SEC14L1P1对口腔鳞状细胞癌细胞增殖和迁移的影响

doi:10.19401/j.cnki.1007-3639.2025.03.007

摘要/Abstract

摘要：

背景与目的：SEC14L1P1是SEC14家族的一种假基因,已被发现与多种肿瘤的发生、发展密切相关,但其在口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）中的作用尚未明确。本研究旨在深入了解SEC14L1P1在OSCC细胞内的表达特征和亚细胞定位,以及其对OSCC细胞增殖和迁移的影响。方法：通过ENCORI数据库对SEC14L1P1在头颈部鳞状细胞癌（head and neck squamous cell carcinoma,HNSCC）组织中的表达进行分析；利用GDC和UCSC Xena数据库进一步分析SEC14L1P1在HNSCC中的表达及其与患者预后之间的关系。利用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）检测SEC14L1P1在OSCC细胞系中的表达；采用RNA核质分离实验确定SEC14L1P1在OSCC细胞中的定位。对CAL-27细胞建立SEC14L1P1敲减（SS-SEC14L1P1）组和敲减对照（SS-NC）组,对HN30细胞建立SEC14L1P1过表达（SEC14L1P1）组和过表达对照（Vector）组。通过细胞计数试剂盒-8（cell counting kit-8,CCK-8）和transwell迁移实验评估SEC14L1P1表达变化对各组细胞增殖、迁移能力的影响。采用RTFQ-PCR和蛋白质印迹法（Western blot）检测SEC14L1P1表达改变对上皮-间充质转化（epithelial-mesenchymal transition,EMT）相关基因表达水平的影响。采用裸鼠皮下移植瘤模型观察SEC14L1P1在体内对OSCC细胞增殖的影响,将12只4周龄BALB/c裸鼠随机分为反义寡核苷酸（antisense oligonucleotide,ASO）-NC组和ASO-SEC14L1P1组,每组6只,每只裸鼠均做标记。进一步的机制研究通过RNAInter数据库分析与SEC14L1P1交互的分子,通过ENCORI数据库查询SEC14L1P1与DHX9的表达相关性。采用Western blot检测SEC14L1P1表达改变对磷脂酰肌醇3-激酶（phosphoinositide 3-kinase,PI3K）/蛋白激酶B（protein kinase B,AKT）通路的影响。结果：数据库分析显示,SEC14L1P1在HNSCC组织中的表达高于正常组织,且与患者的不良预后密切相关。RTFQ-PCR结果表明,SEC14L1P1在6种OSCC细胞系中均高表达；RNA核质分离实验结果显示,在CAL-27和HN30细胞中SEC14L1P1主要定位于细胞核内。与SS-NC组相比,SS-SEC14L1P1组中的相对表达水平明显降低,且能显著抑制细胞增殖和迁移能力；与Vector组相比,SEC14L1P1组中的相对表达水平明显升高,细胞增殖和迁移能力也明显升高。SEC14L1P1的下调伴随E-钙粘蛋白（E-cadherin）的mRNA表达和蛋白水平升高,N-钙粘蛋白（N-cadherin）和波形蛋白（vimentin）的mRNA表达和蛋白水平降低,SEC14L1P1过表达后的结果则相反。裸鼠皮下移植瘤模型实验显示,与ASO-NC组比较,ASO-SEC14L1P1组皮下移植瘤体积和重量均减小。进一步的机制研究发现SEC14L1P1与DHX9表达呈正相关性,且已有研究表明DHX9能激活PI3K/AKT信号通路；敲减SEC14L1P1导致磷酸化PI3K（phosphorylated-PI3K,p-PI3K）和磷酸化AKT（phosphorylated-AKT,p-AKT）的蛋白表达减少,过表达SEC14L1P1则显示p-PI3K和p-AKT的蛋白表达增加。结论：SEC14L1P1在OSCC细胞和组织中呈现出较高的表达水平,并且能促进OSCC细胞增殖及迁移,这一现象可能与SEC14L1P1调控PI3K/AKT信号通路,进而促进EMT有关。

关键词: SEC14L1P1, 口腔鳞状细胞癌, 细胞增殖, 细胞迁移, 上皮-间充质转化

Abstract:

