Circular RNA CircPVT1 Promotes Nasopharyngeal Carcinoma Metastasis Via The β-TrCP/c-Myc/SRSF1 Positive Feedback Loop

ObjectiveNasopharyngeal Carcinoma(NPC)is a malignant tumor that originates from the epithelium of nasopharynx and is common in Southeast Asia and Southern China.Due to its insidious site of origin,inconspicuous early symptoms and high susceptibility to metastasis,patients usually present with advanced nasopharyngeal carcinoma.Recurrence and distant metastasis are the major causes of death in patients with NPC.Therefore,exploring the molecular mechanism of NPC metastasis and finding new molecular markers or therapeutic targets are important guidelines for early diagnosis and precise treatment of NPC.Circular RNAs(circRNAs)are a class of non-coding RNA molecules that have attracted much attention in recent years.They are formed by back splicing of the 3’ and 5’ ends of precursor RNAs.Due to their unique structure,circRNAs are more stable than linear RNAs,making them an ideal molecular diagnostic marker and therapeutic target.Circ RNAs have been shown to play an important role in tumorigenesis.In this study,we identified a circular RNA circPVT1 that plays an important role in the metastasis of NPC and investigated its molecular mechanism of pro-invasive metastasis during the development of NPC.MethodsThe expression profiles of circRNAs in NPC cells within different metastatic potential were analyzed.Quantitative reverse transcription PCR and in situ hybridization were used to detect the expression level of circPVT1 in NPC cells and tissue samples.RNase R digestion assay were used to detect circPVT1 stability.RNA nucleoplasm isolation assay and fluorescence in situ hybridization assay were used to detect the location of circPVT1 in NPC cells.The effects of circPVT1 on NPC metastasis were investigated by scratch healing assay and Transwell invasion assay.A lung metastasis tumor model and an inguinal lymph node metastasis model were established to investigate the effect of circPVT1 on NPC metastasis in nude mice.RNA pulldown assay and mass spectrometry analysis were used to detect the circPVT1 binding proteins.RNA immunoprecipitation,RNA pulldown assay and western blotting were performed to confirm the interaction between circPVT1 and β-TrCP in NPC cells.Coimmunoprecipitation and western blotting were performed to confirm the interaction between β-TrCP and c-Myc in NPC cells.Protein stability assay and ubiquitination assay were used to detect the effects of circPVT1 and β-TrCP on c-Myc.Downstream genes regulated by circPVT1 were analyzed by the whole proteomics mass spectrometry and western blotting experiments.RNA immunoprecipitation,luciferase reporter gene assay and ChIP-q PCR assay were performed to confirm the effects of c-Myc and SRSF1 on circPVT1.Finally,the correlation between circPVT1,β-TrCP and c-Myc expression was detected in NPC tissues by in situ hybridization assay and immunohistochemistry assay.Results1.circPVT1 is highly expressed in NPC and associated with poor prognosis.Four circRNAs,including circCDYL,circCAMSAP1,circHIPK3 and circPVT1,were found to be highly expressed in NPC and may have prometastatic ability in NPC according to the high-throughput RNA sequencing data(GSE137543 and PRJNA391554).Further study showed that circPVT1 was highly expressed in NPC and positively correlated with clinical stage and TNM stage of NPC patients,and negatively correlated with patients survival.The Sanger sequencing showed that circPVT1 was spliced from exon 2 of the PVT1 gene precursor RNA.RNase R digestion assay showed that circPVT1 was significantly more stable than lnc RNA PVT1 in NPC cells.RNA nucleoplasmic separation assay and fluorescence in situ hybridization assay showed that circPVT1 was mainly located in the cytoplasm of NPC cells.2.Circ PVT1 promotes invasion and metastasis of NPCScratch healing assay and transwell invasion assay showed that circPVT1 significantly promoted the migration and invasion of NPC cells.The lung metastasis tumor model showed that circPVT1 promoted the metastasis of NPC cells.The inguinal lymph node metastasis model showed that overexpression of circPVT1 significantly promoted the metastasis of NPC cells to lymph nodes.3.Circ PVT1 binds to β-TrCP protein in NPC cellsTo investigate the molecular mechanism of circPVT1 promoting NPC invasion and metastasis,we searched for proteins interacting with circPVT1 by RNA pulldown assay combined with high-resolution mass spectrometry and found that circPVT1 had a higher probability of binding to β-TrCP protein.RNA pulldown and RIP experiments demonstrated the interaction between circPVT1 and β-TrCP protein.The stem-loop structure formed at 230-280 nt of circPVT1 was the core site for binding to β-TrCP.Mutation of 230-280 nt of circPVT1 significantly reduced the invasion and metastatic abilities of NPC cells.In addition,the WD40 structural domain of β-TrCP play a critical role in the combination of β-TrCP protein with circPVT1.