LRRC8A Expression Influences Growth of Esophageal Squamous Cell Carcinoma
2019; Elsevier BV; Volume: 189; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2019.06.006
ISSN1525-2191
AutoresTomoki Konishi, Atsushi Shiozaki, Toshiyuki Kosuga, Michihiro Kudou, Katsutoshi Shoda, Tomohiro Arita, Hirotaka Konishi, Shuhei Komatsu, Takeshi Kubota, Hitoshi Fujiwara, Kazuma Okamoto, Mitsuo Kishimoto, Eiichi Konishi, Yoshinori Marunaka, Eigo Otsuji,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe volume-regulated anion channel is composed of leucine-rich repeat–containing protein A (LRRC8A) and is activated by hypotonic conditions to implement the process of regulatory volume decrease. The role of LRRC8A in regulating genes related to progression of esophageal squamous cell carcinoma (ESCC) was investigated, as well as the prognostic significance of LRRC8A expression in this tumor. Knockdown experiments were conducted using ESCC cell lines and LRRC8A siRNA to assess the influence of this protein on tumor function. In addition, the gene expression profile of ESCC was determined by microarray analysis. Immunohistochemistry was performed on 64 primary tumor samples from ESCC patients receiving radical esophagectomy. It was found that depletion of LRRC8A decreased cell proliferation and migration and also promoted apoptosis. Microarray data demonstrated G1/S checkpoint regulation and up-regulation or down-regulation of phosphatidylinositol 3-kinase/AKT signaling, matrix metalloproteinase, and integrin signaling–related genes (including p21, p27, MMP1, and ITGAV) in LRRC8A-depleted cells. Immunohistochemistry showed that LRRC8A expression was related to the pathologic N and T stage categories, and strong LRRC8A expression was correlated with a worse prognosis of ESCC. These findings indicate that LRRC8A modulates tumor progression by influencing cell cycle, apoptosis, and migration, providing new insights into its function as an effector or biomarker of ESCC. The volume-regulated anion channel is composed of leucine-rich repeat–containing protein A (LRRC8A) and is activated by hypotonic conditions to implement the process of regulatory volume decrease. The role of LRRC8A in regulating genes related to progression of esophageal squamous cell carcinoma (ESCC) was investigated, as well as the prognostic significance of LRRC8A expression in this tumor. Knockdown experiments were conducted using ESCC cell lines and LRRC8A siRNA to assess the influence of this protein on tumor function. In addition, the gene expression profile of ESCC was determined by microarray analysis. Immunohistochemistry was performed on 64 primary tumor samples from ESCC patients receiving radical esophagectomy. It was found that depletion of LRRC8A decreased cell proliferation and migration and also promoted apoptosis. Microarray data demonstrated G1/S checkpoint regulation and up-regulation or down-regulation of phosphatidylinositol 3-kinase/AKT signaling, matrix metalloproteinase, and integrin signaling–related genes (including p21, p27, MMP1, and ITGAV) in LRRC8A-depleted cells. Immunohistochemistry showed that LRRC8A expression was related to the pathologic N and T stage categories, and strong LRRC8A expression was correlated with a worse prognosis of ESCC. These findings indicate that LRRC8A modulates tumor progression by influencing cell cycle, apoptosis, and migration, providing new insights into its function as an effector or biomarker of ESCC. Esophageal cancer is an extremely malignant tumor that is an important cause of cancer death.1Yuequan J. Shifeng C. Bing Z. Prognostic factors and family history for survival of esophageal squamous cell carcinoma patients after surgery.Ann Thorac Surg. 2010; 90: 908-913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 2Tachimori Y. Ozawa S. Numasaki H. Ishihara R. Matsubara H. Muro K. Oyama T. Toh Y. Udagawa H. Uno T. Registration Committee for Esophageal Cancer of the Japan Esophageal SocietyComprehensive registry of esophageal cancer in Japan, 2010.Esophagus. 2017; 14: 180-214Crossref Scopus (68) Google Scholar, 3Kamangar F. Dores G.M. Anderson W.F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world.J Clin Oncol. 2006; 24: 2137-2150Crossref PubMed Scopus (3044) Google Scholar, 4Torre L.A. Bray F. Siegel R.L. Ferlay J. Lortet-Tieulent J. Jemal A. Global cancer statistics, 2012.CA Cancer J Clin. 2015; 65: 87-108Crossref PubMed Scopus (23667) Google Scholar In Asia, the most frequent histologic finding of esophageal cancer is squamous cell carcinoma (ESCC). Despite advances in perioperative chemotherapy and/or radiotherapy, surgical procedures, and perioperative management, ESCC still has a poor prognosis because of its high invasiveness and frequent metastasis.1Yuequan J. Shifeng C. Bing Z. Prognostic factors and family history for survival of esophageal squamous cell carcinoma patients after surgery.Ann Thorac Surg. 2010; 90: 908-913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 2Tachimori Y. Ozawa S. Numasaki H. Ishihara R. Matsubara H. Muro K. Oyama T. Toh Y. Udagawa H. Uno T. Registration Committee for Esophageal Cancer of the Japan Esophageal SocietyComprehensive registry of esophageal cancer in Japan, 2010.Esophagus. 