title: Overexpression of RBM5 induces autophagy in human lung adenocarcinoma cells.
abstract: BACKGROUND
  Dysfunctions in autophagy and apoptosis are closely interacted and play an important role in cancer development. RNA binding motif 5 (RBM5) is a tumor suppressor gene, which inhibits tumor cells' growth and enhances chemosensitivity through inducing apoptosis in our previous studies. In this study, we investigated the relationship between RBM5 overexpression and autophagy in human lung adenocarcinoma cells.
  
  
  METHODS
  Human lung adenocarcinoma cancer (A549) cells were cultured in vitro and were transiently transfected with a RBM5 expressing plasmid (GV287-RBM5) or plasmid with scrambled control sequence. RBM5 expression was determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Intracellular LC-3 I/II, Beclin-1, lysosome associated membrane protein-1 (LAMP1), Bcl-2, and NF-κB/p65 protein levels were detected by Western blot. Chemical staining with monodansylcadaverine (MDC) and acridine orange (AO) was applied to detect acidic vesicular organelles (AVOs). The ultrastructure changes were observed under transmission electron microscope (TEM). Then, transplanted tumor models of A549 cells on BALB/c nude mice were established and treated with the recombinant plasmids carried by attenuated Salmonella to induce RBM5 overexpression in tumor tissues. RBM5, LC-3, LAMP1, and Beclin1 expression was determined by immunohistochemistry staining in plasmids-treated A549 xenografts.
  
  
  RESULTS
  Our study demonstrated that overexpression of RBM5 caused an increase in the autophagy-related proteins including LC3-I, LC3-II, LC3-II/LC3-I ratio, Beclin1, and LAMP1 in A549 cells. A large number of autophagosomes with double-membrane structure and AVOs were detected in the cytoplasm of A549 cells transfected with GV287-RBM5 at 24 h. We observed that the protein level of NF-κB/P65 was increased and the protein level of Bcl-2 decreased by RBM5 overexpression. Furthermore, treatment with an autophagy inhibitor, 3-MA, enhanced RBM5-induced cell death and chemosensitivity in A549 cells. Furthermore, we successfully established the lung adenocarcinoma animal model using A549 cells. Overexpression of RBM5 enhanced the LC-3, LAMP1, and Beclin1 expression in the A549 xenografts.
  
  
  CONCLUSIONS
  Our findings showed for the first time that RBM5 overexpression induced autophagy in human lung adenocarcinoma cells, which might be driven by upregulation of Beclin1, NF-κB/P65, and downregulation of Bcl-2. RBM5-enhanced autophagy acts in a cytoprotective way and inhibition of autophagy may improve the anti-tumor efficacy of RBM5 in lung cancer.
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. su_zhenzhong@163.com.
authors.forename: Zhenzhong
authors.initials: Z
authors.lastname: Su
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. kewangm1@hotmail.com.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Urinary Surgery, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. ranwei1968@yahoo.com.
authors.forename: Ranwei
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. yjz6437@163.com.
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. hao.yu.qi.u@163.com.
authors.forename: Yuqiu
authors.initials: Y
authors.lastname: Hao
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. lvxuejiao0311@163.com.
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Lv
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. 13943175477@126.com.
authors.forename: Junyao
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Pathophysiology, Norman Bethune College of Medicine of Jilin University, Changchun, Jilin, 130021, People's Republic of China. zhao_lj@jlu.edu.cn.
authors.forename: Lijing
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Pathophysiology, Norman Bethune College of Medicine of Jilin University, Changchun, Jilin, 130021, People's Republic of China. 1079389886@qq.com.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Paediatrics, The Second Affiliated Hospital of Jilin University, Chuangchun, Jilin, 130041, People's Republic of China. lypinglp@163.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, 130041, People's Republic of China. 18643111766@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C473448:RBM5 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins