敲低Sec31A对头颈鳞状细胞癌恶性表型的影响

doi:10.13591/j.cnki.kqyx.2024.07.002

摘要/Abstract

摘要：

目的探讨敲低Sec31A对头颈鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)恶性表型的影响及可能机制。方法从TCGA数据库中获得HNSCC组织与癌旁组织的转录组测序数据并比较Sec31A的表达水平,免疫组化染色分析Sec31A在HNSCC组织中的表达情况,利用Kaplan-Meier生存分析比较Sec31A与HNSCC患者预后的关系;在HNSCC细胞系HN6、HN4中转染小干扰质粒si-Sec31A、si-NC,通过CCK-8实验、平板克隆实验、划痕愈合实验和Transwell实验检测Sec31A敲低后对HNSCC细胞生物学行为的影响;通过蛋白免疫印迹(Western Blot,WB)实验检测HN6及HN4敲低Sec31A后细胞中PI3K/AKT/mTOR通路相关蛋白表达水平的变化;构建HN6-siSec31A和HN6-siNC的转染稳定细胞株接种于裸鼠皮下进一步验证Sec31A在体内的成瘤作用。结果 TCGA数据显示Sec31A在HNSCC组织中高于癌旁正常组织(P<0.01),且Sec31A高表达与患者的预后不良显著相关(P<0.05),免疫组化染色显示Sec31A在HNSCC中表达强于正常组织;在HN6和HN4细胞中,敲低Sec31A后,细胞的增殖、迁移和侵袭能力明显弱于对照组;通过WB实验发现,HN6、HN4转染si-Sec31A后,p-PI3K、p-AKT、p-mTOR蛋白表达水平显著降低;HN6敲低Sec31A后在裸鼠皮下的移植瘤体积明显小于对照组。结论敲低Sec31A后可以抑制HNSCC细胞增殖、迁移和侵袭的能力,可能是通过PI3K/AKT/mTOR通路发挥作用。

关键词: Sec31A, 头颈鳞状细胞癌, 分子靶向治疗, PI3K/AKT/mTOR通路

Abstract:

Objective To explore the impact of knocking down Sec31A on the malignant phenotype of head and neck squamous cell carcinoma(HNSCC) and its possible mechanisms. Methods Transcriptome sequencing data of HNSCC tissues and adjacent tissues were obtained from the TCGA database, and the expression levels of Sec31A were compared. Immunohistochemical staining was used to analyze the expression of Sec31A in HNSCC tissues. Kaplan-Meier survival analysis was used to compare the relationship between Sec31A and the prognosis of HNSCC patients. Small interfering plasmids si-Sec31A and si-NC were transfected into HNSCC cell lines HN6 and HN4, and the impact of knocking down Sec31A on the biological behavior of HNSCC cells was detected through CCK-8 experiments, plate cloning experiments, scratch healing experiments, and Transwell experiments. Changes in the expression levels of PI3K/AKT/mTOR pathway related proteins in cells were detected after knocking down Sec31A with HN6 and HN4 through Western Blot(WB)experiments. Stable transfected cell lines of HN6 siSec31A and HN6 siNC were constructed and inoculated subcutaneously in nude mice to further verify the tumorigenic effect of Sec31A in vivo. Results TCGA data showed that Sec31A was higher in HNSCC tissues than in adjacent normal tissues(P<0.01), and high expression of Sec31A was significantly correlated with poor prognosis in patients(P<0.05). Immunohistochemical staining showed that Sec31A was expressed stronger in HNSCC tissues than in normal tissues. In HN6 and HN4 cells, knocking down Sec31A resulted in significantly weaker proliferation, migration, and invasion abilities compared to the control group. Through WB experiments, it was found that transfection of si-Sec31A with HN6 and HN4 significantly reduced the expression levels of p-PI3K, p-AKT, and p-mTOR proteins. After knocking down Sec31A with HN6, the transplanted tumor volume in nude mice was significantly smaller than that in the control group. Conclusion Knocking down Sec31A can inhibit the proliferation, migration and invasion of HNSCC cells, possibly through the PI3K/AKT/mTOR pathway.

Key words: Sec31A, head and neck squamous cell carcinoma, molecular targeted therapy, PI3K/AKT/mTOR pathway

中图分类号:

R739.8

何垚, 赵振远, 高腾, 林鹏, 陈以任, 宋晓萌. 敲低Sec31A对头颈鳞状细胞癌恶性表型的影响[J]. 口腔医学, 2024, 44(7): 487-493.

HE Yao, ZHAO Zhenyuan, GAO Teng, LIN Peng, CHEN Yiren, SONG Xiaomeng. The effect of knocking down Sec31A on the malignant phenotype of HNSCC[J]. Stomatology, 2024, 44(7): 487-493.

