GPR19基因在前列腺癌中的表达及意义

doi:10.11958/20220419

天津医药 ›› 2022, Vol. 50 ›› Issue (11): 1128-1133.doi: 10.11958/20220419

GPR19基因在前列腺癌中的表达及意义

刘帅兵(), 闫墨, 王楷斌, 杨阔, 王玉琢^△()

天津医科大学第二医院泌尿外科，天津市泌尿外科研究所（邮编300211）

收稿日期:2022-03-19 修回日期:2022-07-09 出版日期:2022-11-15 发布日期:2022-11-11
通讯作者: 王玉琢 E-mail:liushuaibing@tmu.edu.cn;460576427@qq.com
作者简介:刘帅兵（1995），男，硕士在读，主要从事泌尿系肿瘤研究。E-mail：liushuaibing@tmu.edu.cn
基金资助:
天津市科技计划项目(18ZXDBSY00020)

The expression and significance of GPR19 gene in prostate cancer

LIU Shuaibing(), YAN Mo, WANG Kaibin, YANG Kuo, WANG Yuzhuo^△()

Department of Urology, Tianjin Institute of Urology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China

Received:2022-03-19 Revised:2022-07-09 Published:2022-11-15 Online:2022-11-11
Contact: WANG Yuzhuo E-mail:liushuaibing@tmu.edu.cn;460576427@qq.com

刘帅兵, 闫墨, 王楷斌, 杨阔, 王玉琢. GPR19基因在前列腺癌中的表达及意义[J]. 天津医药, 2022, 50(11): 1128-1133.

LIU Shuaibing, YAN Mo, WANG Kaibin, YANG Kuo, WANG Yuzhuo. The expression and significance of GPR19 gene in prostate cancer[J]. Tianjin Medical Journal, 2022, 50(11): 1128-1133.

摘要/Abstract

摘要：

目的分析G蛋白偶联受体19（GPR19）在前列腺癌（PCa）中的表达情况，探究GPR19参与PCa进展的机制。方法利用TCGA、Oncomine数据库分析GPR19在PCa中的表达水平及临床病理信息。qPCR及Western blot检测PCa细胞系中GPR19的mRNA及蛋白的表达水平并验证相关通路中的关键分子，EdU实验验证敲低GPR19对肿瘤细胞增殖的影响。结果生物信息学分析显示GPR19基因在PCa样本中表达增高，且GPR19基因表达与患者的年龄、无进展生存率、Gleason评分、TNM分期有关。单因素Cox分析显示Gleason评分、TNM分期和GPR19表达是PCa患者无进展生存的影响因素。qPCR和Western blot显示PCa细胞系中GPR19的表达高于正常前列腺上皮细胞RWPE-1，敲低GPR19后的PCa细胞增殖能力减弱。通路富集分析发现GPR19参与了PCa的细胞周期调控，实验证实敲低GPR19后PCa细胞中G2M信号检查点上的关键分子细胞周期蛋白依赖性激酶1（CDK1）表达降低，通路相关性分析显示GPR19同细胞周期蛋白依赖性激酶抑制剂3（CDKN3）基因相关性最高。结论 GPR19在PCa中表达升高，并与多种临床信息相关，通过G2M信号检查点影响肿瘤细胞增殖，有望成为PCa的潜在治疗靶点。

关键词: 前列腺肿瘤, 受体, G-蛋白偶联, 细胞增殖, 细胞周期, G2期细胞周期检查点

Abstract:

Objective To analyze the expression of G protein-coupled receptor 19 (GPR19) in prostate cancer (PCa), and explore the mechanism of GPR19 in prostate cancer. Methods TCGA and Oncomine online databases were used to analyze the expression level and clinicopathological information of GPR19 in PCa. Expression levels of GPR19 mRNA and protein in PCa cell lines were detected by qPCR and Western blot assay, and the key molecules in related pathways were verified. EdU assay was used to verify the effect of GPR19 knockdown on tumor cell proliferation. Results Database analysis showed that GPR19 gene expression was increased in PCa samples. GPR19 gene expression was related to patient's age, disease-free survival rate, Gleason score and TNM stage. Univariate Cox analysis showed that Gleason score, TNM stage and GPR19 expression were the influencing factors of progression-free survival in PCa patients. qPCR and Western blot assay showed that GPR19 was expressed higher in PCa cell lines than that of normal prostate epithelial cell line, RWPE-1. EdU experiments verified that the proliferation ability of PCa cells was reduced after GPR19 knockdown. Pathway enrichment analysis showed that GPR19 was involved in cell cycle regulation of PCa. It was confirmed that knockdown of GPR19 reduced the expression of cyclin dependent kinase 1 (CDK1), a key molecule at the G2M signaling checkpoint in PCa cells. Pathway correlation analysis showed that GPR19 had the highest correlation with CDKN3 gene. Conclusion The expression of GPR19 is increased in PCa and related to a variety of clinical information, which affects tumor cell proliferation through the G2M signaling checkpoint. GPR19 is expected to be a potential therapeutic target for PCa.

