长链非编码RNA FLJ30679对口腔鳞状细胞癌细胞增殖和迁移的影响

doi:10.19401/j.cnki.1007-3639.2024.05.001

摘要/Abstract

摘要：

背景与目的： 长链非编码RNA（long noncoding RNA，lncRNA）能够调控基因转录、mRNA剪切、稳定和翻译，是多种生物学过程中的重要调节因子。本研究旨在探讨lncRNA FLJ30679在口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）中的表达、与临床病理学特征的关系及对OSCC恶性生物学行为的影响。方法： 通过UCSC Xena数据库分析FLJ30679在头颈部鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）组织中的表达及与临床病理学特征的关系。通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测FLJ30679在OSCC细胞系中的表达，通过RNA核质分离实验明确其亚细胞定位；采用FLJ30679 Smart Silencer建立FLJ30679敲减组（SS-FLJ30679），采用过表达质粒建立FLJ30679过表达组（FLJ30679）。通过细胞计数试剂盒-8（cell counting kit-8，CCK-8）和transwell迁移实验检测FLJ30679表达改变对OSCC细胞增殖和迁移能力的影响。通过RTFQ-PCR和蛋白质印迹法（Western blot）检测OSCC细胞中的FLJ30679表达改变对上皮-间充质转化（epithelial-mesenchymal transition，EMT）相关基因表达的影响。通过Western blot检测FLJ30679表达改变对磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase，AKT）通路的影响。结果： 数据库分析显示，FLJ30679在HNSCC组织中的表达高于正常组织（P<0.01），且其表达与不良预后有关（P<0.01）。FLJ30679在6种OSCC细胞系中均高表达，且主要定位于细胞核。与对照组相比，FLJ30679敲减组的OSCC细胞增殖和迁移能力显著降低（P<0.01），FLJ30679过表达组的OSCC细胞增殖和迁移能力显著升高（P<0.001）。FLJ30679敲减导致E-钙黏蛋白（E-cadherin）的mRNA和蛋白表达水平上调（P<0.01），而N-钙黏蛋白（N-cadherin）和波形蛋白（vimentin）的mRNA和蛋白表达水平下调（P<0.01）；FLJ30679过表达导致E-cadherin的蛋白表达水平下调（P<0.001），而N-cadherin和vimentin的mRNA和蛋白表达水平上调（P<0.05）。FLJ30679敲减导致磷酸化PI3K（phosphorylated-PI3K，p-PI3K）和磷酸化AKT（phosphorylated-AKT，p-AKT）的蛋白表达水平下调（P<0.001），FLJ30679过表达导致p-PI3K和p-AKT的蛋白表达水平上调（P<0.01）。结论： FLJ30679在OSCC细胞和组织中表达上调，能够促进OSCC细胞增殖和迁移，这可能与FLJ30679激活PI3K/AKT通路，促进EMT的发生有关。

关键词: 口腔鳞状细胞癌, 长链非编码RNA, FLJ30679, 细胞增殖, 细胞迁移, 上皮-间充质转化

Abstract:

Background and purpose: Long noncoding RNA (lncRNA) can regulate gene transcription, mRNA shear, stabilization and translation, and it is an important regulatory factor in a variety of biological processes. This study aimed to investigate the expression and clinical features of lncRNA FLJ30679 in oral squamous cell carcinoma (OSCC) and its effect on the malignant biological behavior of OSCC. Methods: The expression of FLJ30679 in head and neck squamous cell carcinoma (HNSCC) tissues and normal tissues was analyzed by the UCSC Xena database for expression and prognosis. The expression of FLJ30679 in OSCC cell lines was detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). The subcellular localization of FLJ30679 in OSCC cells was detected by RNA nuclear-cytoplasmic fractionation assays. FLJ30679 Smart Silencer was used to establish the FLJ30679 knockdown group (SS-FLJ30679), and overexpression plasmid of FLJ30679 was used to establish FLJ30679 overexpression group (FLJ30679). The effects of altered FLJ30679 expression on the proliferative and migration capacity of OSCC cells were examined by cell counting kit-8 (CCK-8) and transwell migration assays. RTFQ-PCR and Western blot were used to determine the effect of altered FLJ30679 expression on the expression of epithelial-mesenchymal transition (EMT)-related genes in OSCC cells. The effects of altered FLJ30679 expression on the phosphoinositide 3-kinase (PI3K)/protein kinase (AKT) pathway were detected by Western blot. Results: Online query of database showed that FLJ30679 expression was higher in HNSCC tissues compared to normal tissues (P<0.01). HNSCC patients with higher FLJ30679 expression had lower overall survival (P<0.01). The RTFQ-PCR results showed that FLJ30679 was expressed at a higher level in six OSCC cell lines compared with normal cells, and was predominantly localized in the nucleus. The ability of OSCC cells in the SS-FLJ30679 group to proliferate and migrate was significantly lower compared with the SS-NC group (P<0.01). OSCC cells in the FLJ30679 overexpression group had significantly higher proliferative and migratory capacities than those in the vector group (P<0.001). RTFQ-PCR and Western blot results showed that FLJ30679 knockdown resulted in upregulation of mRNA and protein expression levels of E-cadherin (P<0.01) and downregulation of mRNA and protein expression levels of N-cadherin and vimentin (P<0.01). FLJ30679 overexpression resulted in downregulation of protein expression levels of E-cadherin (P<0.01) and upregulation of mRNA and protein expression levels of N-cadherin and vimentin (P<0.05). Western blot results showed that knockdown of FLJ30679 resulted in decreased protein expression levels of phosphorylated-PI3K (p-PI3K) and phosphorylated-AKT (p-AKT) (P<0.001), and overexpression of FLJ30679 resulted in increased protein expression levels of p-PI3K and p-AKT (P<0.01). Conclusion: The expression of FLJ30679 was increased in OSCC tissues and cells. It promoted the proliferation and migration ability of OSCC cells, which may be caused by FLJ30679 promoting EMT via PI3K/AKT pathway.

Key words: Oral squamous cell carcinoma, Long noncoding RNA, FLJ30679, Cell proliferation, Cell migration, Epithelial-mesenchymal transition

中图分类号:

R739.85

孙榕婍, 宋宁, 郑文甜, 张馨月, 李敏敏, 公慧, 蒋英英. 长链非编码RNA FLJ30679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, 2024, 34(5): 439-450.

SUN Rongqi, SONG Ning, ZHENG Wentian, ZHANG Xinyue, LI Minmin, GONG Hui, JIANG Yingying. Effect of long noncoding RNA FLJ30679 on proliferation and migration of oral squamous cell carcinoma cells[J]. China Oncology, 2024, 34(5): 439-450.

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Gene	Primer sequence
FLJ30679	Forward: 5’-GCAAATGTGACCCGCCTCCTAC-3’
	Reverse: 5’-GCTACACGCTCTGCCCTTTCTC-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’
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’

Name	Sequence
FLJ30679 Smart Silencer	GGTGTCCGCAACATTAATA
	GAAGTGCCTCAGAGCATTT
	GACCTCCTTAGGACATAAT
	GCCTACTTAACAGATACAGA
	GCTAACTATGTTTCACAGCA
	GCACAGACCGTCACGTCCAT