ABSTRACT
<p><b>OBJECTIVE</b>To study the effects and mechanism of apigenin on the expression of vascular endothelial growth factor (VEGF) in human breast cancer MDA-MB-231 cells.</p><p><b>METHODS</b>MDA-MB-231 cells were used as the study object. MTT assay was used to detect the effect of apigenin on the cell viability. ELISA was used to determine the protein level of VEGF. RT-PCR was used to detect VEGF at mRNA level. A double luciferase system was used to measure the transcription activity of VEGF. pCEP4-HIF-1alpha was transferred to explore the reversing effect of HIF-1alpha on the inhibitory effect of apigenin on the transcription activity of VEGF. Western blotting was used to detect the time-dependent and dose-dependent effect of apigenin on HIF-lalpha, p-AKT, p-ERK, and p53 expression at protein level.</p><p><b>RESULTS</b>Apigenin had no visible inhibitory effect on cell viability. Apigenin reduced the secretion of VEGF, mRNA levels of VEGF and transcription activity of VEGF. Furthermore, the inhibitory effect of apigenin on the transcription activity of VEGF could be reversed by transferring pCEP4-HIF-1alpha into the cells. Additionally, apigenin inhibited the expression of HIF-1alpha and p-AKT, induced the expression of p53, but had no effect on the expression of p-ERK1/2.</p><p><b>CONCLUSION</b>Apigenin can inhibit VEGF expression in breast cancer cells, and this effect may be achieved through decreasing the expression of HIF-1alpha.</p>
Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apigenin , Pharmacology , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt , Metabolism , RNA, Messenger , Metabolism , Transcriptional Activation , Tumor Suppressor Protein p53 , Metabolism , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
Objective:To construct NBS1 microRNA expressing eukaryotic recombinants,and identify biological activity of recombinants in Hela cell after transfection.Methods:According to sequence of NBS1mRNA,the NBS1 pre-microRNA was designed and synthesized,then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cell line.To detect integrity of inset fragment through colony PCR and sequencing analysis.The biological activity of recombinants through identify interference efficiency of NBS1 microRNA recombinants by way of Real-Time PCR and Western blot were determined.Results:Sequences of inset fragment in four microRNA expressing recombinants were correct.NBS1 mRNA and protein expression of four microRNA recombinants were decrease,which is the lowest in the NBS1mi-2 group.Conclusion:Four NBS1-targeting microRNA expressing recombinants all have biological activity in Hela cell line,and NBS1mi-2 recombinant has the most interference efficiency.The microRNA expressing plasmid which were successfully constructed and lay foundation for the studies on the tumor gene therapy of microrna targeting NBS1.
