分选链接蛋白1抑制结直肠癌细胞增殖和迁移的作用和机制研究

doi:10.3969/j.issn.1674-8115.2024.09.007

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (9): 1124-1135.doi: 10.3969/j.issn.1674-8115.2024.09.007

• 论著 · 基础研究 • 上一篇

分选链接蛋白1抑制结直肠癌细胞增殖和迁移的作用和机制研究

钱立恒(), 温凯玲, 廖颖娜, 李书鑫, 聂惠贞()

上海交通大学医学院附属仁济医院上海市肿瘤研究所，肿瘤系统医学全国重点实验室，上海 200240

收稿日期:2024-04-28 接受日期:2024-07-16 出版日期:2024-09-28 发布日期:2024-10-09
通讯作者: 聂惠贞 E-mail:shandianqianliheng@sjtu.edu.cn;hznie@shsci.org
作者简介:钱立恒（1998—），男，硕士生；电子信箱：shandianqianliheng@sjtu.edu.cn。
基金资助:
国家自然科学基金(82273228);上海市自然科学基金(21ZR1461400)

Study on the effect and mechanism of sorting nexin 1 on inhibiting the proliferation and migration of colorectal cancer cells

QIAN Liheng(), WEN Kailing, LIAO Yingna, LI Shuxin, NIE Huizhen()

Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine; State Key Laboratory of Systems Medicine for Cancer, Shanghai 200240, China

Received:2024-04-28 Accepted:2024-07-16 Online:2024-09-28 Published:2024-10-09
Contact: NIE Huizhen E-mail:shandianqianliheng@sjtu.edu.cn;hznie@shsci.org
Supported by:
National Natural Science Foundation of China(82273228);Natural Science Foundation of Shanghai(21ZR1461400)

摘要/Abstract

摘要：

目的·分析分选链接蛋白1（sorting nexin 1，SNX1）在结直肠癌（colorectal cancer，CRC）中的表达，探索其对CRC细胞增殖及迁移的影响及潜在的分子机制。方法·基于癌症基因组图谱（The Cancer Genome Atlas，TCGA）、基因型-组织表达（Genotype-Tissue Expression，GTEx）以及基因表达综合（Gene Expression Omnibus，GEO）数据库中CRC相关的转录组数据和临床病理信息，利用基因集富集分析（Gene Set Enrichment Analysis，GSEA）软件进行富集分析。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qPCR）、蛋白质印迹法（Western blotting）、免疫组织化学染色（immunohistochemistry staining，IHC）检测SNX1在CRC组织和细胞中的表达。使用小干扰RNA（small interfering RNA，siRNA）敲低SNX1的表达，观察SNX1对肿瘤细胞增殖和迁移能力的影响。通过相关性分析探索SNX1影响CRC细胞迁移的潜在分子机制，在SNX1敲低细胞株中进行mRNA水平的初步验证。结果·根据对TCGA中CRC患者以及组织芯片样本数据的分析，发现SNX1在CRC组织中表达下调，且与肿瘤的直径以及是否发生远处转移具有相关性。敲低SNX1能够促进肿瘤细胞的增殖和迁移。在CRC中，SNX1的表达与结肠癌转移相关因子1（metastasis associated in colon cancer 1，MACC1）、间质-上皮转换因子（mesenchymal to epithelial transition factor，MET）以及Notch负相关，敲低SNX1后上述基因表达上调；敲低SNX1后，上皮-间质转化（epithelial to mesenchymal transition，EMT）标志物钙黏蛋白1（cadherin 1，CDH1）表达下调，波形蛋白（vimentin，VIM）、Snail家族转录因子1（Snail family transcriptional repressor 1，SNAI1）表达上调。结论·SNX1在CRC组织中表达显著降低，且与患者预后正相关；SNX1低表达能够促进CRC细胞的增殖和迁移，与MACC1-MET通路、EMT相关；SNX1可作为CRC不良预后的潜在生物标志物和新的治疗靶点。

关键词: 分选链接蛋白1, 结直肠癌, 细胞增殖, 细胞迁移

Abstract:

