绒毛样蛋白VILL通过与LMO7蛋白相互作用抑制鼻咽癌细胞的增殖

doi:10.12122/j.issn.1673-4254.2025.05.07

摘要/Abstract

摘要：

目的探索绒毛样蛋白VILL抑制鼻咽癌增殖的分子机制。方法收集136例鼻咽癌和67例鼻咽非癌组织，采用免疫共沉淀（CO-IP）、质谱、Western blotting、免疫荧光和GST pull down实验在鼻咽癌细胞株中筛查并验证VILL相互作用丰度最高蛋白。采用转基因实验，在体外（鼻咽癌细胞株）和体内（裸鼠）验证VILL与靶蛋白对鼻咽癌增殖的调控作用。采用免疫组化实验在临床组织标本中验证VILL与靶蛋白表达水平与鼻咽癌临床特征的相关性。结果在鼻咽癌细胞（HONE1 EBV、S18）中，VILL与E3泛素连接酶LMO7相互作用的丰度最高，二者共定位于细胞浆，直接相互作用。过表达LMO7部分逆转VILL对鼻咽癌细胞增殖的抑制作用。与67例鼻咽非癌组织比较，VILL表达在136例鼻咽癌组织中下调（P<0.0001）且与临床T分期（P=0.04）、N分期（P=0.01）和M分期（P=0.013）显著相关，而LMO7在鼻咽癌普遍高表达。结论 VILL可能通过抑制LMO7的促癌功能而抑制鼻咽癌增殖。

关键词: 鼻咽癌, VILL蛋白, LMO7蛋白, 蛋白相互作用, 增殖

Abstract:

Objective To elucidate the molecular mechanism by which villin-like protein VILL (VILL) inhibits proliferation of nasopharyngeal carcinoma (NPC) cells. Methods Co-immunoprecipitation (CO-IP) assay, mass spectrometry, Western blotting, immunofluorescence staining, and GST pull-down assay were employed to identify and confirm the protein interacting with VILL that had the highest abundance in NPC cell lines. Transgenic experiments were conducted in both NPC cell lines and nude mice to validate the regulatory role of VILL and its target protein in NPC proliferation. Immunohistochemistry was utilized to assess the correlation of the expression levels of VILL and its target protein in clinical tissue specimens of NPC with the clinical features of the patients. Results In NPC cell lines (HONE1 EBV and S18), VILL was found to interact most abundantly with the E3 ubiquitin ligase LMO7, and both proteins co-localized in the cytoplasm with direct interactions. Overexpression of LMO7 partially counteracted the inhibitory effect of VILL on NPC cell proliferation. The expression of VILL was significantly downregulated in 136 NPC tissue samples compared to 67 non-cancerous nasopharyngeal tissues (P<0.00001) with close correlation with clinical T stage (P=0.04), N stage (P=0.01), and M stage (P=0.013), whereas LMO7 was highly expressed in all the NPC tissues. Conclusion VILL overexpression inhibits NPC proliferation probably by suppressing the oncogenic function of LMO7.

Key words: nasopharyngeal carcinoma, villin-like protein VILL, LMO7 protein, protein interaction, cell proliferation

曾玉梅, 李继科, 黄仲曦, 周毅波. 绒毛样蛋白VILL通过与LMO7蛋白相互作用抑制鼻咽癌细胞的增殖[J]. 南方医科大学学报, 2025, 45(5): 954-961.

Yumei ZENG, Jike LI, Zhongxi HUANG, Yibo ZHOU. Villin-like protein VILL suppresses proliferation of nasopharyngeal carcinoma cells by interacting with LMO7 protein[J]. Journal of Southern Medical University, 2025, 45(5): 954-961.

图/表 8

图1 LMO7与VILL蛋白直接相互作用

Fig.1 LMO7 directly interacts with VILL protein. A: Verification of successful construction of NPC cell lines (HONE1-EBV and S18) overexpressing VILL+Flag. B: CO-IP assay and Western blotting, using VILL as the bait, for verification of the proteins interacting with VILL. C: CO-IP assay and Western blotting, using LMO7, VIM and DHX9 as the decoys, for verification of the proteins interacting with VILL. D: GST-pull down experiment for verifying direct interaction between LMO7 and VILL. E: Immunofluorescence assay showing co-localization of LMO7 and VILL in NPC cell lines (HONE1 and SUNE1).

