouse models were established bearing breast cancer xenografts derived from MCF-7/PR cells or MCF-7/PR cells over-expressing lncRNA-GAS5, and the inhibitory effect of paclitaxel on tumor growth was evaluated.</p><p><b>RESULTS</b>The transcriptional levels of lncRNA-GAS5 were significantly lower in breast cancer tissues than in the adjacent non-tumor tissues (P<0.05), and decreased lncRNA-GAS5 expression was significantly correlated with TNM stage and lymph node metastasis of breast cancer (P<0.05). lncRNA-GAS5 expression was also significantly lowered in paclitaxel-resistant breast cancer cells and showed a positive correlation with P21 expression and a negative correlation with CDK6. MCF-7 cells during EMT presented with a lowered expression of lncRNA-GAS5, whereas lncRNA-GAS5 over-expression strongly suppressed MCF-7/PR cell migration and invasion, and increased the susceptibility of the cells to paclitaxel. In the tumor-bearing nude mouse models, lncRNA-GAS5 overexpression in the tumor cells obviously enhanced the inhibitory effect of paclitaxel on tumor growth and lung metastasis by reversing the EMT marker proteins.</p><p><b>CONCLUSION</b>A decreased expression of lncRNA-GAS5 promotes lung metastasis of breast cancer by inducing EMT, suggesting the potential of lncRNA-GAS5 as a therapeutic target in breast cancer.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells.</p><p><b>METHODS</b>DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay.</p><p><b>RESULTS</b>The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.</p>
Subject(s)
Animals , Female , Humans , COS Cells , Chlorocebus aethiops , Chemokines , Genetics , Cloning, Molecular , Genetic Vectors , Plasmids , Genetics , Receptors, CXCR4 , Genetics , Transfection , Tumor Cells, CulturedABSTRACT
Objective To clarify the combined ability of viral macrophage inflammatory protein (vMIP) to receptors through comparison research of vMIP binding to chemokine receptors of peripheral blood macrophages(PBMCs). Methods Combined ability of vMIP to receptors was measured with radioligand assay and identified with saturation, kinetic and specificity analyses. Results Kd is 11.1 nmol/L to human receptors of PBMC and 14.3 nmol/L to monkey receptors. The IC50 is 3.4 nmol/L to CCR5 and 4.5 nmol/L to CXCR4. Conclusion vMIP has high affinity with receptors and high specificity to CCR5 and CXCR4.This may be of great significance in the prevention and treatment of inflammation and HIV infection.