[Villin-like protein VILL suppresses proliferation of nasopharyngeal carcinoma cells by interacting with LMO7 protein]

Abstract

Objectives: 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.

Conclusions: VILL overexpression inhibits NPC proliferation probably by suppressing the oncogenic function of LMO7.

Keywords: LMO7 protein; cell proliferation; nasopharyngeal carcinoma; protein interaction; villin-like protein VILL.

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).

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.

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.

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.