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

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

1.
广西医科大学公共卫生学院，南宁 530021
2.
广西医科大学公共卫生学院儿少卫生与妇幼保健学教研室，南宁 530021
3.
广西医科大学公共卫生学院流行病学与卫生统计学教研室，南宁 530021

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
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- 被引次数: 0
出版历程
- 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

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

1.
School of Public Health, Guangxi Medical University, Nanning 530021, China
2.
Teaching and Research Section of Pediatric and Maternal and Child Health, School of Public Health, Guangxi Medical University, Nanning 530021, China
3.
Teaching and Research Section of Epidemiology and Health Statistics, School of Public Health, Guangxi Medical University, Nanning 530021, China

Funds:

Guangxi Natural Science Foundation Key Program 2020GXNSFDA238002

More Information

Corresponding author: ZENG Xiaoyun, E-mail: zengxiaoyun@gxmu.edu.cn

摘要

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。
- TRPV1 /
- 肝癌 /
- 增殖 /
- 迁移 /
- 细胞周期
Abstract: Objective To clarify the expression of transient receptor potential vanilloid subfamily 1 (TRPV1) in hepatocellular carcinoma (HCC) and its relationship with prognosis, and to explore its effect on the biological behavior of HCC cells. Methods The cancer genome atlas (TCGA), genotype-tissue expression (GTEx) and gene expression ominbus (GEO) databases were utilized to verify the expression of TRPV1 in HCC and its relationship with prognosis. The effects of TRPV1 on the proliferation, migration, invasion, and cell cycle of HCC cells were detected by CCK-8 assay, scratch assay, Transwell assay, and flow cytometry. The expression of cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) was detected by Western blot, and the tumor proliferation ability was detected by subcutaneous tumor formation assay in nude mice. Finally, the TRPV1 co-expression network was constructed and analyzed for functional enrichment. Results The expression level of TRPV1 in HCC tissues was down-regulated in TCGA, GTEx, and GEO databases (P < 0.001) and was associated with a shorter overall survival time of patients (P=0.032), with an area under the ROC curve of 0.91. Overexpression of TRPV1 suppressed the proliferation, migration, and invasion ability of HCC cells, blocked cell cycle progression, and CDK4 and CDK6 protein expression levels decreased (both P < 0.05), and the opposite was true for knockdown of TRPV1 (both P < 0.05). After stable overexpression of TRPV1, the subcutaneous tumor volume of nude mice was reduced (P=0.015), the mass of nude mice was reduced (P=0.033), and the positive expression level of Ki-67 protein was down-regulated (P=0.010). TRPV1 co-expressed genes were mainly involved in the processes of RNA splicing, cell cycle protein binding, and cell proliferation. Conclusions TRPV1 inhibited the proliferation, migration, and invasion of HCC cells and blocked cell cycle progression, which exerted an oncogenic role in HCC and provided a new direction for the prognostic analysis and treatment of HCC.
- Transient receptor potential vanilloid 1 /
- Hepatocellular carcinoma /
- Proliferation /
- Migration /
- Cell cycle

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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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