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Journal of Southern Medical University logoLink to Journal of Southern Medical University
. 2022 Jul 20;42(7):957–965. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2022.07.01

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

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

Shiyu NING 1, Chunmei HE 2, Zehao GUO 1, Hao ZHANG 1, Zhijing MO 1,*
PMCID: PMC9308875  PMID: 35869757

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.

Keywords: transcription factor AP-2α, hepatocellular carcinoma, methylation, tumor growth, vasoactive intestinal peptide receptor 1

原发性肝癌是全球第6大最常见的癌症和第3大癌症死亡原因，2020年约有新发病例906 000例和死亡病例830 000例[1]。在我国肝癌是第5大癌症类型，每年新发病例约41万，死亡病例约39.1万[2]。原发性肝癌中最主要的类型就是肝细胞癌（HCC），占到了总例数的75%~85%。尽管目前已经广泛研究了HCC中一些分子的变化，但其发生、发展及转移的分子机制尚未得到很好的探索，寻找与HCC发生发展相关的靶分子并研究其分子机制十分重要。

血管活性肠肽受体是一组G蛋白偶联受体，有VIPR1和VIPR2两种亚型。有研究发现VIPR1与一些肿瘤的发生、发展及预后密切相关。如VIPR1在前列腺癌组织中过表达，有可能作为诊断前列腺癌的靶标[3]；VIPR1过表达与结肠癌较差的分化有关；此外，VIPR1在肿瘤血管和巨噬细胞中的过表达也可能在侵袭性癌症的发展中发挥重要作用[4]。但也有一些研究报道了VIPR1的抗肿瘤作用。如神经母细胞瘤中VIPR1的高表达会诱导癌细胞分化并降低其致瘤性[5]；滤泡状甲状腺癌中VIPR1表达下调[6]；肺腺癌中VIPR1表达下调以及存在VIPR1基因丢失[7]；VIPR1过表达后可显著抑制人肺腺癌细胞H1299的生长，迁移和侵袭[8]；在淋巴结转移患者中VIPR1下调，与非转移性肺癌相比，转移性肺癌中的VIPR1降低[9]。

我们近期报道了VIPR1在肝细胞癌组织中显著低表达，VIPR1低表达的肝细胞癌患者总生存率降低，并且其表达下调是由VIPR1启动子甲基化介导的[10]。但VIPR1上游调控机制及其生物学功能尚不清楚，有必要作更深入的研究。DNA甲基化是一种重要的表观遗传修饰，可通过促进或抑制转录因子与DNA结合来调节基因表达[11]。而VIPR1的启动子甲基化对转录因子的结合起什么作用，目前尚无相关研究报道。在本研究中，我们检测转录因子AP-2α及甲基化修饰对VIPR1基因转录的调控作用，通过体内和体外实验检测了VIPR1的生物学功能，探索了VIPR1在HCC中的作用机制。

1. 材料和方法

1.1. 细胞培养和DNA甲基化转移酶抑制剂DAC处理

Hep3B和Huh7细胞系由本研究室保留并传代。在37 ℃，5% CO2下用含有10%胎牛血清和抗生素的DMEM培养。细胞系加入5 µmol/L DNA甲基化转移酶抑制剂DAC培养72 h，每24 h更换1次培养基。

1.2. 使用慢病毒过表达VIPR1

VIPR1过表达慢病毒（LV-VIPR1）以及阴性对照（LV-NC）慢病毒均购自GenePharma。用慢病毒感染Huh7细胞株48 h，然后用嘌呤霉素（Santa Cruz）筛选。

1.3. siRNA转染

本研究中使用的siRNA靶序列如下：

VIPR1 sense 5'-CCGAAUCCUGCUUCAGAAA TT-3'；

antisense5'-UUUCUGAAGCAGGAUUCGGTT-3'