表1 质谱预测与VILL发生相互作用的蛋白

Tab.1 Proteins predicted to interact with villin-like protein VILL by mass spectrometry

Gene	hit_num	Score	Matches	Sequences	Cover	pi
MYH3	2	1770	58	34	19.5	5.62
ACTB	8	642	39	12	35.8	5.23
PLEC	13	524	23	23	5.6	5.74
HNRNPK	14	522	20	10	22.5	5.39
ACTN2	17	463	17	12	17	5.31
DHX9	19	419	25	20	16.4	6.41
LMO7	21	398	21	10	16.9	8.73
VIM	22	394	17	12	28.5	5.06
PABPC1	24	374	14	11	18.6	9.52
RCN1	27	345	14	8	19.3	4.86
DDX3X	32	286	14	9	14.2	6.73
DDX5	54	180	7	5	6.5	5.25

图2 CCK-8实验显示LMO7部分逆转VILL对NPC细胞增殖的抑制作用

Fig.2 CCK-8 assay showing that LMO7 partially reverses the inhibitory effect of VILL on NPC cell proliferation. A,B: Western blotting for verification of successful construction of NPC cell lines (HONE1-EBV and S18) with LMO7 overexpression (A) and VILL+LMO7 double overexpression (B). C, D: CCK-8 assay showing that LMO7 partially reverses the inhibitory effect of VILL on proliferation of NPC cell lines HONE1-EBV (C) and S18 (D). ***P<0.001.

图3 裸鼠皮下成瘤实验显示LMO7部分逆转VILL对NPC细胞增殖的抑制作用

Fig.3 Subcutaneous tumor formation in nude mice showing that LMO7 partially reverses the inhibitory effect of VILL on NPC cell proliferation in vivo. A: Tumors dissected 40 days after subcutaneous implantation of NPC cells (S18) in nude mice. B: Time curve of subcutaneous tumor growth in nude mice. C: Tumor weight 40 days after subcutaneous implantation of NPC cells (S18) in nude mice. *P<0.05, ***P<0.001.

表2 VILL在67例鼻咽非癌组织标本和136例 NPC组织标本中的表达谱

Tab.2 Expression profiles of VILL in 67 non-cancerous naso-pharyngeal tissues and 136 NPC tissues (n, %)

Variable	n	VILL expression		P (Fisher's Exact Test)
Variable	n	Low	High	P (Fisher's Exact Test)
Nasopharyngeal tissues	67	0 (0.0)	67 (100.0)	P<0.001
NPC	136	99 (72.8)	37 (27.2)	P<0.001

图4 VILL在NPC组织标本上的表达谱及与临床病理特征的相关性

Fig.4 Expression profile of VILL in NPC tissue specimens and its correlation with clinicopathological features of the patients. A, B: Immunohistochemistry showing significant down-regulation of VILL expression in NPC tissues. C: Representative maps of VILL expression in NPC tissues in different TNM stages. D: Positivity rate of VILL in NPC tissues in different TNM stages. *P<0.05.

表3 VILL表达与NPC临床病理特征的相关性

Tab.3 Correlation between VILL expression level and clinico-pathological features of NPC patients (n, %)

Characteristics	Case	VILL expression		χ²	P
Characteristics	Case	Low	High	χ²	P
Gender
Female	39	27 (69.2)	12 (30.8)	0.144	0.705
Male	97	72 (74.2)	25 (25.8)	0.144	0.705
Age (years)
<50	62	44 (71.0)	18 (29.0)	0.060	0.807
≥50	74	55 (74.3)	19 (25.7)	0.060	0.807
T classification
T1-T2	78	51 (65.4)	27 (34.6)	4.231	0.040
T3-T4	58	48 (82.8)	10 (17.2)	4.231	0.040
N classification
N0-N1	55	33 (60.0)	22 (40.0)	6.587	0.010
N2-N3	81	66 (81.5)	15 (18.5)	6.587	0.010
M classification
M0	110	75 (68.2)	35 (31.8)	5.023	0.013
M1	26	24 (92.3)	2 (7.7)	5.023	0.013
Clinical stage
Ⅰ-Ⅱ	38	22 (57.9)	16 (42.1)	4.913	0.027
III-IV	98	77 (78.6)	21 (21.4)

图5 免疫组化显示VILL和LMO7在NPC组织标本上的表达相关性

Fig.5 Immunohistochemistry showing the correlation between the expressions of VILL and LMO7 in NPC tissue specimens. VILL is lowly expressed (A) while LMO7 is highly expressed (B) in a NPC tissue specimen. VILL (C) and LMO7 (D) are both highly expressed in a NPC tissue specimen.

