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TRPV1对肝细胞性肝癌生物学行为的影响及可能的机制预测

韦丽铃 刘美良 李德远 杨钰 肖苏洋 黎婵华 吴思黔 宋安华 曾小云

韦丽铃, 刘美良, 李德远, 杨钰, 肖苏洋, 黎婵华, 吴思黔, 宋安华, 曾小云. TRPV1对肝细胞性肝癌生物学行为的影响及可能的机制预测[J]. 中华疾病控制杂志, 2024, 28(6): 691-701. doi: 10.16462/j.cnki.zhjbkz.2024.06.012
引用本文: 韦丽铃, 刘美良, 李德远, 杨钰, 肖苏洋, 黎婵华, 吴思黔, 宋安华, 曾小云. TRPV1对肝细胞性肝癌生物学行为的影响及可能的机制预测[J]. 中华疾病控制杂志, 2024, 28(6): 691-701. doi: 10.16462/j.cnki.zhjbkz.2024.06.012
WEI Liling, LIU Meiliang, LI Deyuan, YANG Yu, XIAO Suyang, LI Chanhua, WU Siqian, SONG Anhua, ZENG Xiaoyun. Effects of TRPV1 on biological behavior of hepatocellular carcinoma and prediction of possible mechanisms[J]. CHINESE JOURNAL OF DISEASE CONTROL & PREVENTION, 2024, 28(6): 691-701. doi: 10.16462/j.cnki.zhjbkz.2024.06.012
Citation: WEI Liling, LIU Meiliang, LI Deyuan, YANG Yu, XIAO Suyang, LI Chanhua, WU Siqian, SONG Anhua, ZENG Xiaoyun. Effects of TRPV1 on biological behavior of hepatocellular carcinoma and prediction of possible mechanisms[J]. CHINESE JOURNAL OF DISEASE CONTROL & PREVENTION, 2024, 28(6): 691-701. doi: 10.16462/j.cnki.zhjbkz.2024.06.012

TRPV1对肝细胞性肝癌生物学行为的影响及可能的机制预测

doi: 10.16462/j.cnki.zhjbkz.2024.06.012
基金项目: 

广西自然科学基金重点项目 2020GXNSFDA238002

详细信息
    通讯作者:

    曾小云,E-mail:zengxiaoyun@gxmu.edu.cn

  • 中图分类号: R735.7

Effects of TRPV1 on biological behavior of hepatocellular carcinoma and prediction of possible mechanisms

Funds: 

Guangxi Natural Science Foundation Key Program 2020GXNSFDA238002

More Information
  • 摘要:   目的  明确瞬时受体电位香草醛亚家族1(transient receptor potential vanilloid 1, TRPV1)在肝细胞性肝癌(hepatocellular carcinoma, HCC)中的表达及与预后的关系,探讨其对HCC细胞生物学行为的影响。  方法  利用癌症基因组图谱(the cancer genome atlas, TCGA)、基因型组织表达(genotype-tissue expression, GTEx)及基因表达综合数据库(gene expression omnibus, GEO)验证TRPV1在HCC中的表达及与预后的关系。通过CCK-8实验、划痕实验、Transwell实验、流式细胞术检测TRPV1对HCC细胞增殖、迁移、侵袭和细胞周期的影响,蛋白质印迹法(western blot, WB)检测细胞周期调节蛋白激酶4(cyclin-dependent kinase 4, CDK4)、细胞周期素依赖激酶6(cyclin-dependent kinase 6, CDK6)的表达,裸鼠皮下成瘤实验检测肿瘤增殖能力。最后,构建TRPV1共表达网络并进行富集分析。  结果  TCGA、GTEx和GEO数据库中,TRPV1在HCC组织中表达水平均下调(均P<0.001),且与患者总生存时间更短有关(P=0.032),受试者工作特征(receiver operating characteristic, ROC)曲线下面积为0.91。过表达TRPV1可抑制HCC细胞增殖、迁移和侵袭能力,阻滞细胞周期进展,CDK4和CDK6蛋白表达水平下降(均P < 0.05),敲低TRPV1则相反(均P < 0.05)。稳定过表达TRPV1后裸鼠皮下瘤体体积减小(P=0.015),质量减轻(P=0.033),Ki-67蛋白阳性表达水平下调(P=0.010)。TRPV1共表达基因主要参与核糖核酸(ribonucleic acid, RNA)剪接、细胞周期蛋白结合、细胞增殖等过程。  结论  TRPV1抑制了HCC细胞的增殖、迁移和侵袭能力,并阻滞细胞周期的进程,在HCC中发挥抑癌作用,为HCC预后分析、治疗提供了新方向。
  • 图  1  TCGA、GTEx、GEO数据库中TRPV1在HCC中的表达与预后情况