AP-2α sense 5'-GGAGACGUAAAGCUGCCAAT T-3'；

antisense 5'-UUGGCAGCUUUACGUCUCCTT- 3'，所有siRNA均购自GenePharma。根据制造商的说明，使用siRNA-Mate（Genepharma）转染Hep3B细胞株。

1.4. 双荧光素酶报告基因启动子活性检测

使用正常人DNA作为模板扩增人VIPR1基因-433 bp到+67 bp的野生型序列，以及突变了-371 bp到-354 bp序列的VIPR1启动子突变型序列1，突变了-22 bp到-12 bp序列的VIPR1启动子突变型序列2，同时突变了上述2个序列的VIPR1启动子突变型序列1 + 2，将上述序列分别克隆到pGL3- Basic（VT1554, YouBio）中。从人DNA样品中扩增人AP-2α基因并克隆到pcDNA3.1中。转染前1 d，将Hep3B和Huh7细胞用胰酶消化铺96孔板，控制第2天细胞汇合度约为70% ~80%，使用Hieff TransTM脂质体转染试剂（上海翊圣生物科技有限公司），在转染后48 h，裂解细胞，96微孔板发光检测仪（Promega）测定荧光素酶活性，检测值比值=萤火虫荧光素酶检测值/海肾荧光素酶检测值。

1.5. 焦磷酸测序

焦磷酸测序如前所述进行[10]。测序引物：5'-AGAT AAGTGGTATTTTTTGTTT-3'，F：5'-GGATGAATTG GGGTTGTTGAAAA-3'，R：5'-ACCTTCCCAAACTC CCACCTCTATA-3'。焦磷酸测序序列为VIPR1基因- 423 bp到-388 bp区。

1.6. 染色质免疫共沉淀

使用Hep3B和Huh7细胞按照Millipore提供的方案进行染色质免疫沉淀测定。将稀释的DNA-蛋白质复合物与等量的抗AP-2α抗体（Abcam）或小鼠IgG于4 ℃温育过夜，磁珠吸附，纯化染色体DNA并通过qPCR分析。用于扩增目的区域VIPR1基因启动子区序列的PCR引物如下：

F：5'GAACCCTCGGAGACGCACAG3'，R：5'GG CGCTCCTCATGTCTACCC3'。

1.7. Western blot实验

Western blot如前所述进行[12]。抗VIPR1抗体（1∶1000），抗AP-2α抗体（1∶1000），抗β-actin抗体（1∶ 1000），均购自Abcam。

1.8. 实时荧光定量PCR（qRT-PCR）

RNA提取，cDNA合成和qRT-PCR分析如前所述进行[12]。VIPR1和GAPDH的引物序列显示如下：

VIPR1上游引物F：5'-CCTCCATTCAAGGCCG CAAT-3'，下游引物R：5'-GCTGCTCATCCAAACTCG CT-3'；GPPDH为内参，上游引物F：5'-TGCACCACCA ACTGCTTAGC-3'，下游引物R：5'-GGCATGGACTG TGGTCATGAG-3'。

1.9. 流式细胞术分析细胞周期和细胞凋亡

对于细胞周期分析，将细胞进行血清饥饿以诱导细胞周期同步。收获对数生长期的细胞，并在-20 ℃下在70%乙醇中固定1夜。第2天，洗涤细胞，在PI中孵育并通过流式细胞术分析。对于细胞凋亡分析，使用BD公司Annexin V/PI凋亡检测试剂盒进行Annexin V/PI染色，并用流式细胞仪分析。

1.10. 细胞增殖

HCC细胞的增殖能力通过Cell Counting Kit-8测试。将细胞接种于96孔板中，2500细胞/孔，分别在铺板0~5 d后吸去培养基，每孔换成150 µL含有10%的CCK8的DMEM完全培养基继续培养1 h。使用酶标仪检测吸光值A450 nm。

1.11. 体内实验

5周龄BALB/c裸鼠购自江苏集萃药康生物科技有限公司。将总共14只BALB/c裸鼠随机分为两组（LV-NC和LV-VIPR1，7只/组）。皮下接种Huh7细胞4×106/ 只。每周2次测量瘤体体积，并在35 d后注射戊巴比妥钠，该研究方案得到了桂林医学院实验动物伦理委员会的批准。

