SFRP1对头颈鳞状细胞癌增殖和侵袭的影响及其机制研究

杨中玉; 李薇; 朱光正; 赵文懿; 徐静

doi:10.16766/j.cnki.issn.1674-4152.003285

SFRP1对头颈鳞状细胞癌增殖和侵袭的影响及其机制研究

doi: 10.16766/j.cnki.issn.1674-4152.003285

杨中玉^{1, 2},
李薇¹,
朱光正²,
赵文懿^{1, 2},
徐静^1, ,

1.
蚌埠医学院第一附属医院整形外科，安徽蚌埠 233004
2.
蚌埠医学院组织移植实验室，安徽蚌埠 233000

基金项目:

安徽高校自然科学研究项目 KJ2020A0576

蚌埠医学院科技项目 2020byzd137

蚌埠医学院研究生科研创新计划项目 Byycxz21086

详细信息

通讯作者:
徐静，E-mail: xj.69@163.com

中图分类号: R739.91 R730.43
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- 被引次数: 0
出版历程
- 收稿日期: 2023-02-11
- 网络出版日期: 2024-01-29

Effect of SFRP1 on the proliferation and invasion of head and neck squamous cell carcinoma and its mechanism

YANG Zhongyu^{1, 2},
LI Wei¹,
ZHU Guangzheng²,
ZHAO Wenyi^{1, 2},
XU Jing^{1
, ,}

1.
Department of Plastic Surgery, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China

摘要

摘要: 目的通过研究分泌型卷曲相关蛋白1(SFRP1)对头颈鳞状细胞癌(HNSC)增殖、迁移的影响及机制，观测头颈鳞状细胞癌在体外的生物学活动，对治疗头颈鳞状细胞癌提出新的治疗方案。方法使用生物信息学分析SFRP1在肿瘤中的表达水平及与肿瘤侵袭和增殖的相关性，研究头颈鳞状细胞癌与SFRP1的关系。将头颈鳞状细胞癌细胞SCL-1利用转染敲低SFRP1的表达量，使用干扰SFRP1组(small interfering RNA)和对照组(si-NC)进行CCK-8实验分析、划痕实验分析和Transwell实验分析，分别检测细胞增殖、迁移情况。通过Western blotting实验检测SCL-1细胞中Wnt/β-catenin信号转导通路及C-myc、cyclinD1的表达水平。结果与对照组头颈鳞状癌细胞相比，皮肤癌细胞SFRP1蛋白表达量降低(P < 0.01)；对照组头颈鳞状细胞癌组在24 h迁移面积比为0.602±0.019，而空白组为0.419±0.053、对照组为0.435±0.009，si-SFRP1组迁移能力明显高于对照组和空白组(P < 0.01)；在Transwell实验中，24 h后si-SFRP1组侵袭的细胞数为(723.333±2.048)个，而空白组为(332.002±9.930)个、对照组为(343.332±32.504)个，si-SFRP1组侵袭能力明显高于对照组和空白组(P < 0.01)；SCL-1中敲低SFRP1通过Wnt/β-catenin及下游C-myc、cyclinD1表达水平升高(P < 0.01)。通过生物信息学数据统计分析，发现敲低SFRP1蛋白，可促进头颈鳞状细胞癌的增殖，对临床分析有一定的指导作用。结论 SFRP1在头颈鳞状细胞癌细胞内低表达，同时通过Wnt/β-catenin信号转导通路促进细胞的增殖和迁移能力。
- 分泌型卷曲相关蛋白1 /
- 头颈鳞状细胞癌 /
- Wnt/β-catenin信号转导通路 /
- 肿瘤转移
Abstract: Objective To observe the biological activities of head and neck squamous cell carcinoma (HNSC) in vitro by studying the proliferation, migration and mechanism of secreted frizzled related protein 1 (SFRP1), and to propose new therapeutic options for the treatment of HNSC. Methods The association of SFRP1 with head and neck squamous cell carcinoma was investigated using bioinformatics analysis of the correlation between SFRP1 expression levels in tumors and tumor invasion and proliferation. Skin cancer cells SCL-1 were knocked down the expression of SFRP1 using transfection, and the effect of cell proliferation, migration was examined using interference with SFRP1 group (si-SFRP1) and control group (si-NC) for CCK-8 assay analysis, scratch assay analysis and Transwell assay analysis, respectively. The Wnt/β-catenin signaling pathway and the expression levels of C-myc and cyclinD1 in SCL-1 cells were detected by Western blotting assay. Results Compared with the control head and neck squamous carcinoma cells, skin cancer cells were found to be low expressing SFRP1 protein (P < 0.010); the migration area ratio of the low expressing SFRP1 protein neck squamous cell carcinoma group was 0.602±0.019 at 24 hours, compared with 0.419±0.053 in the blank group and 0.435±0.009 in the control group, and the migration ability of the si-SFRP1 group was significantly higher than that of the control and blank groups (P < 0.01); in the Transwell experiment, the number of cells invaded by the si-SFRP1 group after 24 hours was 723.333±2.048, compared with 332.002±9.930 in the blank group and 343.332±32.504 in the control group, and the invasion ability of the si-SFRP1 group was significantly higher than that of the control and blank group (P < 0.01); knockdown of SFRP1 in SCL-1 was elevated through Wnt/β-catenin and downstream C-myc and cyclinD1 expression levels (P < 0.01). Statistical analysis of bioinformatics data revealed that knockdown of SFRP1 protein had promoted the proliferation of head and neck squamous cell carcinoma, which is a guide for clinical analysis. Conclusion SFRP1 is lowly expressed in skin cancer cells, and also promotes cell proliferation and migration ability through the Wnt/β-catenin signaling pathway.
- Recombinant secreted frizzled related protein 1 /
- Head and neck squamous cell carcinoma /
- Wnt/β-catenin signal transduction pathway /
- Tumor metastasis