    TCGA: 癌症基因组图谱; GTEx,基因型组织表达; GEO: 基因表达综合数据库; TRPV1: 瞬时受体电位香草醛亚家族1; AUC: 曲线下面积; A: TCGA、GTEx数据库中TRPV1表达情况; B: GEO数据库中TRPV1表达情况; C: Kaplan-Meier生存曲线; D: 受试者工作特征曲线; a: P<0.001。

    Figure  1.  Expression and prognosis of TRPV1 in HCC in TCGA, GTEx, and GEO databases

    TCGA: the cancer genome atlas; GTEx: genotype-tissue expression; GEO: gene expression omnibus; TRPV1: transient receptor potential vanilloid 1; AUC: area under curve; A: TRPV1 expression in TCGA and GTEx databases; B: TRPV1 expression in GEO database; C: Kaplan-Meier survival curve; D: receiver operating characteristic curve; a: P < 0.001.

    图  2  瞬时转染效率验证及TRPV1对增殖能力的影响

    DNA:脱氧核糖核酸;TRPV1:瞬时受体电位香草醛亚家族1;A:TRPV1相对表达水平;B:敲低TRPV1效率验证;C:过表达TRPV1效率验证;D:敲低TRPV1促进Hep3B细胞的增殖能力;E:过表达TRPV1抑制HepG2细胞的增殖能力;a:P<0.001。

    Figure  2.  Validation of transient transfection efficiency and effect of TRPV1 on proliferative capacity

    DNA: deoxyribonucleic acid; TRPV1: transient receptor potential vanilloid 1; A: relative exppression level of TRPV1; B: validation of knockdown TRPV1 efficiency; C: validation of the efficiency of overexpression of TRPV1; D: knockdown of TRPV1 promotes proliferative capacity of Hep3B cells; E: overexpression of TRPV1 inhibited the proliferative capacity of HepG2 cells; a: P < 0.001.

    图  3  细胞划痕实验结果

    DNA:脱氧核糖核酸;TRPV1:瞬时受体电位香草醛亚家族1;A:细胞划痕结果图;B:敲低TRPV1促进Hep3B细胞的迁移能力;C:过表达TRPV1抑制HepG2细胞的迁移能力;a:P<0.001;b:P<0.05。

    Figure  3.  Results of cell scratching experiments

    DNA: deoxyribonucleic acid; TRPV1: transient receptor potential vanilloid 1; A : plot of cell scratching results; B: knockdown of TRPV1 promotes migratory capacity of Hep3B cells; C: overexpression of TRPV1 inhibited the migration ability of HepG2 cells; a: P < 0.001; b: P < 0.05.

    图  4  Transwell迁移和侵袭实验结果

    DNA:脱氧核糖核酸;TRPV1:瞬时受体电位香草醛亚家族1;A:Hep3B细胞Transwell迁移实验结果;B:敲低TRPV1促进Hep3B细胞的迁移能力;C:HepG2细胞Transwell迁移实验结果;D:过表达TRPV1抑制HepG2细胞的迁移能力;E:Hep3B细胞Transwell侵袭实验结果;F:敲低TRPV1促进Hep3B细胞的侵袭能力;G:HepG2细胞Transwell侵袭实验结果;H:过表达TRPV1抑制HepG2细胞的侵袭能力;a:P<0.05;b:P<0.01;c:P<0.001。

    Figure  4.  Transwell migration and invasion assay results

    DNA: deoxyribonucleic acid; TRPV1: transient receptor potential vanilloid 1; A: results of Transwell migration assay of Hep3B cells; B: knockdown of TRPV1 promotes migratory capacity of Hep3B cells; C: results of Transwell migration assay of HepG2 cells; D: overexpression of TRPV1 inhibits the migration ability of HepG2 cells; E: results of Transwell invasion assay of Hep3B cells; F: knockdown of TRPV1 promotes the invasive ability of Hep3B cells; G: results of Transwell invasion assay of HepG2 cells; H: overexpression of TRPV1 inhibited the invasive ability of HepG2 cells; a: P < 0.05; b: P < 0.01; c: P < 0.001.

