ABSTRACT
OBJECTIVE@#To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC).@*METHODS@#We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice.@*RESULTS@#Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05).@*CONCLUSION@#VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.
Subject(s)
Animals , Humans , Mice , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Luciferases/genetics , Methylation , Mice, Nude , Receptors, Vasoactive Intestinal Polypeptide, Type I/metabolism , Transcription Factor AP-2/metabolismABSTRACT
Bronchial asthma is a heterogeneous disease characterized by airway inflammation and airway hyperresponsiveness.In addition to adrenergic and cholinergic innervation,bronchial asthma is also regulated by non-adrenergic non-cholinergic nervous system.As a non-adrenergic non-cholinergic nerve medium,vasoactive intestinal peptide has many biological functions,such as anti-inflammatory and anti-oxidation.It plays an important role in airway hyperresponsiveness,airway inflammation and airway remodeling.
ABSTRACT
PURPOSE: We investigated the expression of vasoactive intestinal peptide (VIP), VIP receptor 1 (VPAC1), VIP receptor 2 (VPAC2) genes in the human umbilical cord blood CD34 cells, and the ability of VIP to stimulate human primitive as well as monopotent hematopoietic progenitors. METHODS: We isolated RNA from umbilical cord blood CD34 cells, and then performed RT-PCR, and sequencing. The umbilical cord blood CD34 cells were cultured with the various concentrations of VIP for burst-forming unit of erythrocyte (BFU-E), colony-forming unit of granulocyte/monocyte (CFU-GM), colony-forming unit of graulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM), and colony-forming unit of megakaryocyte (CFU-Mk). RESULTS: The RNA coding for VPAC1 was detected in human umbilical cord blood CD34 cells. VIP significantly stimulated the growth of CFU-GEMM and CFU-Mk. CONCLUSION: The present results suggest that VIP is an important neuropeptide in the early proliferation of human primitive as well as megakaryocyte progenitors.
Subject(s)
Humans , Clinical Coding , Erythrocytes , Fetal Blood , Megakaryocyte Progenitor Cells , Megakaryocytes , Myeloid Progenitor Cells , Neuropeptides , Receptors, Vasoactive Intestinal Peptide , RNA , Stem Cells , Vasoactive Intestinal PeptideABSTRACT
PURPOSE: We analyzed the expression of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), VIP receptor 1 (VIPR1), VIP receptor 2 (VIPR 2) and PACAP receptor (PACAPR) genes in human neuroblastoma, neuroblastoma cell line, human stomach cancer, and human stomach cancer cell lines using RT-PCR and Sourthern hybridization. The results should permit identification of potential clinical applications for VIP and PACAP. METHODS: We isolated RNA from 1 neuroblastoma cell line, 8 stomach cancer cell lines, 13 neuroblastoma, and 10 stomach cancer tumor specimens. And then we performed RT-PCR, Sourthern hybridization, and sequencing. RESULTS: We detected the RNAs coding for VIP, VIPR1, VIPR2, PACAP, and PACAPR in 1, 11, 2, 12, and 13 out of 13 neuroblastoma tumor specimens, respectively. VIP and PACAPR RNA was expressed in SKNSH. VIPR1 RNA was expressed in 4 of 8 the stomach cancer cell lines and 6 of 10 stomach cancer tumor specimens. CONCLUSION: VIP/PACAP RNA and VIP/PACAP receptors RNA were expressed in SKNSH and neuroblastoma tumor specimens. VIPR1 was expressed in stomach cancer cell lines and tumor specimens. The present results suggested that VIP/PACAP analogues could be a candidate as the growth inhibitor of neuroblastoma and stomach cancer.