2017; 14: 180-214Crossref Scopus (68) Google Scholar, 3Kamangar F. Dores G.M. Anderson W.F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world.J Clin Oncol. 2006; 24: 2137-2150Crossref PubMed Scopus (3044) Google Scholar To achieve better outcomes, more investigation of the detailed molecular mechanisms that regulate the development and progression of ESCC is needed. Autoregulation of cell volume occurs when there is an imbalance between intracellular and extracellular osmolarity.5McManus M.L. Churchwell K.B. Strange K. Regulation of cell volume in health and disease.N Engl J Med. 1995; 333: 1260-1266Crossref PubMed Scopus (400) Google Scholar, 6Strange K. Emma F. Jackson P.S. Cellular and molecular physiology of volume-sensitive anion channels.Am J Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar Cell swelling results in activation of K+ and Cl− channels or KCl cotransporters, which is followed by release of KCl into the extracellular space and subsequent release of water.5McManus M.L. Churchwell K.B. Strange K. Regulation of cell volume in health and disease.N Engl J Med. 1995; 333: 1260-1266Crossref PubMed Scopus (400) Google Scholar, 6Strange K. Emma F. Jackson P.S. Cellular and molecular physiology of volume-sensitive anion channels.Am J Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar The volume-regulated anion channel is a type of Cl− channel that may be essential for volume regulation, particularly the regulatory volume decrease (RVD) process.6Strange K. Emma F. Jackson P.S. Cellular and molecular physiology of volume-sensitive anion channels.Am J Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar Leucine-rich repeat–containing protein A (LRRC8A) is a ubiquitous and integral component of the volume-regulated anion channel and has been reported to be required for regulation of cell volume.7Qiu Z. Dubin A.E. Mathur J. Reddy K. Miraglia L.J. Reinhardt J. Orth A.P. Patapoutian A. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.Cell. 2014; 157: 447-458Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar LRRC8A mRNA is expressed in the esophagus (35.7 reads per kb of exon per million mapped reads), the lung (27.9 reads per kb of exon per million mapped reads), and 29 other tissues according to the GTEx data set (The Human Protein Atlas, https://www.proteinatlas.org/ENSG00000136802-LRRC8A/tissue, last accessed December 12, 2018). LRRC8 proteins have also been detected in various other chordates, generally as five paralogues termed LRRC8A (the first protein described), B, C, D, and E.8Abascal F. Zardoya R. LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication.Bioessays. 2012; 34: 551-560Crossref PubMed Scopus (120) Google Scholar However, the role of LRRC8A in cancer remains unclear, including its impact on ESCC, and the clinicopathologic implications of LRRC8A expression in human ESCC have not been assessed. Recent studies have shown an essential role of ion transporters in various cancers, suggesting that physiological intracellular factors may become new targets for cancer treatment.9Shiozaki A. Ichikawa D. Otsuji E. Marunaka Y. Cellular physiological approach for treatment of gastric cancer.World J Gastroenterol. 2014; 20: 11560-11566Crossref PubMed Scopus (19) Google Scholar We previously reported on the importance of ion channels,10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar water channels,14Yamazato Y. Shiozaki A. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Aquaporin 1 suppresses apoptosis and affects prognosis in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 29957-29974Crossref PubMed Scopus (20) Google Scholar, 15Shimizu H. Shiozaki A. Ichikawa D. Fujiwara H. Konishi H. Ishii H. Komatsu S. Kubota T. Okamoto K. Kishimoto M. Otsuji E. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma.J Gastroenterol. 2014; 49: 655-666Crossref PubMed Scopus (40) Google Scholar and pH-regulating channels16Shiozaki A. Kudou M. Ichikawa D. Shimizu H. Arita T. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 17921-17935Crossref PubMed Scopus (22) Google Scholar, 17Shiozaki A. Hikami S. Ichikawa D. Kosuga T. Shimizu H. Kudou M. Yamazato Y. Kobayashi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Anion exchanger 2 suppresses cellular movement and has prognostic significance in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 25993-26006Crossref PubMed Scopus (15) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar, 19Ochi F. Shiozaki A. Ichikawa D. Fujiwara H. Nakashima S. Takemoto K. Kosuga T. Konishi H. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Carbonic anhydrase XII as an independent prognostic factor in advanced esophageal squamous cell carcinoma.J Cancer. 