黏附性G蛋白偶联受体F1在胰腺导管腺癌中的表达及其促进癌症进展的机制研究

doi:10.3969/j.issn.1674-8115.2024.01.003

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (1): 23-34.doi: 10.3969/j.issn.1674-8115.2024.01.003

黏附性G蛋白偶联受体F1在胰腺导管腺癌中的表达及其促进癌症进展的机制研究

陈溯源(), 木司塔巴·木台力甫(), 李冬雪(), 张志刚()

上海交通大学医学院附属仁济医院上海市肿瘤研究所，肿瘤系统医学全国重点实验室，上海 200240

收稿日期:2023-05-31 接受日期:2023-09-18 出版日期:2024-01-28 发布日期:2024-02-28
通讯作者: 李冬雪,张志刚 E-mail:sychen96@sjtu.edu.cn;18139363645@sjtu.edu.cn;dxli@shsci.org;zzhang@shsci.org
作者简介:陈溯源（1996—），男，硕士生，电子信箱：sychen96@sjtu.edu.cn
木司塔巴·木台力甫（1992—），男，维吾尔族，硕士生，电子信箱：18139363645@sjtu.edu.cn第一联系人：陈溯源、木司塔巴·木台力甫为共同第一作者。
基金资助:
国家自然科学基金(82203228);上海市科学技术委员会科技创新行动计划(22YF1445600);上海交通大学医工交叉研究基金(YG2021ZD08);上海交通大学医学院“双百人”项目(20181708)

Expression of adhesion G protein-coupled receptor F1 in pancreatic ductal adenocarcinoma and its mechanism of promoting cancer progression

CHEN Suyuan(), Mutailifu Musitaba(), LI Dongxue(), ZHANG Zhigang()

State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China

Received:2023-05-31 Accepted:2023-09-18 Online:2024-01-28 Published:2024-02-28
Contact: LI Dongxue,ZHANG Zhigang E-mail:sychen96@sjtu.edu.cn;18139363645@sjtu.edu.cn;dxli@shsci.org;zzhang@shsci.org
Supported by:
National Natural Science Foundation of China(82203228);Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission(22YF1445600);Medicine and Engineering Interdisciplinary Research Fund of Shanghai Jiao Tong University(YG2021ZD08);"Two-hundred Talents" Program of Shanghai Jiao Tong University School of Medicine(20181708)

摘要/Abstract

摘要：

目的·分析黏附性G蛋白偶联受体F1（adhesion G protein-coupled receptor F1，ADGRF1）在胰腺导管腺癌（pancreatic ductal adenocarcinoma，PDAC）发生及发展过程中的表达变化，探究ADGRF1对PDAC细胞增殖的影响以及促进PDAC进展的潜在分子机制。方法·基于癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库和基因表达综合（Gene Expression Omnibus，GEO）数据库分析ADGRF1在正常胰腺组织及PDAC组织中的mRNA水平表达。利用实时荧光定量PCR（quantitative real-time PCR，qPCR）和蛋白质印迹法（Western blotting）检测ADGRF1在正常胰腺导管上皮细胞hTERT-HPNE及多种PDAC细胞中的表达情况。利用免疫组织化学染色（immunohistochemistry staining，IHC）检测PDAC患者的癌组织及癌旁组织中ADGRF1的表达差异。转染小干扰RNA（small interfering RNA，siRNA）敲低ADGRF1后，通过CCK8和平板克隆形成实验检测PDAC细胞AsPC-1、SW1990增殖能力的变化。构建稳定过表达ADGRF1的Patu8988细胞，通过CCK8实验检测过表达ADGRF1引起的PDAC细胞增殖变化。利用RNA测序（RNA-sequence，RNA-seq）、基因集富集分析（gene set enrichment analysis，GSEA）和免疫浸润分析预测与ADGRF1促进PDAC癌症进展相关的信号通路。结果·TCGA数据库和GEO数据库的分析结果显示ADGRF1 mRNA在PDAC组织中的表达高于正常胰腺组织（均P=0.000）。qPCR和Western blotting结果显示，与hTERT-HPNE细胞相比，多种PDAC细胞中ADGRF1的mRNA和蛋白水平均有所上调（均P<0.05）。IHC结果显示ADGRF1在PDAC患者癌组织中的表达也高于癌旁组织。此外，下调ADGRF1能够抑制PDAC细胞AsPC-1、SW1990的增殖能力；而过表达ADGRF1则促进Patu8988细胞的增殖能力（均P<0.05）。RNA-seq、GSEA富集分析和免疫浸润的结果显示，ADGRF1的表达与干扰素α（interferon-α，IFN-α）、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）和核因子κB（nuclear factor κB，NF-κB）等信号通路有关。结论·ADGRF1在PDAC细胞和组织中高表达，促进PDAC细胞的增殖，其机制可能与多个免疫相关信号通路有关。