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图 1 TCGA数据库分析细胞癌、癌旁数据及患者预后结果

注：A为SFRP1的差异表达数据通过分析TCGA-HNSC的鳞状细胞癌及癌旁数据，肿瘤组织中位数表达低于癌旁组织(t=8.234, P < 0.001)；B为SFRP1的表达量与患者预后通过survival和survminer包进行分析，高表达SFRP1具有较好的生存周期(χ²=13.202, P < 0.001)；C为SFRP1在不同性别、年龄、分期、原发肿瘤浸润、TMN分型中表达量的热图。

Figure 1. The analysis of cell carcinoma, paracancer data, and patient outcomes using the TCGA database

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图 2 SCL-1细胞实验结果和SFRP1通过TCGA数据库分析

注：A为划痕实验检测SCL-1细胞迁移实验结果图，敲低组si-SFRP1迁移能力增强(F=27.150, P=0.001)；B为Transwell小室实验检测细胞迁移实验结果, 敲低SFRP1后细胞侵袭能力增强(F=256.842, P < 0.001)；C为划痕实验统计图(P < 0.05)；D为Transwell小室实验统计图(P < 0.05)。

Figure 2. Results of SCL-1 cell experiment and TCGA database analysis of SFRP1

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图 3 SCL-1细胞蛋白印迹实验和增殖实验

注：A为Western blotting检测SCL-1细胞β-Catenin、β-Tublin、c-Myc、SFRP1、Cyclin D1蛋白表达情况(F=372.701, P < 0.001)；B为Western blotting实验统计图，敲低组中β-Catenin、β-Tublin、c-Myc、SFRP1、Cyclin D1蛋白表达均升高；C为CCK8检测SCL-1细胞增殖结果图，si-SFRP1组增殖能力增强(F=44.270, P < 0.001)。

Figure 3. The results of western blot and proliferation for SCL-1 cells

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表 1 SFRP1基因表达与各免疫细胞浸润比例的相关性

Table 1. Correlation analysis between SFRP1 expression and immune cell infiltration ratio

细胞	Rho值	P值
B-cells-naive	0.111	0.013
Mast-cells-resting	0.135	0.002
Dendritic-cells-activated	-0.166	< 0.001
CD4T-memory-resting	0.127	0.004

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表 2 SFRP1基因表达与各免疫检查点基因表达的相关性

Table 2. Correlation analysis between the expression of SFRP1 and immune checkpoint genes

免疫检查点基因	Rho值	P值
CTLA4	0.197	< 0.001
TIGIT	0.138	0.002
PDCD1	0.118	0.008

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表 3 SFRP1基因高表达对比低表达的GSEA富集分析

Table 3. GSEA enrichment analysis comparing high versus low expression of SFRP1

通路	富集分数	归一化富集分数	P值
B_CELL_RECEPTOR_SIGNALING_PATHWAY	0.498	1.658	0.003
T_CELL_RECEPTOR_SIGNALING_PATHWAY	0.500	1.694	< 0.001
NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY	0.514	1.878	< 0.001
PRIMARY_IMMUNODEFICIENCY	0.541	1.516	0.029

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参考文献(18)