1.12. 统计学分析

使用GraphPad prism 8.0进行统计分析。数据的结果表示为均数±标准差，方差分析和t检验用于计算组间的统计学差异，P < 0.05认为差异具有统计学意义。

2. 结果

2.1. VIPR1基因启动子区结合AP-2α下调VIPR1表达

基于Cistrome Data Browser[13, 14]分析ChIP-seq公共数据，显示VIPR1基因的CpG岛区有RNA聚合酶Ⅱ（POL2）和H3K4me3富集，转录因子AP-2α也在CpG岛区富集（图 1A）。

图 1.

图 1

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转录因子AP-2α调控VIPR1表达

Transcription factor AP-2α regulates VIPR1 expression. A: VIPR1 gene CpG island enriched in RNA polymerase Ⅱ (POL2), H3K4me3 and transcription factor AP-2α. B: Design of tVIPR1 mutated promoter regions (mut1 and mut2) based on AP-2α binding site. C: Dual luciferase reporter assay revealing the influence of VIPR1 promoter mutations and AP-2α on the expression of VIPR1 in the two cell lines. *P < 0.05, ***P < 0.001.

我们成功构建了针对AP-2α两个结合位点分别突变的VIPR1启动子突变载体1、突变载体2，以及两个结合位点同时突变的VIPR1启动子突变载体1+2（图 1B），并检测了AP-2α表达对启动子活性的影响。VIPR1启动子野生型载体与AP-2α表达质粒共转染后荧光素酶活性显著降低（P < 0.001，图 1C），而启动子突变载体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）。

焦磷酸测序结果显示加入DNA甲基化转移酶抑制剂DAC处理后，Huh7细胞中7个CpG位点的甲基化均值由58%下降到13.14%，Hep3B细胞中7个CpG位点的甲基化均值由35.43%下降到10.57%（图 2A）。染色质免疫共沉淀结果显示加入DAC后，随着甲基化程度减弱，与VIPR1启动子区结合的转录因子AP-2α显著减少（P < 0.01，图 2B）。GEPIA数据库[15]分析RNA-Seq数据发现AP-2α和VIPR1的表达在肝组织样本中呈显著负相关（P < 0.001，图 2C）。敲减AP-2α后，发现VIPR1表达上升（图 2D）。

图 2.

图 2

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VIPR1基因启动子区甲基化序列结合AP-2α抑制VIPR1表达

Methylation of VIPR1 promoter promotes the binding of AP-2α and inhibits VIPR1 expression. A: Pyrosequencing for detecting the percentage change of methylation at 7 CpG sites in the promoter region of the VIPR1 gene after DAC treatment. B: ChIP analysis of the effect of DAC on AP- 2α binding to the VIPR1 promoter methylated sequence (**P < 0.01).C: Plot diagram of the correlation between AP-2α and VIPR1 RNA expression in HCC tissue samples. D: Western blotting showing the change in VIPR1 protein expression after AP-2α knockdown.

2.2. VIPR1阻滞细胞周期进程

VIPR1蛋白在Huh7细胞株中的表达要低于在Hep3B细胞株中的表达（图 3A），我们选择Huh7细胞株构建VIPR1过表达模型，Hep3B细胞株构建VIPR1敲减模型。q-PCR结果显示，在Huh7细胞中，与空白组相比LV-NC组VIPR1的mRNA表达水平差异无统计学意义，LV-VIPR1组中mRNA表达水平显著上升（P < 0.05）; 在Hep3B细胞中，与空白组相比NC组VIPR1的mRNA表达水平差异无统计学意义，siVIPR1组中mRNA表达水平显著下降（P < 0.001，图 3B），因此后续实验我们采用Huh7细胞中LV-NC组和Hep3B细胞中NC组作为对照组。Western blot实验结果显示，与LV-NC组相比，LV-VIPR1组中VIPR1表达量上升; 与NC组相比，siVIPR1组中VIPR1表达量下降（图 3C）。

图 3.