2015; 6: 922-929Crossref PubMed Scopus (19) Google Scholar in gastrointestinal cancer. Our findings indicated that such physiological factors could be useful biomarkers and that targeting them may lead to novel treatment strategies for ESCC. This study investigated the influence of LRRC8A on tumorigenesis-related genes and its clinicopathologic importance in ESCC. Microarray analysis revealed that knockdown of LRRC8A with siRNA influenced the expression of various genes related to cellular growth and migration. LRRC8A expression was also investigated in human ESCC samples, analyzing relations with clinicopathologic findings and the prognosis. Our findings revealed an important role of LRRC8A in the progression of ESCC. Seven human ESCC cell lines (TE2, TE4, TE5, TE8, TE9, TE13, and TE15) and the PK59 human pancreatic cell line were obtained from the Riken Cell Bank (Tsukuba, Japan), and four other human ESCC cell lines (KYSE70, KYSE150, KYSE170, and KYSE790) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Two cell lines (TE15, KYSE170) were used for in vitro studies, with these cells being cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum under a humidified 5% CO2 atmosphere at 37°C.10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar, 14Yamazato Y. Shiozaki A. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Aquaporin 1 suppresses apoptosis and affects prognosis in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 29957-29974Crossref PubMed Scopus (20) Google Scholar, 15Shimizu H. Shiozaki A. Ichikawa D. Fujiwara H. Konishi H. Ishii H. Komatsu S. Kubota T. Okamoto K. Kishimoto M. Otsuji E. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma.J Gastroenterol. 2014; 49: 655-666Crossref PubMed Scopus (40) Google Scholar, 16Shiozaki A. Kudou M. Ichikawa D. Shimizu H. Arita T. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 17921-17935Crossref PubMed Scopus (22) Google Scholar, 17Shiozaki A. Hikami S. Ichikawa D. Kosuga T. Shimizu H. Kudou M. Yamazato Y. Kobayashi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Anion exchanger 2 suppresses cellular movement and has prognostic significance in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 25993-26006Crossref PubMed Scopus (15) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar For immunohistochemistry, a polyclonal anti-LRRC8A antibody (ab157489) and a monoclonal anti-cytokeratin 13 antibody (ab16112) were purchased from Abcam (Cambridge, MA), whereas the monoclonal anti-LRRC8A antibody (SAB1412855) for the protein assay was from Sigma-Aldrich (St. Louis, MO), and the mouse monoclonal antibody that targeted β-actin was also from Sigma-Aldrich. Horseradish peroxidase–conjugated anti-rabbit or mouse secondary antibodies were from Cell Signaling Technology (Beverly, MA). M-PER lysis buffer (Pierce, Rockford, IL) supplemented with protease inhibitor (Pierce) was used to harvest the cells. The protein concentration was measured by a modification of the Bradford method (Bio-Rad, Hercules, CA). Then cell lysates that contained equal amounts of total protein were subjected to SDS-PAGE, followed by transfer of the proteins to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ). Membranes were probed with the relevant antibodies and proteins were detected by an ECL Plus Western Blotting Detection System (GE Healthcare). β-Actin was used as the loading control.10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar, 14Yamazato Y. Shiozaki A. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Aquaporin 1 suppresses apoptosis and affects prognosis in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 29957-29974Crossref PubMed Scopus (20) Google Scholar, 15Shimizu H. Shiozaki A. Ichikawa D. Fujiwara H. Konishi H. Ishii H. Komatsu S. Kubota T. Okamoto K. Kishimoto M. Otsuji E. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma.J Gastroenterol. 2014; 49: 655-666Crossref PubMed Scopus (40) Google Scholar, 16Shiozaki A. Kudou M. Ichikawa D. Shimizu H. Arita T. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 17921-17935Crossref PubMed Scopus (22) Google Scholar, 17Shiozaki A. Hikami S. Ichikawa D. Kosuga T. Shimizu H. Kudou M. Yamazato Y. Kobayashi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Anion exchanger 2 suppresses cellular movement and has prognostic significance in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 25993-26006Crossref PubMed Scopus (15) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar Cells were transfected with 20 nmol/L LRRC8A siRNA (Stealth RNAi siRNA number HSS125512; number 1: 327013F05; number 2: 327013G01; number 3: 327013B11; Invitrogen, Carlsbad, CA) by using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's directions. After 24 hours, the siRNA-containing medium was removed and replaced with fresh medium. Control siRNA (20 nmol/L) provided by the manufacturer (Stealth RNAi siRNA Negative Control; Invitrogen) was the negative control.10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar, 14Yamazato Y. Shiozaki A. