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.

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.

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.