目的：探讨HOTAIR/CSTF2/CD44轴调控食管癌（esophageal cancer,EC）侵袭转移的机制。方法：收集2020年9月―2022年6月43例非特异性侵袭性EC患者的EC组织样本和邻近正常组织,分为对照组和EC组。培养EC细胞系KYSE150,EC109和TE-1以及人正常食管上皮细胞系HEEC细胞,分为control、si-HOTAIR、CSTF2模拟物和si-HOTAIR+CSTF2模拟物等4组。采用实时荧光定量PCR检测HOTAIR、CD24和CSTF2的表达,CCK8测定细胞活力,流式细胞术检测细胞凋亡,随后检测细胞的迁移和侵袭,蛋白印迹检测细胞凋亡蛋白（Ki67、PCNA、裂解的半胱天冬酶-3和半胱天冬酶-9）和迁移蛋白（VEGF和MMP-9）。监测小鼠模型肿瘤生长的迹象。结果：HOTAIR和CSTF2在EC组织中的表达显著高于正常组织;与HEEC细胞相比,HOTAIR和CSTF2在EC细胞系EC109,KYSE150和TE-1中表达增加;与control组比较,HHOTAIR敲低显著降低了细胞增殖、迁移和侵袭,并加速了细胞凋亡;与CSTF2相互作用的生物信息学分析显示,HOTAIR转录本中存在潜在的结合域,HOTAIR与CSTF2表达之间存在较强的相关关系,引起CSTF2下调的HOTAIR敲低显著抑制了CD24的表达,CSTF2的升高显著增加了CD24的表达。体内研究表明HOTAIR敲低可降低CSTF和CD24表达,进而抑制肿瘤细胞生长和侵袭。结论：结果表明发现HOTAIR和CSTF2在EC细胞和组织中的表达显着增加。体外和体内研究表明,HOTAIR敲低促进细胞凋亡并抑制细胞增殖和侵袭,HOTAIR可能结合CSTF2,并间接调节CD24在EC细胞中的表达。
Abstract
Objective To explore the mechanism of HOTAIR/CSTF2/CD44 axis regulating invasion and metastasis of esophageal cancer. Methods EC tissue samples and adjacent normal tissues from 43 patients with nonspecific invasive EC from September 2020 to June 2022 were collected and divided into control group and EC group. EC cell lines KYSE150, EC109 and TE-1 and human normal esophageal epithelial cell line HEEC cells were cultured and divided into control, si-HOTAIR, CSTF2 mimics and si-HOTAIR+CSTF2 mimics. Real-time fluorescence quantitative PCR was used to detect the expression of HOTAIR, CD24 and CSTF2, CCK8 was used to measure cell viability, flow cytometry was used to detect apoptosis, and then cell migration and invasion were detected. Apoptotic proteins (Ki67, PCNA, lysed caspase 3 and caspase 9) and migration proteins (VEGF and MMP-9) were detected by western blotting. Mouse models were monitored for signs of tumor growth. Results The results showed that the expression of HOTAIR and CSTF2 in EC tissues was significantly higher than that in normal tissues. Compared with HEEC cells, the expressions of HOTAIR and CSTF2 were increased in EC cell lines EC109, KYSE150 and TE-1. Compared with control, HHOTAIR knockdown significantly reduced cell proliferation, migration and invasion, and accelerated cell apoptosis. Bioinformatics analysis of CSTF2 interaction showed that there was a potential binding domain in HOTAIR transcripts, and there was a strong correlation between HOTAIR and CSTF2 expression. HOTAIR knockdown resulting in CSTF2 downregulation significantly inhibited CD24 expression, and CSTF2 elevation significantly increased CD24 expression. In vivo studies have shown that HOTAIR knockdown can reduce the expression of CSTF and CD24, thus inhibiting the growth and invasion of tumor cells. Conclusion The results showed that the expression of HOTAIR and CSTF2 in EC cells and tissues increased significantly. In vitro and in vivo studies have shown that HOTAIR knockdown promotes apoptosis and inhibits cell proliferation and invasion. HOTAIR may bind CSTF2 and indirectly regulate the expression of CD24 in EC cells.
关键词
HOTAIR /
CSTF2 /
CD24 /
食管癌
Key words
HOTAIR /
CSTF2 /
CD24 /
esophageal cancer
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基金
新疆维吾尔自治区卫生健康委员会科研基金项目（20210373）
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