SFXN3在头颈部鳞状细胞癌中的表达及其对细胞增殖的影响

doi:10.3969/j.issn.1674-8115.2024.04.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (4): 427-434.doi: 10.3969/j.issn.1674-8115.2024.04.002

• 肿瘤基础专题 • 上一篇

SFXN3在头颈部鳞状细胞癌中的表达及其对细胞增殖的影响

安俊伊(), 陈必颖, 陈循睿, 尹姗姗, 边洲亮, 刘峰()

上海交通大学医学院附属第九人民医院肿瘤科，上海 201999

收稿日期:2023-11-04 接受日期:2024-01-26 出版日期:2024-04-28 发布日期:2024-04-28
通讯作者: 刘峰 E-mail:anjunyi1998@163.com;nuanliu@126.com
作者简介:安俊伊（1998—），女，硕士生；电子信箱：anjunyi1998@163.com。
基金资助:
上海交通大学医学院附属第九人民医院临床研究助推计划(JYLJ202019)

SFXN3 expression in head and neck squamous cell carcinoma and its effect on cell proliferation

AN Junyi(), CHEN Biying, CHEN Xunrui, YIN Shanshan, BIAN Zhouliang, LIU Feng()

Department of Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China

Received:2023-11-04 Accepted:2024-01-26 Online:2024-04-28 Published:2024-04-28
Contact: LIU Feng E-mail:anjunyi1998@163.com;nuanliu@126.com
Supported by:
Clinical Research Promotion Program of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine(JYLJ202019)

摘要/Abstract

摘要：

目的·分析线粒体相关基因SFXN3（sideroflexin 3）在头颈部鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）中的表达及其对细胞增殖的影响。方法·从公共数据库获取与线粒体相关的基因和TCGA-HNSCC数据集，使用生物信息学方法筛选出目标基因SFXN3。使用UALCAN数据库分析SFXN3在HNSCC患者样本中的表达，并根据TCGA-HNSCC队列和GEO队列（GSE65858、GSE41613和GSE27020）对不同SFXN3表达水平的患者进行生存分析，使用TCGA-HNSCC队列和GEO队列（GSE40020、GSE210287）在治疗有应答和无应答的患者之间比较SFXN3表达水平差异。通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）在收集的HNSCC癌和癌旁组织验证SFXN3的表达情况，并检测SFXN3在人正常口腔上皮细胞和HNSCC肿瘤细胞系中的表达水平。构建稳定敲低SFXN3的HNSCC细胞株，使用Incucyte活细胞成像分析系统和平板克隆形成实验观察SFXN3对HNSCC细胞增殖能力的影响。将稳定敲低SFXN3的细胞和对照组细胞的总RNA进行转录组测序，对敲低组相比对照组差异表达下调的基因进行通路富集分析。结果·SFXN3在HNSCC患者肿瘤组织中高表达（P=0.000），SFXN3高表达组患者预后较差（均P<0.05）；治疗无应答者SFXN3表达水平高于有应答者（P=0.008），提示不良预后。并且在收集的HNSCC肿瘤组织和HNSCC细胞系上验证了SFXN3的高表达（均P<0.05）。SFXN3的表达水平敲低后，HNSCC细胞的增殖能力下降，平板克隆形成数目减少（均P<0.05）。RNA测序显示，SFXN3敲低组的HNSCC细胞差异表达下调的基因在DNA复制、细胞周期、线粒体翻译、线粒体RNA代谢过程、线粒体基因表达等通路富集。结论·SFXN3在HNSCC中高表达，与患者预后负相关。HNSCC细胞敲低SFXN3后增殖能力和平板克隆形成能力受到抑制，可能通过影响线粒体功能发挥作用。

关键词: SFXN3, 头颈部鳞状细胞癌, 线粒体, 细胞增殖

Abstract:

