The Role And Regulatory Mechanism Of Repulsive Guidance Molecules B In The Development And Progression Of Lung Cancer

BackgroundThe alteration of Repulsive guidance molecules B expression in breast cancer,prostate cancer and colorectal cancer is related to the tumor progression.In our previous research,we found that RGMB expression in lung cancer tissues was lower than that in normal tissues.In addition,it was found that the prognosis of cancer patients was better when the level of RGMB expression was higher.However,the functional role and molecular mechanism of RGMB in lung cancer remain undetermined.MethodsRT-PCR was used to detect RGMB mRNA expression lever in 12 lung cancer cell lines.Full-length RGMB was cloned in the pLVX-mCMV-ZsGreen-puro plasmid.The ShRNA targeted RGMB gene was designed and inserted into p LVX-Puro plasmid.Then the recombinant plasmid was transfected into 293 T cells to package lentivirus.We used the packaged lentivirus to infection A549 cells and then used Western blot to detect the over-expression and silencing efficiency of RGMB gene.Scratching assay,transwell invasion and adhesion assay were used to observe the migration,invasion and adhesion ability of cells.Western blot was used to detect the changes of protein in BMPs signaling pathway in RGMB knockdown cells.ResultsA549 lung cancer cells were selected for further experiments.In order to obtain RGMB stable high expression and stable knockout of A549 cell lines,we first cloned full-length RGMB into the pLVX-mCMV-ZsGreen-puro plasmidand and inserted the ShRNA targeted RGMB gene into p LVX-Puro plasmid.Sequence analysis was used to identify the recombinant plasmid.Then the recombinant plasmid was transfected into 293 T cells.After lentivirus packaging and infection A549 cells,we use puromycin to select A549 cells.Then the overexpression and knockdown of RGMB cells were obtained.Scratching assay,transwell invasion and adhesion assay were used to observe the migration,invasion and adhesion ability of cells.The results showed that RGMB suppresses invasion,adhesion and migration of lung cancer cells in vitro.Western blot was used to detect the changes of protein in BMPs signaling pathway in RGMB knockdown cells.We found that an increase in phosphorylated Smad-1/5/8 levels was observed in RGMB knockdown cells.No changes were observed in activation of ERK and JNK in RGMB knockdown cells.ConclusionIn our study,RGMB suppresses cell adhesion,invasion and migration.The tumor-suppressing effect of RGMB was exerted through inhibition of the Smad1/5/8 pathway.Our results demonstrate that RGMB is an important inhibitor of NSCLC metastasis.