南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (7): 957-965.doi: 10.12122/j.issn.1673-4254.2022.07.01

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VIPR1启动子甲基化促进转录因子AP-2α下调VIPR1的表达并促进肝细胞癌的生长

宁诗雨，何春梅，郭泽皓，张 豪，莫之婧   

  1. 桂林医学院智能医学与生物技术学院，基础医学院，广西 桂林 541199
  • 出版日期:2022-07-20 发布日期:2022-07-15

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro

NING Shiyu, HE Chunmei, GUO Zehao, ZHANG Hao, MO Zhijing   

  1. School of Intelligent Medicine and Biotechnology, Faculty of Basic Medical Sciences, Guilin Medical University, Guilin 541199, China
  • Online:2022-07-20 Published:2022-07-15

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摘要： 目的 探讨血管活性肠肽受体1（VIPR1）在肝细胞癌（HCC）组织中低表达的转录调控机制和生物学功能。方法 肝癌细胞系Hep3B和Huh7常规培养，实验分为VIPR1启动子野生型组、启动子突变载体1组、启动子突变载体2组和启动子突变载体1+2组。双荧光素酶报告基因实验检测AP-2α表达对VIPR1启动子活性的影响；DNA甲基化转移酶抑制剂（DAC）处理肝癌细胞，焦磷酸测序法检测VIPR1启动子甲基化水平的变化；染色质免疫共沉淀检测AP-2α与VIPR1启动子的结合能力；Western blot检测AP-2α的敲减作用以及VIPR1的表达；Western blot 检测两种细胞株中VIPR1的差异表达；qPCR和Western blot检测VIPR1过表达和敲减效果；流式细胞术检测细胞周期和细胞凋亡；CCK8实验检测细胞增殖。建立裸鼠移植瘤模型，检测LV-NC组和LV-VIPR1组中肿瘤的体积和质量。结果 与VIPR1启动子野生型组相比，启动子突变载体1+2与AP-2α表达质粒共转染后，荧光素酶活性明显恢复（P<0.05）；DAC处理后，VIPR1启动子甲基化水平降低，并且随着甲基化程度减弱，与VIPR1启动子结合的AP-2α显著减少（P<0.01）；敲减AP-2α后，VIPR1表达上升；Huh7细胞株中VIPR1表达低于Hep3B细胞株，在两种细胞株中成功构建了VIPR1过表达和敲减模型，VIPR1过表达增加了G2/M期的细胞数量（P<0.01），凋亡细胞显著增加（P<0.001），细胞增殖受到抑制（P<0.001），而VIPR1敲低则相反。体内实验表明VIPR1过表达组的肿瘤体积减小（P<0.001），肿瘤量降低（P<0.05）。结论 HCC中VIPR1的启动子甲基化促进了转录因子AP-2α的结合，并且抑制了VIPR1表达，而VIPR1过表达可使细胞周期阻滞在G2/M期，促进细胞凋亡，抑制细胞增殖和肿瘤生长。

关键词: 转录因子AP-2α；肝细胞癌；甲基化；肿瘤生长；血管活性肠肽受体1

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.

Key words: transcription factor AP-2α; hepatocellular carcinoma; methylation; tumor growth; vasoactive intestinal peptide receptor 1