Objective ·To analyze the expression of mitochondrial related gene SFXN3 (sideroflexin 3) in head and neck squamous cell carcinoma (HNSCC) and its effect on cell proliferation. Methods ·Mitochondrial related genes and TCGA-HNSCC dataset were obtained from public databases to identify the target gene SFXN3 by using bioinformatic methods. The expression of SFXN3 in HNSCC patient samples was analysed by using the UALCAN database, and survival analyses of patients with different SFXN3 expression levels were performed according to TCGA-HNSCC cohort and GEO cohorts (GSE65858, GSE41613 and GSE27020). By using TCGA-HNSCC cohort and GEO cohorts (GSE40020, GSE210287), the differences in SFXN3 expression levels between therapeutic responders and non-responders were compared. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of SFXN3 in the collected HNSCC tumor and para-tumor tissues, and the expression level of SFXN3 in human normal oral epithelial cells and HNSCC tumor cell lines was detected. With the construction of stable-SFXN3-knockdown HNSCC cell lines, the effect of SFXN3 on the proliferation ability of HNSCC cells was observed by using the Incucyte live-cell imaging analysis system and plate colony formation assay. Transcriptome sequencing was performed on the total RNA of stably-SFXN3-knockdown cells and control cells, and pathway enrichment analysis was performed on the genes with differentially down-regulated expression in knockdown group compared with control group. Results ·SFXN3 was highly expressed in tumor tissues of HNSCC patients (P=0.000), and the prognosis of patients in the SFXN3-high-expression group was poor (all P<0.05). The expression level of SFXN3 in the non-responders was higher than that in the responders (P=0.008), indicating an unfavorable prognosis. High expression of SFXN3 was verified in the collected HNSCC tumor tissues and HNSCC cell lines (all P<0.05). After the knockdown of SFXN3 expression, the proliferation ability of HNSCC cells decreased, and the number of plate clones decreased (all P<0.05). RNA sequencing showed that the differentially expressed down-regulated genes in HNSCC cells in the SFXN3-knockdown group were enriched in DNA replication, cell cycle, mitochondrial translation, mitochondrial RNA metabolic process and mitochondrial gene expression pathways. Conclusion ·SFXN3 is highly expressed in HNSCC and it has negative correlation with the prognosis of patients. The proliferation ability and plate colony formation ability are inhibited in SFXN3-knockdown HNSCC cells, and these may work by affecting mitochondria function.

Key words: SFXN3, head and neck squamous cell carcinoma, mitochondria, cell proliferation

中图分类号:

安俊伊, 陈必颖, 陈循睿, 尹姗姗, 边洲亮, 刘峰. SFXN3在头颈部鳞状细胞癌中的表达及其对细胞增殖的影响[J]. 上海交通大学学报（医学版）, 2024, 44(4): 427-434.

AN Junyi, CHEN Biying, CHEN Xunrui, YIN Shanshan, BIAN Zhouliang, LIU Feng. SFXN3 expression in head and neck squamous cell carcinoma and its effect on cell proliferation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(4): 427-434.

图/表 7

表1 qRT-PCR引物序列（5′→3′）

Tab 1 Primer sequences for qRT-PCR （5′→3′）

Primer	Forward	Reverse
GAPDH	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG
ACTB	TGACGTGGACATCCGCAAAG	CTGGAAGGTGGACAGCGAGG
SFXN3	GGGTGAATTGCCTTTAGACATCA	GCAGCAGATTTCGAGGATCAGT

表2 shRNA引物序列（5′→3′）

Tab 2 Primer sequences of shRNA （5′→3′）

Primer	Forward	Reverse
sh-SFXN3-1	GATCCGGCTGGGACCAAAGTACTTTCCCTCGAGGGAAAGTACTTTGGTCCCAGCCTTTTTTG	AATTCAAAAAAGGCTGGGACCAAAGTACTTTCCCTCGAGGGAAAGTACTTTGGTCCCAGCCG
sh-SFXN3-2	GATCCGCAGCGTTGAAGTGGTCTACTACTCGAGTAGTAGACCACTTCAACGCTGCTTTTTTG	AATTCAAAAAAGCAGCGTTGAAGTGGTCTACTACTCGAGTAGTAGACCACTTCAACGCTGCG