参考文献 28

1	Chen YP, Chan ATC, Le QT, et al. Nasopharyngeal carcinoma[J]. Lancet, 2019, 394(10192): 64-80.
2	Sung H, Ferlay J, Siegel RL, 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): 209-49.
3	Han BF, Zheng RS, Zeng HM, et al. Cancer incidence and mortality in China, 2022[J]. J Natl Cancer Cent, 2024, 4(1): 47-53.
4	Liang YY, Deng XB, Lin XT, et al. RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner[J]. Cell Death Dis, 2020, 11(10): 855.
5	Zhao Y, Hong XH, Li K, et al. ZNF₅82 hypermethylation promotes metastasis of nasopharyngeal carcinoma by regulating the transcription of adhesion molecules Nectin-3 and NRXN3[J]. Cancer Commun: Lond, 2020, 40(12): 721-37.
6	Chen Y, Zhao Y, Yang X, et al. USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma[J]. Nat Commun, 2022, 13(1): 501.
7	Wang YQ, Wu DH, Wei D, et al. TEAD4 is a master regulator of high-risk nasopharyngeal carcinoma[J]. Sci Adv, 2023, 9(1): eadd0960.
8	Li ZX, Zheng ZQ, Yang PY, et al. WTAP-mediated m⁶A modification of lncRNA DIAPH1-AS1 enhances its stability to facilitate nasopharyngeal carcinoma growth and metastasis[J]. Cell Death Differ, 2022, 29(6): 1137-51.
9	Chen B, Huang R, Xia T, et al. The m6A reader IGF₂BP₃ preserves NOTCH₃ mRNA stability to sustain Notch3 signaling and promote tumor metastasis in nasopharyngeal carcinoma[J]. Oncogene, 2023, 42(48): 3564-74.
10	Zhang B, Li J, Wang Y, et al. Deubiquitinase USP7 stabilizes KDM5B and promotes tumor progression and cisplatin resistance in nasopharyngeal carcinoma through the ZBTB16/TOP2A axis[J]. Cell Death Differ, 2024, 31(3): 309-21.
11	Sun XS, Zhang K, Peng XZ, et al. HDAC4 mediated LHPP deacetylation enhances its destabilization and promotes the proliferation and metastasis of nasopharyngeal carcinoma[J]. Cancer Lett, 2023, 562: 216158.
12	Luo SD, Tsai HT, Hwang CF, et al. Aberrant miR-874-3p/leptin/EGFR/c-Myc signaling contributes to nasopharyngeal carcinoma pathogenesis[J]. J Exp Clin Cancer Res, 2022, 41(1): 215.
13	Wu P, Hou X, Peng M, et al. Circular RNA circRILPL1 promotes nasopharyngeal carcinoma malignant progression by activating the Hippo-YAP signaling pathway[J]. Cell Death Differ, 2023, 30(7): 1679-94.
14	Bruce JP, To KF, Lui VWY, et al. Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape[J]. Nat Commun, 2021, 12(1): 4193.
15	Patel SA, Hirosue S, Rodrigues P, et al. The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer[J]. Nature, 2022, 606(7916): 999-1006.
16	Schrag D, Beer TM, McDonnell CH, et al. Blood-based tests for multicancer early detection (PATHFINDER): a prospective cohort study[J]. Lancet, 2023, 402(10409): 1251-60.
17	Huang H, Liu A, Liang Y, et al. A urinary assay for mutation and methylation biomarkers in the diagnosis and recurrence prediction of non-muscle invasive bladder cancer patients[J]. BMC Med, 2023, 21(1): 357.
18	Jiang W, Liu N, Chen XZ, et al. Genome-wide identification of a methylation gene panel as a prognostic biomarker in nasopharyngeal carcinoma[J]. Mol Cancer Ther, 2015, 14(12): 2864-73.
19	Guo S, Diep D, Plongthongkum N, et al. Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA[J]. Nat Genet, 2017, 49(4): 635-42.
20	Silacci P, Mazzolai L, Gauci C, et al. Gelsolin superfamily proteins: key regulators of cellular functions[J]. Cell Mol Life Sci CMLS, 2004, 61(19): 2614-23.
21	Uhlén M, Fagerberg L, Hallström BM, et al. Proteomics. tissue-based map of the human proteome[J]. Science, 2015, 347(6220): 1260419.
22	Mahajan-Miklos S, Cooley L. The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis[J]. Cell, 1994, 78(2): 291-301.
23	Kang CK, Lee TH. Medaka villin 1-like protein (VILL) is associated with the formation of microvilli induced by decreasing salinities in the absorptive ionocytes[J]. Front Zool, 2014, 11(1): 2.
24	Zeng Q, Jiang T, Wang J. Role of LMO7 in cancer (review)[J]. Oncol Rep, 2024, 52(3): 117.
25	Karlsson T, Kvarnbrink S, Holmlund C, et al. LMO7 and LIMCH1 interact with LRIG proteins in lung cancer, with prognostic implications for early-stage disease[J]. Lung Cancer, 2018, 125: 174-84.
26	Liu X, Yuan H, Zhou J, et al. LMO7 as an unrecognized factor promoting pancreatic cancer progression and metastasis[J]. Front Cell Dev Biol, 2021, 9: 647387.
27	Takahashi M, Lio CJ, Campeau A, et al. The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING-IFN-β pathway[J]. Nat Immunol, 2021, 22(4): 485-96.
28	Dai S, Peng Y, Wang G, et al. LIM domain only 7: a novel driver of immune evasion through regulatory T cell differentiation and chemotaxis in pancreatic ductal adenocarcinoma[J]. Cell Death Differ, 2025, 32(2): 271-90.

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