    图  5  流式细胞术检测TRPV1对细胞周期的影响及相关蛋白表达

    TRPV1: 瞬时受体电位香草醛亚家族1; DNA: 脱氧核糖核酸; CDK4: 细胞周期调节蛋白激酶4; CDK6: 细胞周期素依赖激酶6; A~B: 敲低TRPV1加速Hep3B细胞周期进展; C~D: 过表达TRPV1阻滞HepG2细胞周期进展; E~F: 敲低TRPV1后CDK4和CDK6蛋白表达水平; G~H: 过表达TRPV1后CDK4和CDK6蛋白表达水平; a: P<0.01; b: P<0.001; c: P<0.05。

    Figure  5.  Effect of TRPV1 on cell cycle and related protein expression detected by flow cytometry

    TRPV1: transient receptor potential vanilloid 1; DNA: deoxyribonucleic acid; CDK4: cyclin-dependent kinase 4; CDK6: cyclin-dependent kinase 6; A-B: knockdown of TRPV1 accelerates Hep3B cell cycle progression; C-D: overexpression of TRPV1 blocks HepG2 cell cycle progression; E-F: CDK4 and CDK6 protein expression levels after knockdown of TRPV1; G-H: CDK4 and CDK6 protein expression levels after overexpression of TRPV1; a: P < 0.01; b: P < 0.001; c: P < 0.05.

    图  6  稳定过表达后HepG2细胞中TRPV1的mRNA表达水平

    TRPV1: 瞬时受体电位香草醛亚家族1; mRNA: 信使核糖核酸; a: P<0.001。

    Figure  6.  The mRNA expression levels of TRPV1 in HepG2 cells after stable overexpression

    TRPV1: transient receptor potential vanilloid 1; mRNA: messenger RNA; a: P < 0.001.

    图  7  TRPV1影响HepG2细胞在裸鼠体内的增殖

    TRPV1: 瞬时受体电位香草醛亚家族1; A: 裸鼠皮下肿瘤形成情况; B: 瘤体图; C: 瘤体体积变化曲线; D: 瘤体质量; E: 肿瘤组织Ki-67蛋白IHC染色,放大200倍; F: Ki-67蛋白阳性细胞百分比; a: P<0.05。

    Figure  7.  TRPV1 affects HepG2 cell proliferation in nude mice

    TRPV1: transient receptor potential vanilloid 1; A: subcutaneous tumour formation in nude mice; B: tumour map; C: tumour volume change curve; D: tumour mass; E: IHC staining of tumour tissues for Ki-67 protein, magnified 200x; F: percentage of Ki-67 protein positive cells; a: P < 0.05.

    图  8  TRPV1共表达网络的构建

    TRPV1: 瞬时受体电位香草醛亚家族1; GS: 基因显著性; MM: 模块身份; A:软阈值图;B:基因模块间相关性热图;C:基因模块与临床诊断相关性热图;D:MEturquoise模块基因中GS与MM之间相关性散点图;E:TCGA数据库中差异表达基因;F:MEturquoise模块基因和差异表达基因的韦恩图;G:GO分析。

    Figure  8.  Construction of TRPV1 co-expression network

    TRPV1: transient receptor potential vanilloid 1; GS: gene significance; MM: module membership; A: soft threshold plot; B: heatmap of correlation between gene modules; C: heatmap of correlation between gene modules and clinical diagnosis; D: scatterplot of correlation between GS and MM in the MEturquoise module genes; E: differently expressed genes in the TCGA database; F: Venn diagram of MEturquoise module genes and differentially expressed genes; G: gene ontology analysis.

    表  1  研究选择纳入的基因表达数据集详细信息

    Table  1.   Detailed information on the gene expression datasets selected for inclusion in the study

    数据库 Database 样本类型 Sample type 正常/癌旁肝组织例数 Number of normal/paracancerous liver tissues 肝癌组织例数 Number of liver cancer tissues 来源 Source
    TCGA+GTEx 肝脏组织 Liver tissue 112 369 https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project; https://www.cancer.gov/ccg/research/genome-sequencing/tcga
    GEO(GSE25097) 肝脏组织 Liver tissue 243 268 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25097
    注:TCGA,癌症基因组图谱;GTEx,基因型组织表达;GEO,基因表达综合数据库。
    Note:TCGA,the cancer genome atlas;GTEx,genotype-tissue expression;GEO,gene expression omnibus.
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  • 收稿日期:  2024-02-26
  • 修回日期:  2024-05-17
  • 网络出版日期:  2024-07-13
  • 刊出日期:  2024-06-10

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