Background and purpose: SEC14L1P1, a pseudogene of the SEC14 family, is closely associated with the development of various tumors, but its role in oral squamous cell carcinoma (OSCC) has not been clarified. This study aimed to gain insights into the expression characteristics and subcellular localization of SEC14L1P1 in OSCC cells, as well as its effects on OSCC cell proliferation and migration. Methods: The expression of SEC14L1P1 in head and neck squamous cell carcinoma (HNSCC) tissues was analyzed by the ENCORI database; The expression of SEC14L1P1 and its relationship with patient prognosis in HNSCC was further analyzed using the GDC and UCSC Xena databases. The expression of SEC14L1P1 in OSCC cell lines was detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR); RNA nucleoplasmic separation assay was performed to determine the localization of SEC14L1P1 in OSCC cells. SEC14L1P1 knockdown (SS-SEC14L1P1) group and knockdown control (SS-NC) group were established for CAL-27 cells, and SEC14L1P1 overexpression (SEC14L1P1) group and overexpression control (Vector) group were established for HN30 cells. The effects of SEC14L1P1 expression on the proliferation and migration abilities of cells in each group were assessed by cell counting kit-8 (CCK-8) and transwell migration assays. RTFQ-PCR and Western blot experiments were used to detect the effects of altered SEC14L1P1 expression on the expression levels of epithelial-mesenchymal transition (EMT)-related genes. To investigate the effects of SEC14L1P1 on the proliferation of OSCC cells in vivo using a subcutaneous xenograft tumor model in nude mice, 12 four-week-old BALB/c nude mice were randomly divided into two groups: the antisense oligonucleotide (ASO)-NC group and the ASO-SEC14L1P1 group, with 6 mice in each group. All mice were individually labeled. Further mechanistic studies were performed by analyzing molecules interacting with SEC14L1P1 through the RNAInter database, and the ENCORI database was queried for expression correlation between SEC14L1P1 and DHX9. The effect of altered SEC14L1P1 expression on the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway was detected by Western blot assay. Results: Database analysis showed that the expression of SEC14L1P1 was higher in HNSCC tissues than in normal tissues, and was strongly associated with poor patient prognosis. The RTFQ-PCR results showed that SEC14L1P1 was highly expressed in all six OSCC cell lines; RNA nucleoplasmic separation showed that SEC14L1P1 was mainly localized in the nucleus in CAL-27 and HN30 cells. Compared with SS-NC, the relative expression of SEC14L1P1 in the SS-SEC14L1P1 group was significantly lower and significantly inhibited cell proliferation and migration, while the relative expression of SEC14L1P1 in the SEC14L1P1 group was significantly higher compared with the Vector group, which also significantly increased cell proliferation and migration. The down-regulation of SEC14L1P1 was accompanied by increased mRNA and protein levels of E-cadherin, and decreased mRNA and protein levels of N-cadherin and vimentin, with the opposite result after SEC14L1P1 overexpression. In vivo experiments showed that the xenograft tumor weight and volume of the ASO-SEC14L1P1 group were significantly reduced. Further mechanistic studies revealed a positive correlation between SEC14L1P1 and DHX9 expressions, and DHX9 has been shown to activate the PI3K/AKT signaling pathway. Knockdown of SEC14L1P1 resulted in decreased protein expressions of phosphorylated-PI3K (p-PI3K) and phosphorylated-AKT (p-AKT), and overexpression of SEC14L1P1 increased protein expressions of p-PI3K and p-AKT. Conclusion: SEC14L1P1 showed high expression levels in OSCC cells and tissues and promoted the proliferation and migration of OSCC cells, a phenomenon that may be related to the regulation of the PI3K/AKT signaling pathway by SEC14L1P1, which in turn promotes EMT.

Key words: SEC14L1P1, Oral squamous cell carcinoma, Cell proliferation, Cell migration, Epithelial-mesenchymal transition

中图分类号:

R739.85

郑文甜, 公慧, 张馨月, 郝嘉仪, 王亚杰, 蒋英英. SEC14L1P1对口腔鳞状细胞癌细胞增殖和迁移的影响[J]. 中国癌症杂志, 2025, 35(3): 309-319.

ZHENG Wentian, GONG Hui, ZHANG Xinyue, HAO Jiayi, WANG Yajie, JIANG Yingying. Effects of SEC14L1P1 on proliferation and migration of oral squamous cell carcinoma cells[J]. China Oncology, 2025, 35(3): 309-319.

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Gene	Primer sequence
SEC14L1P1	Forward: 5‘-GGCCTGCTGGATTACATTGATAG-3‘
	Reverse: 5‘-CCAGTAGAGAGATTTGGGGACC-3‘
β-actin	Forward: 5‘-CATGTACGTTGCTATCCAGGC-3‘
	Reverse: 5‘-CTCCTTAATGTCACGCACGAT-3‘
GAPDH	Forward: 5‘-GAACGGGAAGCTCACTGG-3‘
	Reverse: 5‘-GCCTGCTTCACCACCTTCT-3‘
U6	Forward: 5‘-CTCGCTTCGGCAGCACATATACT-3‘
	Reverse: 5‘-ATTTGCGTGTCATCCTTGCGCA-3‘
E-cadherin	Forward: 5‘-CGAGAGCTACACGTTCACGG-3‘
	Reverse: 5‘-GGGTGTCGAGGGAAAAATAGG-3‘
N-cadherin	Forward: 5‘-TGCGGTACAGTGTAACTGGG-3‘
	Reverse: 5‘-GAAACCGGGCTATCTGCTCG-3‘
Vimentin	Forward: 5‘-AGTCCACTGAGTACCGGAGAC-3‘
	Reverse: 5‘-CATTTCACGCATCTGGCGTTC-3‘

Name	Target sequences
SEC14L1P1 Smart Silencer	GATTATCCTTGGCAAGACA
	CCATTGGACTCCACCTTCC
	GCATCCCTGATAACAAACT
	TCAGTTCCTATGTGCATGGG
	TTGCTACCTGGGCCGTTCCA
	GGCCAGTTACCCTAAGAGAC