图 3

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VIPR1调控细胞周期

VIPR1 regulates cell cycle in HCC cells. A: VIPR1 protein levels in two HCC cell lines determined by Western blotting. B: qPCR confirms VIPR1 overexpression and knockdown in Huh7 and Hep3B HCC cells, respectively. C: Western blotting confirms VIPR1 overexpression and knockdown in Huh7 and Hep3B HCC cells, respectively. D: Cell cycle analysis in Huh7 cells overexpressing VIPR1 (**P < 0.01). E: Cell cycle analysis in Hep3B cells with VIPR1 knockdown (*P < 0.05, ***P < 0.001).

细胞周期实验结果显示，VIPR1过表达增加了G2/M期的细胞数量，P < 0.01，同时减少了S期的细胞数量，P < 0.01;而在VIPR1敲减的Hep3B细胞中则相反，S期的细胞数量增加，P < 0.001，G2/M期的细胞数量减少（P < 0.05，图 3D~E）。

2.3. VIPR1促进细胞凋亡抑制细胞增殖

细胞凋亡实验结果显示，与对照组相比，LV-VIPR1组的凋亡细胞显著增加（P < 0.001）。siVIPR1组的凋亡细胞数显著降低（P < 0.05，图 4A、B）。

图 4.

图 4

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VIPR1调节细胞凋亡和细胞增殖

VIPR1 regulates apoptosis and cell proliferation. A: Apoptosis in Huh7 cells overexpressing VIPR1 (***P < 0.001).B: Apoptosis in Hep3B cells with VIPR1 knockdown (*P < 0.05).C: CCK8 detection of cell proliferation after VIPR1 overexpression or knockdown in Huh7 and Hep3B cells.

CCK8实验结果显示，与对照组相比，LV-VIPR1组的细胞增殖受到抑制，而siVIPR1组的细胞增殖显著（图 4C）。

2.4. VIPR1在体内抑制肿瘤生长

裸鼠移植瘤实验结果显示，与LV-NC组相比，LV-VIPR1组肿瘤的体积明显减小（P < 0.001），质量显著降低（P < 0.05，图 5A、B）。

图 5.

图 5

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VIPR1在体内抑制肿瘤的生长

VIPR1 inhibits tumor growth in nude mice. A: Gross observation of the tumors in LV- VIPR1 and LV-NC groups. B: Tumor volume and weight in LV-VIPR1 and LV-NC groups (*P < 0.05, ***P < 0.001). C: A possible mechanism by which AP-2α inhibits VIPR1 expression and thereby inhibits tumor growth.

3. 讨论

RNA聚合酶Ⅱ（POL2）和H3K4me3通常在强启动子处高度富集[16-18]，我们通过Cistrome Data Browser分析发现VIPR1基因的CpG岛区有POL2和H3K4me3富集，提示该CpG岛区为VIPR1基因的启动子。同时分析发现转录因子AP-2α也在此CpG岛区富集，表明转录因子AP-2α可能参与VIPR1基因转录，并且与启动子的甲基化相关。之前的研究发现转录因子AP-2α既有转录激活作用，也有转录抑制作用。已有研究表明AP-2α可转录激活HER2、ESR1和CDKN1A[19]，还可转录抑制FGFR1[20]和SAA1[21]。而转录因子AP-2α对VIPR1基因的转录起哪种作用尚未见报道。在本研究中我们通过双荧光素酶实验，发现转染AP-2α可明显抑制VIPR1启动子野生型组荧光素酶活性，对于VIPR1启动子突变型1+2组，则荧光素酶活性明显恢复，提示VIPR1启动子突变型序列1和序列2可影响AP-2α结合，并且AP-2α结合后抑制其转录活性。

