Yuanli Dong1,2, Yun Sun2,3, Yangle Huang1,2, Xumeng Fang1,2, Pian Sun1,2, Bilikere Dwarakanath2,3, Lin Kong1,2, Jiade Jay Lu2,4. 1. Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Fudan University Cancer Hospital, Shanghai 201321, China. 2. Shanghai Engineering Research Center of Proton and Heavy Ion Radiation Therapy, Shanghai 201321, China. 3. Division of Research and Development, Shanghai Proton and Heavy Ion Center, Shanghai 201321, China. 4. Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, Shanghai 201321, China.
Abstract
BACKGROUND: To examine whether MLKL participated in the invasion of radiosensitive nasopharyngeal carcinoma (NPC) cell (CNE-2) and radioresistant NPC cell (CR) through regulating epithelial-mesenchymal transition (EMT). METHODS: siRNA and CRISPR/Cas9 technique were used to decrease MLKL expression in NPC cell (CNE-2 and CR). Trans-well assay was conducted to evaluate invasion. Gene expression profiling was performed using Human U133 2.0 plus arrays (Affymetrix). Kyoto Encyclopedia of Genes and Genomes (KEGG) was adopted to analyze gene expression profiling. Hub genes at a functional level were accessed by protein-to-protein network (PPI). Quantitative real-time PCR and Western blot were used to access EMT markers. RESULTS: Invasion of CR was about 3~fold change higher than that of CNE-2. Silencing MLKL by siRNA inhibited invasion of CR, not CNE-2. Further, depleting MLKL by CRISPR-Cas9 in CR (CR-MLKL KO) also inhibited its invasion. KEGG pathway analysis showed invasion-related pathways were altered, such as adherent junction, TGF-β signaling pathway. PPI demonstrated that compared with CNE-2, CR showed 9 elevated hub genes including EGFR, JUN, CD44, SPP1, VIM, IL-8, BCL2, WDFY2, PIK3CD and 1 downregulated hub gene CDH1. After MLKL depletion, 8 hub genes were downregulated (EGFR, JUN, CD44, SPP1, VIM, FGF13, PLAU, MMP1) and 2 hub genes were upregulated (MMP9, CDH1). Quantitative real-time PCR results showed that compared with CNE-2, CR displayed decreased epithelial markers significantly (E-Cadherin) and increased mesenchymal markers significantly (Vimentin, N-Cadherin, Zeb1), indicating irradiation-induced EMT. After depletion of MLKL in CR, the expression of E-Cadherin, Vimentin, N-Cadherin, Zeb1 was reversed to the level of CNE-2. Western blot confirmed the results from qRT-PCR. CONCLUSIONS: Depletion of MLKL efficiently inhibits invasion of radioresistant NPC by suppressing EMT. MLKL may be an important target to suppress distant metastasis of NPC patients who relapsed after radiotherapy. 2019 Annals of Translational Medicine. All rights reserved.
BACKGROUND: To examine whether MLKL participated in the invasion of radiosensitive nasopharyngeal carcinoma (NPC) cell (CNE-2) and radioresistant NPC cell (CR) through regulating epithelial-mesenchymal transition (EMT). METHODS: siRNA and CRISPR/Cas9 technique were used to decrease MLKL expression in NPC cell (CNE-2 and CR). Trans-well assay was conducted to evaluate invasion. Gene expression profiling was performed using Human U133 2.0 plus arrays (Affymetrix). Kyoto Encyclopedia of Genes and Genomes (KEGG) was adopted to analyze gene expression profiling. Hub genes at a functional level were accessed by protein-to-protein network (PPI). Quantitative real-time PCR and Western blot were used to access EMT markers. RESULTS: Invasion of CR was about 3~fold change higher than that of CNE-2. Silencing MLKL by siRNA inhibited invasion of CR, not CNE-2. Further, depleting MLKL by CRISPR-Cas9 in CR (CR-MLKL KO) also inhibited its invasion. KEGG pathway analysis showed invasion-related pathways were altered, such as adherent junction, TGF-β signaling pathway. PPI demonstrated that compared with CNE-2, CR showed 9 elevated hub genes including EGFR, JUN, CD44, SPP1, VIM, IL-8, BCL2, WDFY2, PIK3CD and 1 downregulated hub gene CDH1. After MLKL depletion, 8 hub genes were downregulated (EGFR, JUN, CD44, SPP1, VIM, FGF13, PLAU, MMP1) and 2 hub genes were upregulated (MMP9, CDH1). Quantitative real-time PCR results showed that compared with CNE-2, CR displayed decreased epithelial markers significantly (E-Cadherin) and increased mesenchymal markers significantly (Vimentin, N-Cadherin, Zeb1), indicating irradiation-induced EMT. After depletion of MLKL in CR, the expression of E-Cadherin, Vimentin, N-Cadherin, Zeb1 was reversed to the level of CNE-2. Western blot confirmed the results from qRT-PCR. CONCLUSIONS: Depletion of MLKL efficiently inhibits invasion of radioresistant NPC by suppressing EMT. MLKL may be an important target to suppress distant metastasis of NPC patients who relapsed after radiotherapy. 2019 Annals of Translational Medicine. All rights reserved.
Authors: Wai Tong Ng; Michael C H Lee; Wai Man Hung; Cheuk Wai Choi; Kin Chung Lee; Oscar S H Chan; Anne W M Lee Journal: Int J Radiat Oncol Biol Phys Date: 2010-05-06 Impact factor: 7.038
Authors: Hiroshi Tanaka; Evelyn Kono; Chau P Tran; Hideyo Miyazaki; Joyce Yamashiro; Tatsuya Shimomura; Ladan Fazli; Robert Wada; Jiaoti Huang; Robert L Vessella; Jaibin An; Steven Horvath; Martin Gleave; Matthew B Rettig; Zev A Wainberg; Robert E Reiter Journal: Nat Med Date: 2010-11-07 Impact factor: 53.440