图1 目标基因 SFXN3 的鉴定Note： A. The identification of DEGs between the tumor and normal tissues in TCGA-HNSCC dataset. The red dots represent up-regulated DEGs of tumor tissues and the blue dots represent down-regulated DEGs of tumor tissues, compared with the normal tissues. |log2FC|>1, Padjust<0.01. B. Venn plot of the common genes between TCGA-HNSCC DEGs (up-regulated DEGs/down-regulated DEGs) and the mitochondrial related genes. C. The result of univariate Cox regression.

Fig 1 Identification of targeted gene SFXN3

图2 SFXN3 在HNSCC中高表达且与不良预后显著相关Note： A. SFXN3 mRNA and protein expression between HNSCC tumor tissues and normal tissues analysed by the UALCAN database. B. Five-year survival curves of HNSCC patients with high SFXN3 expression and low SFXN3 expression in TCGA-HNSCC cohort and GEO cohorts (GSE65858, GSE41613 and GSE27020). C. Differences in SFXN3 expression between therapeutic responders (R) and non-responders (NR) based on TCGA-HNSCC cohort, GSE40020 cohort and GSE210287 cohort. D. Relative SFXN3 expression in para-tumor and tumor tissues from HNSCC patients detected by qRT-PCR. E. Relative SFXN3 expression in different HNSCC cell lines detected by qRT-PCR. Mean±SEM. ①P=0.000, compared with the normal tissues. ②P=0.000, ③P=0.005, ④P=0.010, ⑤P=0.007, compared with the SFXN3-low-expression patients. ⑥P=0.008, compared with the R group. ⑦P=0.003, compared with the para-tumor tissues. ⑧P=0.014, ⑨P=0.011, ⑩P=0.000, compared with the HOK cells.

Fig 2 High expression of SFXN3 in HNSCC and its significant association with poor prognosis

图3 稳定敲低 SFXN3 HNSCC细胞株的构建和验证Note： A. Detection of relative SFXN3 expression in SCC9, CAL27 and HN30 cells after SFXN3 knockdown by qRT-PCR. B. Detection of SFXN3 expression in SCC9, CAL27 and HN30 cells after SFXN3 knockdown by Western blotting. Mean±SD. ①P=0.000, compared with the sh-Ctrl group.

Fig 3 Establishment and verification of stable-SFXN3-knowdown HNSCC cell lines

图4 敲低 SFXN3 对HNSCC细胞增殖的影响Note： A. Growth curves of SCC9, CAL27 and HN30 cells after SFXN3 knockdown detected by Incucyte system. B The results and statistical analysis of plate colony formation assay in SCC9, CAL27 and HN30 cells after SFXN3 knockdown. Mean±SEM. ①P=0.010, ②P=0.009, ③P=0.000, ④P=0.002, compared with the sh-Ctrl group.

Fig 4 Effect of SFXN3 knockdown on the proliferation of HNSCC cells

图5 敲低 SFXN3 后SCC9细胞差异下调基因的通路富集分析Note： A/B. KEGG (A) and GSEA enrichment analysis (B) of differentially expressed down-regulated genes in SCC9 cells after SFXN3 knockdown.

Fig 5 Pathway enrichment analysis of differentially expressed down-regulated genes in SFXN3-knockdown SCC9 cells

