SRSF1通过调控VEGFA mRNA可变剪接促进食管鳞状细胞癌Eca9706细胞增殖、侵袭和迁移

doi:10.19401/j.cnki.1007-3639.2022.03.001

摘要/Abstract

摘要：

背景与目的：丝氨酸/精氨酸富集剪接因子1（serine/arginine-rich splicing factor 1,SRSF1）与肿瘤的发生、发展密切相关。食管癌是常见的消化系统恶性肿瘤,但SRSF1在食管癌中的作用罕有报道。检测SRSF1在食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）组织中的表达及其对ESCC细胞增殖、迁移和侵袭的影响,并初步探讨其作用机制。方法：收集2020年1月—2021年12月于河北医科大学第四医院行手术切除的40例ESCC患者的癌及癌旁组织标本,采用免疫组织化学染色法检测ESCC组织中SRSF1的水平。采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）和蛋白质印迹法（Western blot）检测ESCC细胞系中SRSF1 mRNA表达和蛋白水平。筛选SRSF1高表达的Eca9706细胞进行研究。采用小干扰RNA（small interfering RNA,siRNA）技术降低SRSF1 mRNA表达。采用细胞计数试剂盒-8（cell counting kit-8,CCK-8）、transwell和Matrigel基质胶实验分别检测Eca9706细胞增殖、迁移和侵袭能力。采用数据库分析ESCC组织中血管内皮生长因子A（vascular endothelial growth factor A,VEGFA）的表达,并分析SRSF1与VEGFA表达的相关性。采用RTFQ-PCR检测Eca9706细胞VEGFA Iso8a和Iso8b亚型的表达水平,以及敲低SRSF1后VEGFA Iso8a和Iso8b亚型的表达变化。结果：SRSF1在ESCC组织中的表达水平高于癌旁组织（P<0.05）。SRSF1 mRNA表达和蛋白水平在ESCC Eca9706细胞系中最高（P<0.01）。转染siRNA-SRSF1组中SRSF1 mRNA表达和蛋白水平显著低于siRNA-NC组（P<0.01）。与siRNA-NC组相比,siRNA-SRSF1组Eca9706细增殖、迁移和侵袭能力显著降低（P<0.05）。ESCC组织中VEGFA表达明显高于食管正常组织（P<0.05）,且与SRSF1表达呈正相关（P<0.01）。Eca9706细胞VEGFA Iso8a亚型表达明显高于VEGFA Iso8b亚型（P<0.01）,且敲低SRSF1后VEGFA Iso8a亚型表达降低,VEGFA Iso8b亚型表达升高（P<0.01）。结论：SRSF1可通过作用于VEGFA可变剪接促进ESCC Eca9706细胞增殖、侵袭和迁移。

关键词: 食管鳞状细胞癌, 丝氨酸/精氨酸富集剪接因子1, 血管内皮生长因子A, 细胞增殖, 细胞侵袭, 细胞迁移

Abstract:

Background and purpose: Serine/arginine-rich splicing factor 1 (SRSF1) is closely related to the development of tumor. Esophageal carcinoma is the common malignant tumor of digestive system. However, the role of SRSF1 in esophageal carcinoma is rarely reported. This study aimed to detect the expression of SRSF1 in esophageal squamous cell carcinoma (ESCC) and its effects on the proliferation, invasion and migration of ESCC cells, and to explore its mechanism. Methods: Forty pairs of ESCC tissues and paracancerous tissues resected at the Fourth Hospital of Hebei Medical University from January 2020 to December 2021 were collected for this study. The expression of SRSF1 in ESCC was detected by immunohistochemistry. The mRNA expression and protein level of SRSF1 in ESCC cells were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot, respectively. Eca9706 cell with high expression of SRSF1 was selected. The mRNA expression of SRSF1 was reduced by small interfering RNA (siRNA). The proliferation, migration and invasion of Eca9706 cells were detected by cell counting kit-8 (CCK-8), transwell and Matrigel matrix gel assays, respectively. Database was used to analyze the expression of vascular endothelial growth factor A (VEGFA) in ESCC tissues, and the correlation between expressions of SRSF1 and VEGFA was also analyzed. RTFQ-PCR was used to detect the expression levels of VEGFA Iso8a and Iso8b isoform in Eca9706 cells, and the change in VEGFA Iso8a and Iso8b isoform expressions was also detected after knocking down SRSF1. Results: The expression of SRSF1 was significantly higher in ESCC tissues than in normal esophageal tissues (P<0.05). SRSF1 mRNA and protein were highly expressed in Eca9706 cells (both P<0.01). The mRNA expression and protein level of SRSF1 was significantly lower in siRNA-SRSF1 group than in siRNA-NC group (P<0.01). After knocking down SRSF1, the proliferation, migration and invasion abilities of Eca9706 cells were significantly lower compared with siRNA-NC group (all P<0.05). The expression of VEGFA was significantly higher in ESCC tissue than in normal esophageal tissue (P<0.05), and it was positively correlated with the expression of SRSF1 (P<0.01). RTFQ-PCR showed that the level of VEGFA Iso8a expression in Eca9706 cells was significantly higher compared with VEGFA Iso8b (P<0.01). Moreover, the level of VEGFA Iso8a expression was significantly decreased, while the level of VEGFA Iso8b expression increased after knocking down SRSF1 (P<0.01). Conclusion: SRSF1 can promote proliferation, invasion and migration of ESCC Eca9706 cells by possibly regulating VEGFA mRNA splicing.

Key words: Esophageal squamous cell carcinoma, Serine/arginine-rich splicing factor 1, Vascular endothelial growth factor A, Cell proliferation, Cell migration, Cell invasion

中图分类号:

R735.1

段玉青, 夏宁, 贾云泷, 郑文雅, 刘丽华. SRSF1通过调控VEGFA mRNA可变剪接促进食管鳞状细胞癌Eca9706细胞增殖、侵袭和迁移[J]. 中国癌症杂志, 2022, 32(3): 191-199.

DUAN Yuqing, XIA Ning, JIA Yunlong, ZHENG Wenya, LIU Lihua. SRSF1 promotes proliferation, invasion and migration of esophageal squamous cell carcinoma Eca9706 cells by regulating VEGFA mRNA alternative splicing[J]. China Oncology, 2022, 32(3): 191-199.

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SRSF1 expression	ESCC tissues (n=40)	Matched paracancerous tissues (n=40)	χ²	P value
Low	13 (32.5)	24 (60.0)	6.084	0.013 6
High	27 (67.5)	16 (40.0)