Objective ·To explore the expression of sorting nexin 1 (SNX1) in colorectal cancer (CRC) and its impact on the proliferation and migration of CRC cells. Methods ·Transcriptomic data and clinical pathological information of CRC were obtained from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Gene Expression Omnibus (GEO) databases for enrichment analysis with Gene Set Enrichment Analysis (GSEA) software. The expression of SNX1 in CRC tissues and cells was detected by quantitative real-time polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry staining (IHC). Small interfering RNA (siRNA) was used to knock down the expression of SNX1 to observe its effect on tumor cell proliferation and migration. Correlation analysis was conducted to explore the potential molecular mechanisms underlying SNX1-mediated CRC cell migration, and mRNA level validation was performed in SNX1 knockdown cell lines. Results ·Analysis of CRC patients data in TCGA and tissue microarrays revealed that SNX1 expression was downregulated in CRC tissues and correlated with tumor diameter and distant metastasis. Knockdown of SNX1 enhanced tumor cell proliferation and migration. The expression of SNX1 was negatively correlated with metastasis associated in colon cancer 1 (MACC1), mesenchymal to epithelial transition factor (MET), and Notch; knockdown of SNX1 led to upregulation of these genes. Silencing SNX1 resulted in the downregulation of the epithelial marker cadherin 1 (CDH1) and the upregulation of vimentin (VIM) and Snail family transcriptional repressor 1 (SNAI1). Conclusion ·SNX1 expression was significantly downregulated in CRC tissues and correlated with patient prognosis. Low expression of SNX1 enhanced the proliferation and migration of CRC cells and was associated with the MACC1-MET pathway and EMT. SNX1 may serve as a potential biomarker for poor prognosis and a novel therapeutic target in CRC.

Key words: sorting nexin 1 (SNX1), colorectal cancer, cell migration, cell metastasis

中图分类号:

R735.3

钱立恒, 温凯玲, 廖颖娜, 李书鑫, 聂惠贞. 分选链接蛋白1抑制结直肠癌细胞增殖和迁移的作用和机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(9): 1124-1135.

QIAN Liheng, WEN Kailing, LIAO Yingna, LI Shuxin, NIE Huizhen. Study on the effect and mechanism of sorting nexin 1 on inhibiting the proliferation and migration of colorectal cancer cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(9): 1124-1135.

图/表 8

表1 siRNA序列

Tab 1 Sequences of siRNA

siRNA	Forward (5′➝3′)	Reverse (5′➝3′)
NC-siRNA	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
SNX1-siRNA1	GAGGAUCAAUUUGAUUUGATT	UCAAAUCAAAUUGAUCCUCTT
SNX1-siRNA2	CAGGCCAACAAUGACUUCUTT	AGAAGUCAUUGUUGGCCUGTT

表2 qPCR引物序列

Tab 2 Primer Sequences for qPCR

Primer	Forward (5′➝3′)	Reverse (5′➝3′)
18s mRNA	TGCGAGTACTCAACACCAACA	GCATATCTTCGGCCCACA
SNX1	AAGCACTCTCAGAATGGCTTC	CGGCCCTCCGTTTTTCAAG
MACC1	GGGTCACAGGTGAACGAGAT	CTGGGTCCTGGCATTCTGTA
MET	ATTTTGCTTTGCCAGTGGTGG	AGCGATGTTGACATGCCACT
NOTCH1	AGAGGCGTGGCAGACTATG	CTGGCACGATTTCCCTGACC
CDH1	GGCTGGACCGAGAGAGTTTC	CGACGTTAGCCTCGTTCTCA
VIM	CTCCCTGAACCTGAGGGAAAC	TTGCGCTCCTGAAAAACTGC
SNAI1	TAGCGAGTGGTTCTTCTGCG	TGCTGGAAGGTAAACTCTGGA

图1 SNX家族在人类肿瘤中的表达情况Note： STAD—stomach adenocarcinoma; LIHC—liver hepatocellular carcinoma; BRCA—breast invasive carcinoma; HNSC—head and neck squamous cell carcinoma; ESCA—esophageal carcinoma; COAD—colon adenocarcinoma; READ—rectum adenocarcinoma; LGG—brain lower grade glioma; GBM—glioblastoma multiforme; THYM—thymoma; DLBC—lymphoid neoplasm diffuse large B-cell lymphoma; PAAD—pancreatic adenocarcinoma; CHOL—cholangiocarcinoma; TGCT—testicular germ cell tumor; LUAD—lung adenocarcinoma; LUSC—lung squamous cell carcinoma; KIRP—kidney renal papillary cell carcinoma; KIRC—kidney renal clear cell carcinoma; SARC—sarcoma; PCPG—pheochromocytoma and paraganglioma; THCA—thyroid carcinoma; SKCM—skin cutaneous melanoma; OV—ovarian serous cystadenocarcinoma; BLCA—bladder urothelial carcinoma; CESC—cervical squamous cell carcinoma and endocervical adenocarcinoma; UCS—uterine carcinosarcoma; UCEC—uterine corpus endometrial carcinoma; KICH—kidney chromophobe; ACC—adrenocortical carcinoma; PRAD—prostate adenocarcinoma.

Fig 1 Expression levels of SNX family in human cancers

图2 SNX1 在CRC数据库中表达情况及其与临床病理信息的相关性分析Note： A. Expression levels of SNX1 mRNA in human cancers. TPM—transcript per million. B. Expression levels of SNX1 mRNA in CRC tissues and normal colon tissues from the TCGA and GTEx databases. C. Expression levels of SNX1 mRNA in CRC tissues and normal colon tissues from the GEO databases. D. Correlation between SNX1 mRNA expression and M stage of patients from the TCGA database. E. Correlation between SNX1 mRNA expression and tumor size in patients from the TCGA database. F. Correlation between SNX1 mRNA expression and overall survival (OS) in patients from the GSE39582 database. G. Correlation between SNX1 mRNA expression and recurrence-free survival (RFS) in patients from the GSE39582 database. ①P=0.000, compared with normal colon tissues; ②P=0.022, compared with M0 groups; ③P=0.041, compared with the ≤1 cm groups.