β-TrCP can inhibit migraion and invasion of NPC cells caused by circPVT1 overexpression.4.β-TrCP binds and promotes ubiquitination of c-Mycβ-TrCP,acting as a type of E3 ligase,can recognize and degrade the specific amino acid sequence of its substrate in cells.The mass spectrometry analysis showed that β-TrCP can bind to c-Myc protein,coIP and immunofluorescence experiments confirmed the interaction between β-TrCP and c-Myc proteins.Further co-IP experiments showed that β-TrCP preferentially interacts with phosphorylated c-Myc protein.Overexpression of β-TrCP increased c-Myc ubiquitination and decreased c-Myc protein in NPC cells.5.Circ PVT1 inhibits ubiquitination of c-Myc through blocking the binding of β-TrCP to c-Myc proteinTo investigate the specific mechanism of circPVT1 in NPC,the β-TrCP expression was detected in NPC cells after overexpression of circPVT1 by western blotting.The results showed that circPVT1 did not affect β-TrCP expression.Further experiments showed that overexpression of circPVT1 inhibited β-TrCP binding to c-Myc,while knockdown of circPVT1 increased β-TrCP binding to c-Myc.Overexpression of circPVT1 increased the protein stability of c-Myc and prolonged the half-life of cMyc.The ubiquitination results showed that overexpression of circPVT1 significantly inhibited the ubiquitination level of c-Myc protein.These data suggest that circPVT1 can block the binding of β-TrCP to c-Myc and thus inhibit the degradation of c-Myc ubiquitination.6.circPVT1 promotes migration and invasion of NPC cells by regulating cell adhesion and cytoskeleton remodelingc-Myc is a well-known transcription factor that affects cancer development.To clarify the specific mechanism by which circPVT1 promotes metastasis,the whole proteome mass spectrometry was performed in NPC cells after overexpression of circPVT1.Differential proteins regulated by circPVT1 was enriched by the pathway analysis.The data revealed that these differential proteins were mainly enriched in cell adhesion junctions and cytoskeletal pathways.The atomic force microscopy showed that overexpression of circPVT1 attenuated the adhesion and stiffness of NPC cells.Further experiments showed that circPVT1 could induce the expression of RhoA,RhoC,and Vimentin,whereas reduce E-cadherin expression.Furthermore,knockdown of c-Myc reversed circPVT1 induced up-regulation of RhoA,RhoC,and Vimentin,as well as circPVT1 reduced E-cadherin expression.These results suggest that circPVT1 promotes metastasis of NPC by regulating cell adhesion and skeletal remodeling.7.c-Myc promotes the biosynthesis of circPVT1 through upregulating and recruiting SRSF1Bioinformatics analysis showed that there are several c-Myc binding sites in the promoter region of PVT1.Luciferase reporter gene experiments and ChIP-q PCR experiments showed that c-Myc can bind to the promoter region of PVT1 and promote PVT1 gene transcription.Bioinformatics analysis showed that there are several splicing factor binding sites on exon2 of PVT1,among these splicing factors,SRSF1 displayed the highest score.RIP experiments showed that SRSF1 could bind to the exon 2 of PVT1 gene,and overexpression of SRSF1 could promote the expression of circPVT1.Co-IP results indicated that c-Myc binds to SRSF1.The above results suggest that c-Myc promote the production of circPVT1 by recruiting SRSF1 to mediate the transcription splicing coupling mechanism.In addition,we also found that c-Myc could upregulate the expression of SRSF1,and jointly promote the generation of circPVT1.ConclusionIn this study,we found that circPVT1 was highly expressed in NPC and positively correlated with clinical stage and TNM staging of patients.circPVT1 inhibits proteasomal degradation of c-Myc by binding to β-TrCP.Stablization of c-Myc by circPVT1 alters the cytoskeleton remodeling and cell adhesion in NPC,which ultimately promotes the invasion and metastasis of NPC cells.In addition,as a transcription factor,c-Myc not only promoted PVT1 gene transcription,but also recruited the splicing factor SRSF1 to mediate transcription splicing coupling to promote circPVT1 generation,forming a positive feedback loop.This study elucidates the mechanism of action of circPVT1 in promoting invasion and metastasis of NPC and provides potential targets for diagnosis and clinical treatment of NPC.