关键词: 胰腺导管腺癌, 黏附性G蛋白偶联受体F1, 免疫, 促癌作用

Abstract:

Objective ·To analyze the expression changes of adhesion G protein-coupled receptor F1 (ADGRF1) in the occurrence and development of pancreatic ductal adenocarcinoma (PDAC), and explore the impact of ADGRF1 on the proliferation of PDAC cells and the potential molecular mechanisms that promote PDAC progression. Methods ·The expression of ADGRF1 at mRNA level was analyzed based on the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database, respectively. The expression of ADGRF1 in normal pancreatic ductal epithelial cells (hTERT-HPNE) and various PDAC tumor cells was detected by using real-time fluorescence quantitative PCR (qPCR) and Western blotting. Immunohistochemical staining (IHC) was used to detect the differential expression of ADGRF1 in cancer tissues and adjacent tissues of PDAC patients. After knocking down ADGRF1 with small interfering RNA (siRNA) transfection, the changes in the proliferation ability of PDAC AsPC-1 and SW1990 cells were detected through CCK8 assay and plate cloning experiment. Stable overexpression of ADGRF1 was constructed in PDAC Patu8988 cell line, and the proliferation changes induced by overexpression of ADGRF1 were evaluated through CCK8 assay. RNA sequencing (RNA-seq), gene set enrichment analysis (GSEA), and immune infiltration analysis were utilized to predict signaling pathways associated with ADGRF1-mediated promotion of PDAC cancer progression. Results ·Analysis of the TCGA database and GEO database revealed higher expression of ADGRF1 mRNA in PDAC tissues compared to normal pancreatic tissues (all P=0.000). qPCR and Western blotting results demonstrated up-regulation of ADGRF1 mRNA and protein levels in various PDAC cells compared to hTERT-HPNE cells (all P<0.05). IHC results confirmed higher ADGRF1 expression in PDAC cancer tissues compared to adjacent tissues. Furthermore, downregulation of ADGRF1 inhibited the proliferation of PDAC AsPC-1 and SW1990 cell lines, while overexpression of ADGRF1 promoted the proliferation of Patu8988 cells (all P<0.05). RNA-seq, GSEA enrichment analysis, and immune infiltration analysis revealed that ADGRF1 expression was related to signaling pathways such as interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), and nuclear factor κB (NF-κB). Conclusion ·ADGRF1 is highly expressed in PDAC cells and tissues, and promotes the proliferation of PDAC cells via immune-related signaling pathways.

Key words: pancreatic ductal adenocarcinoma (PDAC), adhesion G protein-coupled receptor F1 (ADGRF1), immunity, tumor-promoting effect

中图分类号:

R735.9

陈溯源, 木司塔巴·木台力甫, 李冬雪, 张志刚. 黏附性G蛋白偶联受体F1在胰腺导管腺癌中的表达及其促进癌症进展的机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(1): 23-34.

CHEN Suyuan, Mutailifu Musitaba, LI Dongxue, ZHANG Zhigang. Expression of adhesion G protein-coupled receptor F1 in pancreatic ductal adenocarcinoma and its mechanism of promoting cancer progression[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(1): 23-34.

图/表 8

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Primer	Forward (5′→3′)	Reverse (5′→3′)
18s	TGCGAGTACTCAACACCAACA	GCATATCTTCGGCCCACA
ADGRF1	TTCGGCAAGTTCGATGAATTAGC	CACGCATGTTCAGATCCACCT

表2 siRNA序列和过表达引物序列

Tab 2 Sequence of siRNA and overexpression primer sequence

siRNA or gene	Forward (5′→3′)	Reverse (5′→3′)
NC-siRNA	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
ADGRF1-siRNA1	UGGUCACAUGGGCUAAUUATT	UAAUUAGCCCAUGUGACCAUA
ADGRF1-siRNA2	UCUCAUUAUAUCUGUCAUUTT	AAUGACAGAUAUAAUGAGAGG
ADGRF1	CGCAAATGGGCGGTAGGCGTG	AGAGACAGCAACCAGGAT

图1 aGPCR在PDAC组织和正常胰腺组织中的表达情况Note： A. GEPIA analysis showed that eight genes were differentially expressed, namely ADGRL4, ADGRE5, ADGRA2, ADGRC3, ADGRF1, ADGRF4, ADGRG1 and ADGRG6. B. UALCAN analysis showed that three genes were differentially expressed, namely ADGRC3, ADGRF1 and ADGRG6.①P=0.000, ②P=0.000, ③P=0.000, compared with the normal group.

