VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro / 南方医科大学学报

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.

Porcine transcription factor AP-2δ promotes porcine circovirus type 2 replication through enhancing the activity of the rep gene promoter / 生物工程学报

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.

Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro / 肿瘤研究与临床

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.

Expressions of transcription factor activator protein-2α, estrogen receptor-β and matrixmetalloproteinase-9 in lichen planus lesions / 中华皮肤科杂志

Objective To determine the expressions of transcription factor activator protein-2α (AP-2α), estrogen receptor-β (ER-β) and matrixmetalloproteinase-9 (MMP-9) in lichen planus (LP) lesions and their significance. Methods Tissue samples were resected from the lesions of 30 patients with LP and normal skin of 30 human controls. An immunohistochemical method using streptavidin-peroxidase (SP) was performed to detect the expression of ER-β and MMP-9, reverse transcription-PCR and Western blot to measure the mRNA and protein expressions of AP-2α, ER-β and MMP-9 in these specimens. Results There was a significant decrease in the mRNA expressions of AP-2α (0.488 ± 0.039 vs. 1.008 ± 0.023, P < 0.01 ) and ER-β (0.365 ± 0.032 vs. 0.998 ± 0.036, P < 0.01), together with an increase in the expression of MMP-9 (1.237 ± 0.027 vs. 0.567 ± 0.015, P< 0.01) in the LP lesions compared with the control specimens; similar results were observed for the protein expressions of AP-2α, ER-β and MMP-9. In LP lesions, the expression of AP-2α was positively correlated with that of ER-β (R = 0.89, P < 0.01), but negatively correlated with that of MMP-9 (r = -0.91, P < 0.01). Conclusions The down-regulation of ER-β expression and up-regulation of MMP-9 expression may be ascribed to the abnormal regulation of upstream target genes mediated by the decreased expression of AP-2α, which may be involved in the pathogenesis of LP.

The expression of Sp1 and AP-2α and the correlation between the two transcription factors in colon cancer / 肿瘤研究与临床

Objective To study the expression of transcription factor specificity protein1 and activator protein-2α and the correlation between the two transcription factors in the process of occurrence and development of colorectal cancer. Methods To detect expression of Sp1 and AP-2α mRNA by Real-Time PCR in 60 colon cancer tissues and corresponding normal tissues and the results were compared with the clinical features and pathological characters. The relationship between the expression of Sp1 mRNA and AP-2α mRNA in 60 colon cancer tissues was determined. Results The expression rates of Sp1 mRNA was detectable to highly expressed rates in colon cancer tissues than the matched normal tissues (P ＜0.01),whereas AP-2α mRNA in the colon cancer tissues was significantly lower than that in the matched normal tissues (P ＜0.01). Sp1 mRNA and AP-2α mRNA expression rates had no significant difference between the clinical features (sex, age and tumor areas) respectively. Loss expression or down regulation expression of AP-2α mRNA was detected, whereas Sp1 mRNA was detectable to highly expressed in the different histological grade and Dukes stages. The expression of Spl mRNA and AP-2α mRNA were positively correlated with the histological grade in colon cancer. A significant correlation was found between the expression of Sp1 mRNA and AP-2α mRNA (r =-0.849, P ＜0.001). Conclusion Loss or down regulation expression of AP-2α mRNA,whereas Sp1 was detectable to highly expressed in colon cancer. Negative correlation occurred in Sp1 mRNA and AP-2α mRNA indicated that AP-2α and Sp1 provide the new clues of genetic diagnosis and treatment.

Inhibitory effects of AP-2α on proliferation of colon cancer cell of SW620 and its mechanism / 肿瘤研究与临床

Objective To study inhibitory effects of transcription factor activator protein-2α(AP-2α)on proliferation of colon cancer cells in vitro and its mechanism. Methods The peDNA3.1 (+)-AP-2α recombinant plasmid was constructed. Plasmid pcDNA3.1(+)- AP-2α and pcDNA3.1(+)was transfected into SW620 cell by liposome mediation for transient expression, and proliferative activities of SW620 cell were evaluated by MTT assay. The change in the mRNA and protein expression level of ER-β before and after transfection was detected using the methods of Real-Time PCR and Western blotting respectively. Results The mRNA and protein expressions of AP-2α could be enhanced by transfecting of AP-2α gene in SW620 cell. MTT assay indicated: the proliferation velocity of SW620 cell for transfection of the pcDNA3.1(+)-AP-2α plasmid was apparently inhibited. The expression of ER-β in SW620 cell increased significantly after AP-2α gene transfection. Compared with control group, the difference was significant (P＜0.05). Conclusion Overexpression of AP-2α inhibits the proliferation of SW620 cell in vitro, which is probably related with activation of ER-β.

Effect of AP-2α gene transfection on proliferation and invasion potential of human colon cancer SW480 cells in vitro / 肿瘤研究与临床

Objective To evaluate the proliferation and invasion potential of human colon cancer SW480 cells transfected by AP-2α gene in vitro. Methods pcDNA3.1 (+)-AP-2α was created by cloning AP-2α eDNA into the EcoRI site of poDNA3.1 (+). LipofectamineTM 2000 Reagent was used to mediate the transfection of pcDNA3.1(+)-AP-2α and pcDNA3.1 (+), and normal SW480 celia were cultured as a negative control. The mRNA and protein level of AP-2α in the cells of each group were detected 48 h after being transfected with plasmids above by RT-PCR and Western blotting analysis. The cell proliferation and invasion potential were examined by colony formation assay and Transwell invasion assay respectively. Results Lack of AP-2α protein expression in SW480 cells was verified by western blot analysis. After being transfected with AP-2α gene, the mRNA amount and protein expression increased dramatically, while the colony formation efficiency decreased(P <0.05), the cell proliferation in soft agar was inhibited, and the ability of its invasion dropped off(P <0.05) in vitro. Conclusion AP-2α gene suppresses the proliferation and invasion potential of human colon cancer SW480 cells in vitro.