Apelin通过激活 FGF2/FGFR1通路促进膀胱癌增殖、迁移及血管生成

doi:10.12122/j.issn.1673-4254.2025.06.18

摘要/Abstract

摘要：

目的探究Apelin（APLN）对膀胱癌细胞增殖、迁移和血管生成的影响，并分析可能的作用机制。方法使用GEO数据筛选出膀胱癌组织及细胞中的差异表达基因。收集60例膀胱癌组织及癌旁组织样本，分析患者的临床及病理参数。体外培养膀胱癌J82细胞或人脐静脉内皮细胞HUVEC，转染相关质粒。并将其分为对照组、si-NC组、si-APLN组、oe-NC组、oe-APLN组、oe-APLN+si-NC组及oe-APLN+si-FGFR1组。qRT-PCR及Western blotting实验检测APLN在膀胱癌组织及细胞中的表达情况，平板克隆实验检测J82细胞增殖情况，Transwell小室实验检测J82细胞迁移情况，血管生成实验检测HUVEC细胞血管生成情况，Western blotting实验检测FGF2表达以及FGFR1磷酸化水平。结果 APLN mRNA及蛋白表达在膀胱癌组织中的表达较正常组更高（P<0.05）。此外，与SV-HUC-1相比，膀胱癌细胞（T24和J82）中APLN mRNA及蛋白表达上调（P<0.05）。临床病理特征分析结果显示，APLN表达与膀胱癌的侵袭、远端转移及TNM分期密切相关（P<0.05）。si-APLN组J82细胞增殖和迁移率较si-APIN组下降，HUVEC细胞总血管生成长度减少（P<0.05）。与oe-NC组相比，oe-APLN组J82细胞增殖和迁移率上升，FGF2表达以及FGFR1磷酸化水平上升，HUVEC细胞总血管生成长度增加（P<0.05）。与oe-APLN+si-NC组相比，oe-APLN+si-FGF2组J82细胞增殖和迁移率下降，FGF2表达以及FGFR1磷酸化水平下降，HUVEC细胞总血管生成长度减少（P<0.05）。与oe-APLN+si-NC组相比，oe-APLN+si-FGFR1组J82细胞增殖和迁移率下降，FGFR1表达以及FGFR1磷酸化水平下降，HUVEC细胞总血管生成长度减少（P<0.05）。结论 APLN可能通过促进FGF2/FGFR1通路活化，促进J82细胞增殖、迁移及HUVEC血管生成。

关键词: 膀胱癌, Apelin, FGF2/FGFR1通路, J82细胞, 血管生成

Abstract:

Objective To investigate the role of apelin in regulating proliferation, migration and angiogenesis of bladder cancer cells and the possible regulatory mechanism. Methods GEO database was used to screen the differentially expressed genes in bladder cancer tissues and cells. Bladder cancer and paired adjacent tissues were collected from 60 patients for analysis of apelin expressions in relation to clinicopathological parameters. In cultured bladder cancer J82 cells and human umbilical vein endothelial cells (HUVECs), the effects of transfection with an apelin-overexpressing plasmid or specific siRNAs targeting apelin, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) on proliferation and migration of J82 cells and tube formation in HUVECs were examined using plate cloning assay, Transwell assay, and angiogenesis assay; the changes in FGF2 expression and FGFR1 phosphorylation were detected using Western blotting. Results The expression level of apelin was significantly higher in bladder cancer tissues than adjacent tissues, and bladder cancer cell lines (T24 and J82) also expressed higher mRNA and protein levels of apelin than SV-HUC-1 cells. Apelin expression level in bladder cancer tissues was correlated with tumor invasion, distant metastasis and advanced TNM stages. Apelin knockdown significantly suppressed proliferation and migration of J82 cells and decreased the total angiogenic length of HUVECs. In contrast, apelin overexpression significantly promoted proliferation and migration and enhanced FGFR1 phosphorylation in J82 cells, and increased the total angiogenesis length in HUVECs, but this effects were effectively mitigated by transfection of the cells with FGF2 siRNA or FGFR1 siRNA. Conclusion High expression of apelin promotes J82 cell proliferation and migration and HUVEC angiogenesis by promoting activation of the FGF2/FGFR1 pathway.