Fig 2 Expression levels of SNX1 from the CRC database and its correlation with clinical pathological information

图3 SNX1在CRC组织中表达情况及其与临床病理信息的相关性分析Note： A. Expression of SNX1 detected by IHC in CRC tissues and para-carcinoma tissues from CRC patients. B. SNX1 staining was divided into four grades according to the depth of tissue microarray IHC staining. The two lower grades were grouped as the low-expression group, while the two higher grades were classified as the high-expression group. C. Expression of SNX1 was decreased in 54% of CRC tissues in tissue microarrays, by comparing IHC grades of CRC tissues with para-carcinoma tissues D. Correlation between SNX1 protein expression and age of patients in tissue microarrays. E. Correlation between SNX1 protein expression and tumor size of patients in tissue microarrays. F. Correlation between SNX1 protein expression and lymphatic invasion of patients in tissue microarrays. ①P=0.036, compared with the <60 years old group; ②P=0.005, compared with the <5 cm group; ③P=0.045, compared with the no lymphatic invasion group.

Fig 3 Expression levels of SNX1 in CRC tissues and their correlation with clinical pathological information

图4 SNX1相关信号通路的富集分析Note： A. Ridgeline plots of Hallmark enrichment analysis based on SNX1 low-expression and high-expression groups. B/C. GSEA showed that EMT signaling pathway (B) and apical junction pathway (C) were enriched in the SNX1 low-expression group. D. Dot plots of KEGG enrichment analysis based on the SNX1 low-expression and high-expression groups. E?H. GSEA showed that regulation of actin cytoskeleton pathway (E), adherens junction pathway (F), focal adhesion (G) and protein processing in endoplasmic reticulum pathway (H) were enriched in the SNX1 low-expression group.

Fig 4 Enrichment analysis of SNX1-related signaling pathways

图5 SNX1的表达水平对CRC细胞增殖和迁移的影响Note： A. Expression levels of SNX1 detected by qPCR and western blotting in CRC cell lines. B/C. Transfection efficiency of SNX1-siRNA verified by qPCR (top) and western blotting (bottom) in HT29 cells (B) and SW480 cells (C). D/E. Effect of SNX1-siRNA knockdown on HT29 cell (D) and SW480 cell (E) proliferation detected by CCK-8. F/G. Effect of SNX1-siRNA knockdown on HT29 cell (F) and SW480 cell (G) migration (left) detected by wound healing assay and analysis of migration rate (right). H. Effect of SNX1-siRNA knockdown on HT29 cell (top) and SW480 cell (bottom) migration (left) detected by Transwell assay and analysis of migration cells (right). I. Overexpression efficiency of SNX1-OE verified by western blotting in LoVo cells. J. Effect of SNX1-OE overexpression on LoVo cell proliferation detected by CCK-8. K. Effect of SNX1-OE overexpression on LoVo cell migration (left) detected by wound healing assay and analysis of migration rate (right). L. Effect of SNX1-OE overexpression on LoVo cell migration (left) detected by Transwell assay and analysis of migration cells (right). ①P=0.000, ②P=0.003, ③P=0.000, ④P=0.005, compared with the NC-siRNA group; ⑤P=0.000, ⑥P=0.003, compared with the SNX1-vector group.

Fig 5 Effect of SNX1 expression on the proliferation and migration of CRC cells

图6 SNX1 低表达与CRC转移关键基因以及EMT相关Note： A?C. Correlation analysis between SNX1 and MACC1 (A), MET (B), NOTCH1 (C) in GSE41568 database. D/E. Relative mRNA expression of MACC1, MET and NOCTH1 in SNX1-konckdown and control groups in HT29 cells (D) and SW480 cells (E). F/G. Relative mRNA expression of CDH1, VIM and SNAI1 in SNX1-konckdown and control groups in HT29 cells (F) and SW480 cells (G). ①P=0.013, ②P=0.000, ③P=0.015, ④P=0.025, ⑤P=0.048, ⑥P=0.001, ⑦P=0.037, ⑧P=0.047, ⑨P=0.006, ⑩P=0.004, ?P=0.002, compared with the NC-siRNA group.

Fig 6 Correlation between low expression of SNX1 and key genes of CRC metastasis and EMT

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分选链接蛋白1抑制结直肠癌细胞增殖和迁移的作用和机制研究

Study on the effect and mechanism of sorting nexin 1 on inhibiting the proliferation and migration of colorectal cancer cells

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