Fig 1 Expression of aGPCR in PDAC and normal pancreatic tissues

图2 PDAC患者中 ADGRC3 、 ADGRF1 和 ADGRG6 表达升高对患者预后、肿瘤分期及分级的影响Note： A. Correlation between ADGRC3, ADGRF1 and ADGRG6 mRNA expression and survival prognosis of patients in TCGA database. B. Correlation between ADGRC3, ADGRF1, and ADGRG6 mRNA expression and TNM staging in PDAC patients in the TCGA database. C. Correlation between ADGRC3, ADGRF1 and ADGRG6 mRNA expression and the heterotypic grading of PDAC patients. ①P=0.031, ②P=0.000, compared with the normal group.

Fig 2 Impact of increased expression of ADGRC3, ADGRF1 and ADGRG6 on patient prognosis, tumor staging, and grading in PDAC patients

图3 ADGRF1 在PDAC组织和细胞中的表达Note： A. Expression of ADGRF1 mRNA in PDAC tissues were higher compared to para-carcinoma tissues in GSE15471, GSE16515, GSE28735 and Renji cohort datasets. B. Detection of the mRNA expression of ADGRF1 in eight PDAC cells and hTERT-HPNE cells by qPCR. C. Detection of the protein expression of ADGRF1 in eight PDAC cells and hTERT-HPNE cells by Western blotting. D. IHC was used to detect the expression of ADGRF1 in PDAC tissues and adjacent tissues of PDAC patients. ①P=0.000, compared with the non-tumor group; ②P=0.000, compared with the hTERN-HPNE group.

Fig 3 Analysis of the expression a of ADGRF1 in PDAC tissues and cells

图4 ADGRF1 对PDAC细胞增殖的促进作用Note： A/B. Detection of ADGRF1 expression in AsPC-1 (A) and SW1990 (B) cells by Western blotting. C/D. CCK8 was used to detect the effect of ADGRF1 knockdown on the proliferation ability of AsPC-1 (C) and SW1990 (D) cells. E/F. Plate clonal formation experiment was used to detect the effect of ADGRF1 knockdown on the proliferation ability of AsPC-1 and SW1990 cells. Typical images of cell clones stained with crystal violet (E) and statistical analysis results of formed cell clones (F). G. Detection of ADGRF1 expression in Patu8988 cells by Western blotting. H. Detection of the effect of ADGRF1 overexpression on the proliferation capacity of Patu8988 cells by CCK8. ①P=0.002, ②P=0.000, compared with the NC-siRNA group; ③P=0.002, compared with the ADGRF1-Vector group.

Fig 4 Promoting effect of ADGRF1 on the proliferation of PDAC cells

图5 RNA-seq结果的GO和KEGG分析Note： A. GO enrichment analysis predicted the functional roles of target host genes based on three aspects, including biological processes (BP), cellular components (CC) and molecular functions (MF), and the results of CC suggested that ADGRF1 may be related to the NF-κB signaling pathway. B. KEGG analysis predicted the functions of ADGRF1 and the signaling pathways significantly associated with ADGRF1 alterations including NF-κB, IL-17 and TNF signaling pathways.

Fig 5 GO and KEGG analysis of RNA-seq results

图6 ADGRF1 在GSEA富集分析和免疫浸润分析中的结果Note： A. GSEA enrichment analysis showed that the enrichment of ADGRF1 was related to the immune pathway, including IFN-α, TNF-α and Notch signaling pathways. B. Immune infiltration analysis showed that ADGRF1 was related to B cells and neutrophils.

Fig 6 Results of ADGRF1 in GSEA enrichment analysis and immune infiltration analysis

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黏附性G蛋白偶联受体F1在胰腺导管腺癌中的表达及其促进癌症进展的机制研究

Expression of adhesion G protein-coupled receptor F1 in pancreatic ductal adenocarcinoma and its mechanism of promoting cancer progression

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