Study On The Mechanism Of G3BP1 Accelerating The Malignant Progression Via Activating JAK2/STAT3 Signaling Pathway In Nasopharyngeal Carcinoma

Background and Purpose:Nasopharyngeal carcinoma(NPC)is a subtype of head and neck tumor arising from the epithelium of the nasopharynx.It has an ethnical and geographical preference,mainly occurring in East Asia,Southeast Asia,and North Africa.Although with the development of imaging technology,some NPC patients have benefited from early diagnosis and local control,the local recurrence and distant metastasis still lead to poor prognosis.Therefore,it is urgent to find new molecular markers and therapeutic targets for NPC research.Ras-GTPase-activating protein SH3 domain-binding protein1(G3BP1)is a multifunctional RNA-binding protein known for its involvement in the assembly and kinetics of stress granules(SGs).G3 BPs were first found to specifically bind to the SH3 domain of Ras GAP,which only occurred in proliferous cells.Our previous study has confirmed that G3BP1,one of the components of SGs,could rapidly assemble and form SGs and then recruit YB1 proteins under short stimulation with lethal doses of arsenic trioxide.RNA binding protein(RBP)is a class of conserve proteins with potential to bind RNA.RBPs are proved to involve in various important biological functions such as cell transportation,localization,development,differentiation,metabolism,and maintain the homeostasis of gene expression.In addition,RBPs have participated in almost the whole process of post-transcriptional regulation,supervising the formation of transcripts and maintaining cellular homeostasis.Therefore,revealing the mechanism of RBP-RNA interactions may provide new strategy for the diagnosis and treatment of malignant tumor.The JAK/STAT signaling pathway has been reported to regulate a wide range of physiological and pathological processes,such as cell proliferation,differentiation,apoptosis,angiogenesis,immunosuppression,and persistent inflammation.The occurrence and development of malignant tumors including NPC are also closely related to the dysregulation of JAK/STAT signaling pathway.Many factors could modulate JAK/STAT signaling pathway,such as cytokines,non-coding RNA and oncogene expression,among which RBP is an important class.At present,there are no literature about G3BP1 activating JAK/STAT signaling pathway and its role in NPC.This study explored the roles of G3BP1 in malignant phenotypes such as proliferation,invasion,and metastasis of NPC in vitro and in vivo.The study also investigated the expression of G3BP1,JAK2,and p-STAT3 in NPC tissues and their correlations with clinicopathological features.Moreover,the potential molecular mechanism was revealed that G3BP1 promoted malignant phenotype of NPC by activating JAK2/STAT3 signaling pathway.Methods:1.The expression and clinical significance of G3BP1 in NPC and other malignant tumors were analyzed by GEO database and TCGA database.2.The expression of G3BP1 protein in NPC cell lines and immortalized nasopharyngeal epithelial cell line(NP69)was examined by Western blotting(WB).3.The effect of G3BP1 on cell proliferation was determined by CCK8 and clone formation.Invasion and metastasis ability were confirmed by scratch healing experiment,matric gel invasion assay and 3D invasion assay.The effect of G3BP1 on cell cycle and apoptosis was analyzed by flow cytometry.4.The differential expression genes were identified in HNE2 and HONE1 cells after knockdown or overexpression of G3BP1,and potential molecular mechanisms were suspected by GO,KEGG,GSEA analysis as well as further verification via WB.5.The binding of G3BP1 protein and JAK2 m RNA was predicted and validated by bioinformatics,as well as RNA binding protein immunoprecipitation assay(RIP assay)and RNA pulldown experiments.The potential binding sites of G3BP1 protein and JAK2 m RNA were preliminarily verified by ultrasonic interruption of RNA and the structural inhibitor of G3BP1.6.Stattic,the inhibitor of STAT3,was used to recover the malignant phenotype and related protein expression of JAK2/STAT3 signaling pathway,and knockdown or overexpression of JAK2 was also conducted for rescue experiment.7.The