[1]	CHANG M S, AZIN M, DEMEHRI S. Cutaneous squamous cell carcinoma: the frontier of cancer immunoprevention[J]. Annu Rev Pathol, 2022, 17: 101-119. doi: 10.1146/annurev-pathol-042320-120056
[2]	ALYOUSSEF A, TAHA M. Blocking Wnt as a therapeutic target in mice model of skin cancer[J]. Arch Dermatol Res, 2019, 311(8): 595-605. doi: 10.1007/s00403-019-01939-4
[3]	GEORGY S R, RUDIATMOKO D R, AUDEN A, et al. Identification of a novel GRHL3/HOPX/Wnt/beta-Catenin proto-oncogenic axis in squamous cell carcinoma of the esophagus[J]. Cell Mol Gastroenterol Hepatol, 2022, 15(5): 1051-1069.
[4]	LIANG J, LIU L, TANG H, et al. UVB-induced SFRP1 methylation potentiates skin damage by promoting cell apoptosis and DNA damage[J]. Exp Dermatol, 2022, 31(9): 1443-1453. doi: 10.1111/exd.14621
[5]	FANE M E, CHHABRA Y, ALICEA G M, et al. Stromal changes in the aged lung induce an emergence from melanoma dormancy[J]. Nature, 2022, 606(7913): 396-405. doi: 10.1038/s41586-022-04774-2
[6]	BELLEI B, CAPUTO S, MIGLIANO E, et al. Simultaneous targeting tumor cells and cancer-associated fibroblasts with a paclitaxel-hyaluronan bioconjugate: in vitro evaluation in non-Melanoma skin cancer[J]. Biomedicines, 2021, 9(6): 597. DOI: 10.3390/biomedicines9060597.
[7]	BAHARUDIN R, TIENG F, LEE L H, et al. Epigenetics of SFRP1: the dual roles in human cancers[J]. Cancers (Basel), 2020, 12(2): 445. DOI: 10.3390/cancers12020445.
[8]	MOSA M H, MICHELS B E, MENCHE C, et al. A Wnt-Induced phenotypic switch in cancer-associated fibroblasts inhibits EMT in colorectal cancer[J]. Cancer Res, 2020, 80(24): 5569-5582. doi: 10.1158/0008-5472.CAN-20-0263
[9]	DOBRE M, SALVI A, PELISENCO I A, et al. Crosstalk between DNA methylation and gene mutations in colorectal cancer[J]. Front Oncol, 2021, 11: 697409. DOI: 10.3389/fonc.2021.697409.
[10]	MENYHART O, FEKETE J T, GYORFFY B. Gene expression indicates altered immune modulation and signaling pathway activation in ovarian cancer patients resistant to topotecan[J]. Int J Mol Sci, 2019, 20(11): 2750. DOI: 10.3390/ijms20112750.
[11]	MIAO Y, WANG J, LI Q, et al. Prognostic value and immunological role of PDCD1 gene in pan-cancer[J]. Int Immunopharmacol, 2020, 89(Pt B): 107080. DOI: 10.1016/j.intimp.2020.10708.
[12]	CHAUVIN J M, ZAROUR H M. TIGIT in cancer immunotherapy[J]. J Immunother Cancer, 2020, 8(2): e000957. DOI: 10.1136/jitc-2020-000957.
[13]	GAUGER K J, CHENAUSKY K L, MURRAY M E, et al. SFRP1 reduction results in an increased sensitivity to TGF-beta signaling[J]. BMC Cancer, 2011, 11: 59. doi: 10.1186/1471-2407-11-59
[14]	KUBILIUTE R, ZALIMAS A, BAKAVICIUS A, et al. Clinical significance of ADAMTS19, BMP7, SIM1, and SFRP1 promoter methylation in renal clear cell carcinoma[J]. Onco Targets Ther, 2021, 14: 4979-4990. doi: 10.2147/OTT.S330341
[15]	ZHANG H, SUN D, QIU J, et al. SFRP1 inhibited the epithelial ovarian cancer through inhibiting Wnt/beta-catenin signaling[J]. Acta Biochim Pol, 2019, 66(4): 393-400.
[16]	PAN X, MA X. A novel six-gene signature for prognosis prediction in ovarian cancer[J]. Front Genet, 2020, 11: 1006. DOI: 10.3389/fgene.2020.01006.
[17]	SUNKARA R R, SARATE R M, SETIA P, et al. SFRP1 in skin tumor initiation and cancer stem cell regulation with potential implications in epithelial cancers[J]. Stem Cell Reports, 2020, 14(2): 271-284. doi: 10.1016/j.stemcr.2019.12.006
[18]	戴思远, 蒋婕, 韩瑞, 等. 口腔鳞状细胞癌中乙酰肝素酶、E-cadherin、N-cadherin的表达及其意义[J]. 中华全科医学, 2022, 20(6): 948-951. doi: 10.16766/j.cnki.issn.1674-4152.002495 DAI S Y, JIANG J, HAN R, et al. Expression and significance of heparanase, E-cadherin, and N-cadherin in oral squamous cell carcinoma[J]. Chinese Journal of General Practice, 2022, 20(6): 948-951. doi: 10.16766/j.cnki.issn.1674-4152.002495