Key words: bladder cancer, apelin, FGF2/FGFR1 pathway, J82 cells, angiogenesis

苏维, 赖厚桦, 唐昕, 周群, 唐亚纯, 符浩, 陈选才. Apelin通过激活 FGF2/FGFR1通路促进膀胱癌增殖、迁移及血管生成[J]. 南方医科大学学报, 2025, 45(6): 1289-1296.

Wei SU, Houhua LAI, Xin TANG, Qun ZHOU, Yachun TANG, Hao FU, Xuancai CHEN. Apelin promotes proliferation, migration, and angiogenesis in bladder cancer by activating the FGF2/FGFR1 pathway[J]. Journal of Southern Medical University, 2025, 45(6): 1289-1296.

图/表 6

图1 GEO数据库筛选差异表达基因

Fig.1 Identification of differentially expressed genes (DEGs) in bladder cancer tissues and cells from GEO datasets. A: Venn diagram showing common DEGs between SV-HUC-1 and 4 bladder cancer cell lines (5637, UMUC3, T24 and J82) in the GSE231383 dataset. B: Venn diagram identifying common DEGs across two GEO datasets. C: Volcano plot of DEGs from two independent GEO datasets, showing upregulated APLN expression in bladder cancer tissues/cells (screening criteria: |log FC|≥2, P<0.05).

图2 APLN在膀胱癌组织和细胞中的表达情况

Fig.2 Apelin (APLN) expression in bladder cancer tissues and cells. A: qRT-PCR for detecting APLN mRNA expression in bladder cancer tissues and normal tissues (n=60). B: Western blotting for detecting APLN protein expression in bladder cancer tissues and normal tissues (n=5). C, D: qRT-PCR and Western blotting for detecting APLN expression in bladder cancer cells and SV-HUC-1 cells. Each experiment was repeated 3 times. **P<0.01, ***P<0.001.

表1 APLN mRNA表达与膀胱癌患者临床病理特征的关系

Tab.1 Correlation of APLN mRNA expression with clinico-pathological features of patients with bladder cancer (n)

Characteristics	Case	APLN Expression		P
Characteristics	Case	High (n=30)	Low (n=30)	P
Age(year)				0.796
≥60	32	15	13
<60	28	15	17
Gender				0.158
Female	18	6	12
Male	42	24	18
Tumor size (cm)				0.438
<3	28	12	16
≥3	32	18	14
Invasion				0.019
T0-T2	30	10	20
T3-T4	30	20	10
Distant metastasis				0.037
Yes	33	21	22
No	27	9	8
TNM stage				0.035
0-II	35	13	22
III-IV	25	17	8
Histological grade				0.438
High/intermediate	28	16	12
Low	32	14	18

图3 转染后J82细胞中APLN表达情况

Fig.3 APLN expression in J82 cells after transfection. A: qRT-PCR for detecting APLN mRNA expression in each group of J82 cells. B, C: Western blotting for detecting APLN protein expression in each group of J82 cells. Each experiment was repeated 3 times. *P<0.05, **P<0.01, ***P<0.001.

图4 转染后J82细胞增殖、迁移和血管生成情况

Fig.4 Proliferation, migration and angiogenesis of J82 cells after transfection. A: Plate cloning assayfor assessing J82 cell proliferation in each group. B: Transwell assay for evaluating cell migration in each group. C: Angiogenesis assay for assessing HUVEC tube formation in each group. Each experiment was repeated 3 times. Scale bar=100 μm. **P<0.01.

图5 转染后J82细胞增殖、迁移和血管生成情况

Fig.5 Proliferation, migration, angiogenesis and FGF2/FGFR1 signaling in J82 cells after transfection. A, E: Western blotting for detecting FGF2 expression and FGFR1 phosphorylation in different groups. B, F: Plate cloning assay for assessing J82 cell proliferation. C, G: Transwell assay for evaluating cell migration. D, H: Angiogenesis assay for assessing HUVEC tube formation. Each experiment was repeated 3 times. Scale bar=100 μm. *P<0.05, **P<0.01.

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