Key words: prostatic neoplasms, receptors, G-Protein-coupled, cell proliferation, cell cycle, G2 phase cell cycle checkpoints

中图分类号:

R737.25

图/表 11

表1 qPCR引物序列

Tab.1 Primer sequence for qPCR

基因名称	引物序列（5′→3′）	产物大小（bp）
GPR19	上游：AAACAGACCTTCACTATGTGCTG	91
	下游：TTGCCGAAGATAGAAAACAACCA	91
CDK1	上游：CTTGGATTCTATCCCTCCTGGTCAGTAC	118
	下游：GTCAACTGGAGTTGAGTAACGAGCTGAC	118
GAPDH	上游：GGAAGGTGAAGGTCGGAGTCA	101
	下游：GTCATTGATGGCAACAATATCCACT	101

图1 生物信息学分析GPR19在PCa中的表达及与预后的关系 A：TCGA数据库中PCa组织与癌旁组织中GPR19 mRNA表达；B：Oncomine数据库中Luo prostate队列研究中PCa组织与癌旁组织中GPR19 mRNA表达；C：不同GPR19表达水平PCa患者的无进展生存曲线，以GPR19 mRNA表达水平中位数3.32为界分为高表达和低表达组。

Fig.1 Bioinformatic analysis of GPR19 expression in prostate cancer

表2 不同临床病理特征患者GPR19基因表达情况

Tab.2 GPR19 expression in patients with different clinicopathological features

临床病理特征	n	GPR19		χ²
临床病理特征	n	高表达	低表达	χ²
总生存
存活	415	207（49.88）	208（50.12）	0.995
死亡	6	3（50.00）	3（50.00）	0.995
无进展生存
存活	369	176（47.69）	193（52.30）	5.018^＊
死亡	52	34（65.39）	18（34.62）	5.018^＊
Gleason评分
≤7分	255	109（42.75）	146（57.25）	12.460^＊
>7分	166	101（60.84）	65（39.16）	12.460^＊
TNM分期
T1~T2期	172	66（38.37）	106（61.63）	14.640^＊
T3~T4期	249	144（57.83）	105（42.17）	14.640^＊
年龄
≤60岁	192	77（40.11）	115（59.89）	12.787^＊
>60岁	229	133（58.07）	96（41.92）	12.787^＊

表3 PCa无进展生存的单因素Cox回归分析

Tab.3 Univariate Cox regression analysis for prostate cancer relapse-free survival

变量	β	SE	Wald χ²	P	HR	HR 95%CI
年龄	0.011	0.021	0.263	0.608	1.011	0.971~1.052
Gleason评分	0.867	0.152	32.651	<0.001	2.381	1.768~3.206
TNM分期	0.857	0.258	11.076	0.001	2.357	1.422~3.905
GPR19	0.285	0.114	6.195	0.013	1.329	1.062~1.663

表4 PCa无进展生存的多因素Cox回归分析

Tab.4 Multivariate Cox regression analysis for prostate cancer relapse-free survival

变量	β	SE	Wald χ²	P	HR	HR 95%CI
年龄	-0.008	0.021	0.129	0.720	0.992	0.951~1.035
Gleason评分	0.804	0.170	22.443	<0.001	2.234	1.602~3.116
TNM分期	0.244	0.308	0.630	0.428	1.276	0.698~2.333
GPR19	0.034	0.135	0.065	0.799	1.035	0.794~1.349

表5 各细胞系中GPR19 mRNA及其蛋白相对表达 ($\bar{x}\pm s$)

Tab.5 Expression of GPR19 mRNA and GPR19 protein in each cell line

细胞系	n	GPR19 mRNA	GPR19蛋白
RWPE-1	3	0.989±0.029	0.389±0.133
C4-2	3	2.104±0.120^a	0.960±0.129^a
PC3	3	3.208±0.122^a	0.745±0.148^a
LNCaP	3	8.661±0.354^a	0.941±0.013^a
VCaP	3	2.009±0.244^a	0.416±0.297
22RV1	3	1.701±0.052^a	0.329±0.272
DU145	3	3.718±0.451^a	0.724±0.225
F		331.000^＊	5.478^＊

图2 不同PCa细胞系中GPR19蛋白表达结果

Fig.2 Results of GPR19 protein expression in different PCa cell lines

图3 敲低GPR19后EdU增殖实验检测细胞增殖效率

Fig.3 The cell proliferation efficiency after GPR19 knockdown detected by EdU proliferation assay

图4 PCa中GPR19的通路富集分析 A：GPR19高表达组通路富集分数；B：E2F和G2M的GSEA通路富集结果。

Fig.4 Pathway enrichment analysis of GPR19 in prostate cancer

图5 GPR19与G2M检查点关键分子CDK1的相关性 A：GPR19 mRNA与CDK1 mRNA表达的相关性；B：敲低GPR19后LNCaP细胞CDK1蛋白表达

Fig.5 The relationship of GPR19 and CDK1, a key molecule on the G2M checkpoint

图6 GPR19与周期通路中基因的相关性 A：E2F与G2M信号通路基因韦恩图；B：GPR19与73个基因的相关性。

Fig.6 Correlation between GPR19 and genes in the periodic pathway

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GPR19基因在前列腺癌中的表达及意义

The expression and significance of GPR19 gene in prostate cancer

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