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Aquaporin 1 suppresses apoptosis and affects prognosis in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 29957-29974Crossref PubMed Scopus (20) Google Scholar, 15Shimizu H. Shiozaki A. Ichikawa D. Fujiwara H. Konishi H. Ishii H. Komatsu S. Kubota T. Okamoto K. Kishimoto M. Otsuji E. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma.J Gastroenterol. 2014; 49: 655-666Crossref PubMed Scopus (40) Google Scholar, 16Shiozaki A. Kudou M. Ichikawa D. Shimizu H. Arita T. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 17921-17935Crossref PubMed Scopus (22) Google Scholar, 17Shiozaki A. Hikami S. Ichikawa D. Kosuga T. Shimizu H. Kudou M. Yamazato Y. Kobayashi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Anion exchanger 2 suppresses cellular movement and has prognostic significance in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 25993-26006Crossref PubMed Scopus (15) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar TE4 cells were transfected with either the control HaloTag plasmid (G6591; Promega, Madison, WI) or the LRRC8A HaloTag plasmid (FHC00834; Promega) using FuGENE HD transfection reagents (E2311; Promega) according to the instructions of the manufacturer. Vector transfection was confirmed by performing fluorescent microscopy to detect the HaloTag fusion protein stained by the TMR-conjugated HaloTag ligand (G8252; Promega) according to the manufacturer's protocol. Then the proliferation assay was performed using LRRC8A-expressing cells as described previously.13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar After seeding these cells into 6-well plates at 0.4 × 105 cells/well, cells were cultured under a humidified 5% CO2 atmosphere at 37°C. Transfection of control or LRRC8A siRNA was performed after 24 hours. Then the cells were removed from the flasks by using trypsin-EDTA 48 or 72 hours after transfection and were counted with a hemocytometer.10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Ichikawa D. Otsuji E. Functional analysis and clinical significance of sodium iodide symporter expression in gastric cancer.Gastric Cancer. 2019; 22: 473-485Crossref PubMed Scopus (12) Google Scholar, 14Yamazato Y. Shiozaki A. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Aquaporin 1 suppresses apoptosis and affects prognosis in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 29957-29974Crossref PubMed Scopus (20) Google Scholar, 15Shimizu H. Shiozaki A. Ichikawa D. Fujiwara H. Konishi H. Ishii H. Komatsu S. Kubota T. Okamoto K. Kishimoto M. Otsuji E. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma.J Gastroenterol. 2014; 49: 655-666Crossref PubMed Scopus (40) Google Scholar, 16Shiozaki A. Kudou M. Ichikawa D. Shimizu H. Arita T. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 17921-17935Crossref PubMed Scopus (22) Google Scholar, 17Shiozaki A. Hikami S. Ichikawa D. Kosuga T. Shimizu H. Kudou M. Yamazato Y. Kobayashi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Anion exchanger 2 suppresses cellular movement and has prognostic significance in esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 25993-26006Crossref PubMed Scopus (15) Google Scholar, 18Ariyoshi Y. Shiozaki A. Ichikawa D. Shimizu H. Kosuga T. Konishi H. Komatsu S. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Na+/H+ exchanger 1 has tumor suppressive activity and prognostic value in esophageal squamous cell carcinoma.Oncotarget. 2017; 8: 2209-2223Crossref PubMed Scopus (17) Google Scholar At 48 hours after transfection of siRNA, the cell cycle phase was evaluated by fluorescence-activated cell sorting. Briefly, after detachment from the flasks with trypsin-EDTA, the cells were treated with Triton X-100 and RNase, and nuclear staining was performed with propidium iodide (PI). Then the DNA content was measured by using a Becton-Dickinson Accuri C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ), with analysis of at least 1.0 × 105 cells.10Shiozaki A. Nako Y. Ichikawa D. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. Role of the Na+/K+/2Cl− cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.World J Gastroenterol. 2014; 20: 6844-6859Crossref PubMed Scopus (34) Google Scholar, 11Shiozaki A. Takemoto K. Ichikawa D. Fujiwara H. Konishi H. Kosuga T. Komatsu S. Okamoto K. Kishimoto M. Marunaka Y. Otsuji E. The K-Cl cotransporter KCC3 as an independent prognostic factor in human esophageal squamous cell carcinoma.Biomed Res Int. 2014; 2014: 936401Crossref PubMed Scopus (18) Google Scholar, 12Kobayashi T. Shiozaki A. Nako Y. Ichikawa D. Kosuga T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fujiwara H. Okamoto K. Kishimoto M. Konishi E. Marunaka Y. Otsuji E. Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.Oncotarget. 2018; 9: 23237-23252Crossref PubMed Scopus (16) Google Scholar, 13Shiozaki A. Ariyoshi Y. Iitaka D. Kosuga T. Shimizu H. Kudou M. Konishi T. Shoda K. Arita T. Konishi H. Komatsu S. Kubota T. Fu
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