The Molecular Mechanism Of LRP1-SNRNP25 Fusion Gene Promting Human Osteosarcoma Cell Invasion And Migration

ObjectiveThe aim of this study was to find out the structure and breaking site of LRP1-SNRNP25 fusion gene found in our previous study at DNA level,and to clarify its formation mechanism.then,to investigate the effects of LRP1-SNRNP25 fusion gene on the migration,invasion,tumorigenesis and distant metastasis of osteosarcoma cells and to explore the downstream signaling pathway of LRP1-SNRNP25 fusion gene,to determine the molecular mechanism of LRP1-SNRNP25 fusion gene in promoting invasion and migration in osteosarcoma,which lays the foundation for targeted therapy of LRP1-SNRNP25 fusion gene,and explore a new target for targeted therapy of LRP1-SNRNP25 in osteosarcoma.Methods1.Genome-wide sequencing of LRP1-SNRNP25 fusion gene positive sample was performed on the HiSeq X Ten platform.Firstly,the genomic DNA of one osteosarcoma sample with LRP1-SNRNP25 fusion gene was extracted.Secondly,the whole genome was sequenced by HiSeq X Ten platform.Finally,the structure of LRP1-SNRNP25 fusion gene at the DNA level was identified by bioinformatics technology,and the existence of the fusion gene was verified.2.To investigate the effects of LRP1-SNRNP25 fusion gene on osteosarcoma cell migration,invasion,tumorigenesis and distant metastasis.On the one hand,LRP1-SNRNP25 overexpression plasmid was constructed and verified by plasmid sequencing technology,LRP1 and SNRNP25 plasmids were purchased.LRP1-SNRNP25,LRP1,SNRNP25 and empty vector were transfected instantaneously into osteosarcoma cell lines 143 B and SAOS2.The stable cell line selected by G418 was verified by Western blot,and the invasion and migration ability were detected by Wound Healing and Transwell.On the other hand,osteosarcoma cells 143 B with LRP1-SNRNP25 expressing and lentiviruses with empty vectors were inoculated into the ventral side of the right hind leg of nude mice through subcutaneous implantation,and the changes of tumor volume were observed and measured regularly.The expression and location of LRP1-SNRNP25 was further detected by immunohistochemical method(because there was no antibody against LRP1-SNRNP25,so Flag-labeled antibody was used to detect the expression of LRP1-SNRNP25).To evaluate the effect of LRP1-SNRNP25 on the proliferation activity of osteosarcoma cells in vivo by detecting the expression of Ki-67.Meanwhile,osteosarcoma cells 143 B infected with lentiviruses stably expressing LRP1-SNRNP25 and empty vector were injected into nude mice through tail vein.After that,the nude mice were executed and their lungs and livers were dissected.The nodules of their lungs and livers were counted and compared.The tissues of lungs and livers were embedded and then stained with hematoxylin-eosin(HE)to confirm the nature of nodules.3.To explore the molecular mechanism of LRP1-SNRNP25 fusion gene affecting the invasion and migration of osteosarcoma cells.Firstly,Western blot was used to detect signal pathways and molecules related to migration and invasion in osteosarcoma cells overexpressed by 143 B and SAOS2 LRP1-SNRNP25,LRP1,SNRNP25,and empty vector,such as ERK/pERK,JNK/pJNK,PI3K/Akt signaling pathway,cell skeleton,cell movement and cell adhesion related molecules such as FAK,integrin and cadherin in CAMs,Rac1,Rho A and MMPs etc,to explore the molecular mechanism of LRP1-SNRNP25 fusion gene promoting osteosarcoma cell proliferation,invasion and migration.Secondly,the protein interacting with LRP1-SNRNP25 fusion protein was identified by mass spectrometry,and then verified by Western blot,CO-IP and Immunofluorescence.After the target molecule was identified,osteosarcoma cells and nude mice expressing LRP1-SNRNP25 were treated with inhibitors targeting the molecular signaling pathway and siRNA.Result1.HiSeq X Ten genome sequencing confirmed the existence and structure of LRP1-SNRNP25 at the DNA level.LRP1-SNRNP25 fusion gene is produced by fusion of exon 8 of LRP1 gene with exon 2 of SNRNP25 gene.2.LRP1-SNRNP25 fusion gene affects the migration,invasion,tumorigenesis and distant metastasis in osteosarcoma.In vitro,osteosarcoma cells overexpressing LRP1-SNRNP25 had better migration and invasion ability than osteosarcoma cells overexpressing LRP1,SNRNP25 and empty vector.In vivo,the nude mice in LRP1-SNRNP25 group were more likely to have lung and liver metastasis.Moreover,in vivo,LRP1-SNRNP25 fusion gene enhanced the tumorigenicity of nude mice,and the tumors grew rapidly with large volume and weight.Further immunohistochemical analysis showed that the positive rate of Ki-67 cells in LRP1-SNRNP25 group was significantly increased.3.LRP1-SNRNP25 fusion gene plays biological roles through pJNK/37LRP/MMP2 signaling pathway.The expression of pJNK,37 LRP and MMP2 in osteosarcoma cells overexpressing LRP1-SNRNP25 was higher than that in osteosarcoma cells overexpressing LRP1,SNRNP25 and empty control group.LRP1-SNRNP25 fusion protein interacts with pJNK and 37LRP(37 KD laminin receptor).Phosphorylation of JNK promoteed the expression of 37 LRP.The binding of 37 LRP with its ligand laminin will change its conformation and promote the release of MMP2.After treated with different concentration gradients of pJNK inhibitor SP600125,the expression levels of 37 LRP and MMP2 in osteosarcoma cells overexpressing LRP1-SNRNP25 fusion gene decreased accordingly.The expression of MMP2 in osteosarcoma cells overexpressing LRP1-SNRNP25 also decreased after the treatment with 37 LRP siRNA.Moreover,the migration and invasion ability of osteosarcoma cells overexpressing LRP1-SNRNP25 decreased significantly after the treatment of siRNA of 37 LRP and SP600125 and Marimastat(inhibitor of MMPs).In vivo,the expression of pJNK and MMP2 in LRP1-SNRNP25 group increased significantly,and the tumorigenic ability of nude mice in LRP1-SNRNP25 group decreased significantly after SP600125 was used.ConclusionLRP1-SNRNP25 fusion gene can promote the invasion and migration,tumorigenesis and distant metastasis in osteosarcoma.LRP1-SNRNP25 fusion gene promotes the invasion and migration of osteosarcoma cells through pJNK/37LRP/MMP2 signaling pathway.The results of this study provide significant molecular therapeutic targets for osteosarcoma patients with over-expression of LRP1-SNRNP25 fusion gene.