我们前期的研究发现VIPR1基因的启动子存在DNA甲基化修饰[10]，结合本研究，我们推测转录因子AP-2α与VIPR1启动子的结合可能与甲基化有关。通常认为当启动子甲基化后会阻碍转录因子结合，导致转录抑制，一些转录因子可能在其结合位点促进DNA去甲基化[22]；而有些研究认为DNA甲基化也可以为转录因子创造一个新的结合位点，一些转录因子可以结合甲基化的DNA进而调控基因表达[23, 24]。我们首先用DNA甲基化转移酶抑制剂处理肝癌细胞系，再通过焦磷酸测序检测甲基化水平，染色质免疫共沉淀实验检测AP-2α与VIPR1启动子序列的结合情况。结果发现位于AP-2α结合位点附近的甲基化水平降低后，与启动子区结合的转录因子AP-2α显著减少。这表明除了VIPR1启动子上的AP-2α结合位点（VIPR1启动子突变型序列1和序列2）影响其结合外，结合位点附近的甲基化修饰也能影响转录因子AP-2α的结合。一项在痛风中的研究发现，NRBP1基因启动子的低甲基化抑制AP-2α结合，上调NRBP1表达，更加验证了本实验的结论[25]。而在AP-2α与VIPR1启动子序列的结合过程中，结合位点和甲基化修饰两者之间谁起主导作用，还需后续实验进一步研究。我们通过GEPIA分析发现AP-2α和VIPR1的表达呈显著负相关，敲减AP-2α后，VIPR1表达上升。以上研究表明VIPR1基因启动子区序列的甲基化促进了转录因子AP-2α结合，并且抑制了VIPR1表达。

细胞周期调控是一个复杂而精确的生理过程，细胞周期的异常会导致细胞分裂、增殖、分化等一系列过程紊乱，还会影响免疫反应与自噬，其失控在肿瘤发病中处于极其重要的中心环节[26, 27]。我们的细胞周期实验结果显示VIPR1过表达增加了G2/M期的细胞数量，同时减少了S期的细胞数量，VIPR1敲减的结果则相反。G2/M检查点可阻止带有DNA损伤的细胞进入有丝分裂期，在维持染色体完整性方面发挥着重要作用。研究报道，芹菜素通过G2/M期细胞周期阻滞抑制肾细胞癌细胞的增殖[28]。抑制17β-HSD7可阻滞G2/M周期并阻止卵巢癌细胞增殖[29]。这些结果提示VIPR1过表达可能阻滞了G2/M期，抑制HCC细胞的增殖。细胞周期阻滞后往往会诱发细胞凋亡，如18β-甘草次酸通过诱导细胞凋亡和G2/M细胞周期阻滞，抑制肺癌细胞迁移[30]。氯丙嗪通过诱导G2/M细胞周期停滞和细胞凋亡抑制结直肠癌[31]。我们在本研究中同样发现VIPR1过表达后凋亡细胞显著增加，VIPR1敲减的结果则相反。CCK8实验显示与对照相比，LV-VIPR1组的细胞增殖受到抑制，而siVIPR1组的细胞增殖显著。此外，我们还进一步通过体内实验验证了VIPR1过表达使肿瘤体积减小，且质量减轻。这些数据表明VIPR1抑制肿瘤生长的作用，可能是通过阻滞细胞周期进程和促进细胞凋亡来实现的。

综上所述，本研究发现VIPR1基因启动子区的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（图 5C）。本研究揭示了VIPR1在肝细胞癌中低表达的分子机制及其生物学功能，该发现可为肝细胞癌治疗提供新策略。

Biography

宁诗雨，硕士，E-mail: 93119008@qq.com

Funding Statement

国家自然科学基金地区科学基金（32060159）；广西自然科学基金（2019JJA140462）

Supported by National Natural Science Foundation of China (32060159)

Contributor Information

宁 诗雨 (Shiyu NING), Email: 93119008@qq.com.

莫 之婧 (Zhijing MO), Email: mozhijing@glmc.edu.cn.

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