roles of G3BP1 on cell growth in vivo were verified by tumorigenesis assay in nude mice.Stattic and knockdown of JAK2 were applied for rescue assay.8.The effects of G3BP1 on the metastasis in vivo were confirmed by tail vein metastasis model,and stattic was injected for rescue assay.9.The expression of G3BP1,JAK2,and p-STAT3 in NPC patients was examined by immunohistochemistry(IHC)to analyzing their relationship with clinicopathological features and exploring the influence of the prognosis of NPC patients.Results:1.G3BP1 is up-regulated in tumor tissues(1)G3BP1 was up-regulated in various tumor tissues including NPC compared with normal tissues in TCGA and GEO database.(2)The expression of G3BP1 in NPC cells was higher than NP69 cells.2.Effect of G3BP1 on malignant phenotype of NPC(1)G3BP1 promoted proliferation of NPC cells.(2)G3BP1 increased migration and invasion ability of NPC cells.(3)G3BP1 induced the rapid passage of S phase and affected the cell cycle in NPC cells.(4)G3BP1 reduced apoptosis of NPC cells.3.G3BP1 regulated JAK/STAT3 signaling pathway(1)It was predicted that G3BP1 might influence JAKs/STAT3 signaling pathway by bioinformatics.(2)G3BP1 regulated the expression of key molecules and downstream genes of JAK/STAT3 signaling pathway.G3BP1 could positively regulate the expression of p-STAT3 rather than total STAT3.The expression of proteins related to apoptosis,cell cycle,and EMT process was affected by knockdown or overexpression of G3BP1.(3)Stattic,the inhibitor of STAT3,could recover the protein regulation of G3BP1,meanwhile,stattic could restore the cell proliferation,invasion and metastasis caused by overexpression of G3BP1.4.G3BP1 could bind to JAK2 m RNA and regulate JAK2 expression(1)Knockdown of G3BP1 could decrease JAK2 protein expression rather than m RNA.(2)There were potential binding sites between G3BP1 and JAK2 m RNA by bioinformatics.(3)It was confirmed that G3BP1 could bind to JAKs m RNA by RIP assay and RNA pulldown assay.(4)Natural product EGCG could block the binding of G3BP1 protein to JAKs m RNA.(5)SGs formation was not observed in the binding of G3BP1 protein to JAKs m RNA.5.JAK2 could recover the malignant phenotype caused by G3BP1 in NPC cells(1)JAK2 played an oncogenic role in NPC cells.(2)Knockdown of JAK2 restored malignant phenotype and JAK2/STAT3 pathway activation caused by G3BP1 overexpression.(3)Overexpression of JAK2 recovered the phenotype and JAK2/STAT3 pathway inhibition induced by G3BP1 knockdown.6.G3BP1 promoted tumor growth and metastasis by regulating JAK2/STAT3 signaling pathway in vivo(1)It was confirmed that G3BP1 promoted tumor growth by subcutaneous tumor formation model,and stattic could restore the proliferative effect.(2)Knockdown of G3BP1 inhibited tumor growth,while overexpression of JAK2 promoted tumor growth,and overexpression of JAK2 could recover the proliferative effect.(3)It was verified that G3BP1 promoted tumor metastasis by tail vein metastasis model,and stattic could restore the metastatic effect.7.Expression and clinical significance of G3BP1,JAK2,and pSTAT3 proteins in NPC(1)There was a significant difference between NPC and nasopharyngeal mucosal tissue in the expression of G3BP1,JAK2,and p-STAT3 respectively and simultaneously.(2)The expression of G3BP1,JAK2,and p-STAT3 proteins was associated with clinicopathological features of NPC patients.(3)Higher expression of G3BP1,JAK2,and p-STAT3 indicated poor prognosis of NPC patients.(4)G3BP1 might act as an independent prognostic marker for patients with NPC.Conclusion:(1)The expression of G3BP1 is higher in NPC.G3BP1 acts as an oncogene that promotes proliferation,invasion,and metastasis in vitro and in vivo.(2)G3BP1 regulates the expression of JAK2 protein by binding to JAK2 m RNA,thus activates the JAK2/STAT3 pathway and augments the malignant progression of NPC.(3)Stattic and knockdown or overexpression of JAK2 can reverse the malignant phenotype caused by G3BP1.(4)G3BP1,JAK2,and p-STAT3 proteins are highly expressed in NPC tissues,and correlated with clinicopathological characteristics of NPC patients.The expression of G3BP1 might acts as an independent prognostic biomarker for NPC patients.