图/表 7

图1

图2

图3

图4

图5

图6

图7

参考文献 26

[1]	Siegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023[J]. CA A Cancer J Clinicians, 2023, 73(1):17-48.
[2]	Gangane NM, Ghongade PV, Patil BU, et al. Oral cavity cancer incidence and survival trends: A population-based study[J/OL]. J Cancer Res Ther, (2024-01-22)[2024-03-17]. https://pubmed.ncbi.nlm.nih.gov/38261454/.
[3]	Chamoli A, Gosavi AS, Shirwadkar UP, et al. Overview of oral cavity squamous cell carcinoma: Risk factors, mechanisms, and diagnostics[J]. Oral Oncol, 2021, 121: 105451.
[4]	Tran Q, Maddineni S, Arnaud EH, et al. Oral cavity cancer in young, non-smoking, and non-drinking patients: A contemporary review[J]. Crit Rev Oncol Hematol, 2023, 190: 104112.
[5]	Bhatia A, Burtness B. Treating head and neck cancer in the age of immunotherapy: A 2023 update[J]. Drugs, 2023, 83(3):217-248. doi: 10.1007/s40265-023-01835-2 pmid: 36645621
[6]	Afshari K, Sohal KS. Potential alternative therapeutic modalities for management head and neck squamous cell carcinoma: A review[J]. Cancer Control, 2023, 30: 10732748231185003.
[7]	Johnson DE, Burtness B, Leemans CR, et al. Head and neck squamous cell carcinoma[J]. Nat Rev Dis Primers, 2020, 6(1):92. doi: 10.1038/s41572-020-00224-3 pmid: 33243986
[8]	Bhat GR, Hyole RG, Li J. Head and neck cancer: Current challenges and future perspectives[J]. Adv Cancer Res, 2021, 152: 67-102. doi: 10.1016/bs.acr.2021.05.002 pmid: 34353444
[9]	Halperin D, Kadir R, Perez Y, et al. SEC31A mutation affects ER homeostasis, causing a neurological syndrome[J]. J Med Genet, 2019, 56(3):139-148. doi: 10.1136/jmedgenet-2018-105503 pmid: 30464055
[10]	Long LY, Wei J, Lim SA, et al. CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity[J]. Nature, 2021, 600(7888):308-313.
[11]	Cheng FF, Yu J, Zhang XY, et al. CircSEC31A promotes the malignant progression of non-small cell lung cancer through regulating SEC31A expression via sponging miR-376a[J]. Cancer Manag Res, 2020, 12: 11527-11539.
[12]	Jin MM, Shi CZ, Hua Q, et al. High circ-SEC31A expression predicts unfavorable prognoses in non-small cell lung cancer by regulating the miR-520a-5p/GOT-2 axis[J]. Aging (Albany NY), 2020, 12(11):10381-10397.
[13]	阴艺娜, 苏民, 林梓航, 等. 环状RNA-SEC31A对胰腺癌细胞侵袭、迁移的影响及其机制研究[J]. 中华胰腺病杂志, 2023, 9(2):99-107.
[14]	喻言, 钟红珊. 环状RNA SEC31A促进肝癌细胞增殖和有氧糖酵解[J]. 中国医科大学学报, 2021, 50(4):302-306, 311.
[15]	Ong YS, Tang BL, Loo LS, et al. p125A exists as part of the mammalian Sec13/Sec31 COPII sub complex to facilitate ER-Golgi transport[J]. J Cell Biol, 2010, 190(3):331-345.
[16]	Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[J]. Nat Cell Biol, 2004, 6(11):1122-1128. pmid: 15467718
[17]	Kim DH, Sarbassov DD, Ali SM, et al. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR[J]. Mol Cell, 2003, 11(4):895-904.
[18]	Dibble CC, Cantley LC. Regulation of mTORC1 by PI3K signaling[J]. Trends Cell Biol, 2015, 25(9):545-555. doi: 10.1016/j.tcb.2015.06.002 pmid: 26159692
[19]	Savova MS, Mihaylova LV, Tews D, et al. Targeting PI3K/AKT signaling pathway in obesity[J]. Biomed Pharmacother, 2023, 159: 114244.
[20]	董硕, 汤春波. PI3K/AKT信号通路在2型糖尿病患者种植体骨结合中作用机制的研究进展[J]. 口腔医学, 2022, 42(11):1026-1030,1035.
[21]	Xu F, Na LX, Li YF, et al. Roles of the PI3K/AKT/mTOR signalling pathways in neurodegenerative diseases and tumours[J]. Cell Biosci, 2020, 10(1):54. doi: 10.1186/s13578-020-00416-0 pmid: 32266056
[22]	Porta C, Paglino C, Mosca A. Targeting PI3K/akt/mTOR signaling in cancer[J]. Front Oncol, 2014, 4: 64. doi: 10.3389/fonc.2014.00064 pmid: 24782981
[23]	Xu ZR, Han X, Ou DM, et al. Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy[J]. Appl Microbiol Biotechnol, 2020, 104(2):575-587. doi: 10.1007/s00253-019-10257-8 pmid: 31832711
[24]	Ersahin T, Tuncbag N, Cetin-Atalay R. The PI3K/AKT/mTOR interactive pathway[J]. Mol Biosyst, 2015, 11(7):1946-1954. doi: 10.1039/c5mb00101c pmid: 25924008
[25]	Glaviano A, Foo ASC, Lam HY, et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer[J]. Mol Cancer, 2023, 22(1):138. doi: 10.1186/s12943-023-01827-6 pmid: 37596643
[26]	Tewari D, Patni P, Bishayee A, et al. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: A novel therapeutic strategy[J]. Semin Cancer Biol, 2022, 80: 1-17.