XIAP Mediated-PTEN Neddylation Promotes Proliferation and Migration of Colon Cancer Cells

doi:10.13523/j.cb.2112042

China Biotechnology

2022, Vol. 42

Issue (5): 27-36 DOI: 10.13523/j.cb.2112042

XIAP Mediated-PTEN Neddylation Promotes Proliferation and Migration of Colon Cancer Cells

YUAN Shu-hui^1,³,LI Shao-hua²,FANG Wei²,PENG Zhi-qiang³,ZHANG Ling-qiang^1,^3,^**(

)

1 School of Basic Medicine, Qingdao 266071, China
2 Shanghai Fengxian Central Hospital, The Third School of Clinical Medicine, Southern Medical University, Shanghai 201499, China
3 State Key Laboratory of Proteomics, Beijing National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 100850, China

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Abstract

Objective: To explore the functional role of XIAP-PTEN neddylation axis in colorectal cancer at cell line and clinical level. Methods: The expression level of XIAP was analyzed in tissue microarray of colorectal cancer by immunohistochemical staining, and the protein level of XIAP was detected in human colorectal cancer and adjacent normal tissues by Western blot. Co-IP was performed to analyze the interaction between endogenous PTEN and XIAP in SW480 cell line. CRISPR Cas9 technology was used to construct XIAP-knockout SW480 cell line, and the neddylation level of PTEN was analyzed in sg-XIAP cells by immunoprecipitation and Western blot. XIAP knockout or wild type SW480 cell lines were co-transfected with FLAG-Vector or FLAG-PTEN-Nedd8 plasmid, and then CCK8 and Transwell experiments were used to assay the proliferation and migration of XIAP-PTEN neddylation axis in SW480 cells, respectively. Results: The expression levels of XIAP were up-regulated in colon and rectum cancer tissues compared with adjacent normal tissues. XIAP interacted with PTEN in SW480 colon cancer cells. Deletion of XIAP inhibited PTEN neddylation in SW480 cells. The level of PTEN neddylation was elevated in colorectal cancer tissues compared with adjacent normal tissues. Deletion of XIAP inhibited the proliferation and migration of SW480 cells significantly, while PTEN-Nedd8 fusion protein rescued the phenotypes of XIAP deletion in SW480 cells. Conclusion: XIAP-PTEN neddylation axis promotes SW480 colon cancer cell proliferation and migration.

Key words： Colorectal cancer XIAP PTEN neddylation Proliferation Migration

Received: 19 December 2021 Published: 17 June 2022

ZTFLH:

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Corresponding Authors: Ling-qiang ZHANG E-mail: zhanglq@nic.bmi.ac.cn

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	Shu-hui YUAN
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	Ling-qiang ZHANG

Cite this article:

YUAN Shu-hui,LI Shao-hua,FANG Wei,PENG Zhi-qiang,ZHANG Ling-qiang. XIAP Mediated-PTEN Neddylation Promotes Proliferation and Migration of Colon Cancer Cells. China Biotechnology, 2022, 42(5): 27-36.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2112042 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I5/27

Fig.1 XIAP is highly expressed in colorectal cancer (a) Analysis of XIAP protein expression levels in normal colorectal tissues and colorectal cancer tissues from CPTAC database (b) Immunohistochemical staining for XIAP in colon cancer and adjacent tissues (left), analysis of XIAP immunohistochemical staining in colon cancer and adjacent tissues by Image Pro Plus (right) (c) Immunohistochemical staining for XIAP in rectal cancer and adjacent tissues (left), analysis of XIAP immunohistochemical staining in rectal cancer and adjacent tissues by Image Pro Plus (right) (d) Analysis of XIAP immunohistochemical staining in different stages of colorectal cancer by Image Pro Plus (e) Western blot analysis of XIAP expression in colorectal cancer and adjacent tissues. N(normal), C(cancer). Scale bars, 100 μm.^* P<0.05

Fig.2 There was a significant positive correlation between XIAP expression and PTEN neddylation level in colorectal cancer (a) Co-immunoprecipitation analysis the interaction between XIAP and PTEN in SW480 cells (b) Immunoprecipitation analysis of the level of PTEN neddylation in WT and XIAP knockout SW480 cells (c) Immunoprecipitation analysis the expression of XIAP and AKT pathway in colorectal cancer and adjacent tissues. N(normal), C(cancer)

Fig.3 XIAP promotes the proliferation and migration of colorectal cancer cells by mediating PTEN neddylation modification (a)Western blot analysis of XIAP knockout efficiency and FLAG-PTEN-Nedd8(N8) transfection efficiency (b) The proliferation ability of WT (sg-NC) and XIAP knockout (sg-XIAP) SW480 cells after transfecting PTEN-Nedd8(N8) or re-NC was measured by CCK8 (c) The migration ability of WT (sg-NC) and XIAP knockout (sg-XIAP) SW480 cells after transfecting PTEN-Nedd8(N8) or re-NC was measured by Transwell assay. Scale bars, 500 μm.^** P<0.01, ^*** P<0.001; ns: Non significance

Fig.4 The combination of MLN4924 and C75 synergistically inhibited the proliferation and migration of SW480 cells (a) Cell treated with no agent (control),1 μmol/L MLN4924, 10 μmol/L C75 or 1 μmol/L MLN4924 plus 10 μmol/L C75. Western blot analysis of the efficacy of MLN4924 and C75 (b) CCK8 assay was used to detect the effects of MLN4924 and C75 on SW480 cell proliferation (c) Transwell assay was used to detect the effects of MLN4924 and C75 on SW480 cell migration. Scale bars, 500 μm. ^* P<0.05, ^** P<0.01, ^*** P<0.001, ^**** P<0.000 1


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