参考文献 22

1	MODY M D, ROCCO J W, YOM S S, et al. Head and neck cancer[J]. Lancet, 2021, 398(10318): 2289-2299.
2	JOHNSON D E, BURTNESS B, LEEMANS C R, et al. Head and neck squamous cell carcinoma[J]. Nat Rev Dis Primers, 2020, 6(1): 92.
3	LEEMANS C R, BRAAKHUIS B J M, BRAKENHOFF R H. The molecular biology of head and neck cancer[J]. Nat Rev Cancer, 2011, 11(1): 9-22.
4	SUNG H, FERLAY J, SIEGEL R L, et al. Global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.
5	KORDBACHEH F, FARAH C S. Current and emerging molecular therapies for head and neck squamous cell carcinoma[J]. Cancers, 2021, 13(21): 5471.
6	PORPORATO P E, FILIGHEDDU N, PEDRO J M B S, et al. Mitochondrial metabolism and cancer[J]. Cell Res, 2018, 28(3): 265-280.
7	CHANG J J, WU H, WU J, et al. Constructing a novel mitochondrial-related gene signature for evaluating the tumor immune microenvironment and predicting survival in stomach adenocarcinoma[J]. J Transl Med, 2023, 21(1): 191.
8	FERLAY J, COLOMBET M, SOERJOMATARAM I, et al. Estimating the global cancer incidence and mortality in 2018: globocan sources and methods[J]. Int J Cancer, 2019, 144(8): 1941-1953.
9	ALSAHAFI E, BEGG K, AMELIO I, et al. Clinical update on head and neck cancer: molecular biology and ongoing challenges[J]. Cell Death Dis, 2019, 10(8): 540.
10	ABATE M, FESTA A, FALCO M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence[J]. Semin Cell Dev Biol, 2020, 98: 139-153.
11	KIM H K, NOH Y H, NILIUS B, et al. Current and upcoming mitochondrial targets for cancer therapy[J]. Semin Cancer Biol, 2017, 47: 154-167.
12	FLEMING M D, CAMPAGNA D R, HASLETT J N, et al. A mutation in a mitochondrial transmembrane protein is responsible for the pleiotropic hematological and skeletal phenotype of flexed-tail (f/f) mice[J]. Genes Dev, 2001, 15(6): 652-657.
13	TIFOUN N, DE LAS HERAS J M, GUILLAUME A, et al. Insights into the roles of the sideroflexins/SLC56 family in iron homeostasis and iron-sulfur biogenesis[J]. Biomedicines, 2021, 9(2): 103.
14	KORY N, WYANT G A, PRAKASH G, et al. SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism[J]. Science, 2018, 362(6416): eaat9528.
15	JIN T F, GE L Q, CHEN J Q, et al. Identification of iron metabolism-related genes as prognostic indicators for papillary thyroid carcinoma: a retrospective study[J]. PeerJ, 2023, 11: e15592.
16	MURASE R, ABE Y, TAKEUCHI T, et al. Serum autoantibody to sideroflexin 3 as a novel tumor marker for oral squamous cell carcinoma[J]. Proteomics Clin Appl, 2008, 2(4): 517-527.
17	CHEN K L, GONG S, FANG X L, et al. Non-coding RNA-mediated high expression of SFXN3 as a prognostic biomarker associated with paclitaxel resistance and immunosuppressive microenvironment in head and neck cancer[J]. Front Immunol, 2022, 13: 920136.
18	ZHENG D Y, LUO S W, WANG S, et al. Construction of a competing endogenous RNA network in head and neck squamous cell carcinoma by pan-cancer analysis[J]. Transl Cancer Res, 2022, 11(9): 3050-3063.
19	CRISCUOLO D, AVOLIO R, MATASSA D S, et al. Targeting mitochondrial protein expression as a future approach for cancer therapy[J]. Front Oncol, 2021, 11: 797265.
20	PU M, WANG J L, HUANG Q K, et al. High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes[J]. Tumour Biol, 2017, 39(7): 1010428317709127.
21	ZHU P, LIU Y Z, ZHANG F L, et al. Human elongation factor 4 regulates cancer bioenergetics by acting as a mitochondrial translation switch[J]. Cancer Res, 2018, 78(11): 2813-2824.
22	DELAUNAY S, PASCUAL G, FENG B H, et al. Mitochondrial RNA modifications shape metabolic plasticity in metastasis[J]. Nature, 2022, 607(7919): 593-603.

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SFXN3在头颈部鳞状细胞癌中的表达及其对细胞增殖的影响

SFXN3 expression in head and neck squamous cell carcinoma and its effect on cell proliferation

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