Circular RNA CircCAMSAP1 Accelerates Nasopharyngeal Carcinoma Tumorigenesis Via A SERPINH1/c-Myc Positive Feedback Loop

Research purposeNasopharyngeal carcinoma(NPC)is a malignant tumor of the head and neck that occurs frequently in southern China.The pathological type is mostly non-keratinizing squamous carcinoma.The onset of the disease is insidious and the early symptoms are not obvious,patients are mostly progressing to the middle and late stages when they visit the clinic.Local recurrence and distant metastasis are the main reasons for the poor prognosis of patients.Therefore,exploring the molecular mechanisms of nasopharyngeal carcinoma proliferation and metastasis and finding new molecular markers or targets are important guides for the early diagnosis and clinical treatment strategies of NPC.Circular RNA(circRNA)is a noncoding RNA that has recently received much attention.It is formed by reverse splicing of the 3’ and 5’ ends of the precursor mRNA with the assistance of splicing factors.Due to its special structure,circRNA is more stable than linear RNA.There have been numerous reports indicating that circRNAs play important functions in tumor development.This study aims to explore the molecular mechanism of circRNAs that play a role in promoting metastasis and proliferation in NPC,as well as the mechanism of circRNA that are highly expressed in NPC.Research MethodsIn this study,we analyzed a set of high-throughput RNA sequencing data(GSE68799)including NPC tissues and non-tumor nasopharyngeal epithelial(NPE)tissues to find the differentially expressed circRNA among them.The expression of circCAMSAP1 in NPC tissues and cell lines was detected by qRT-PCR assay and in situ hybridization assay,and the constitutive sequence of circCAMSAP1 was confirmed by Sanger sequencing.The basic characteristics of circCAMSAP1 was explored by RNase R exonuclease digestion assay,actinomycin D assay,RNA nuclear and cytoplasmic isolation and RNA Fluorescence in situ hybridization analysis.The effects of circCAMSAP1 on NPC cell metastasis and proliferation in vitro were explored by Wound-healing assay,Transwell assay,MTT assay and cell cycle analysis.The nude mouse lung metastasis model and the nude mouse subcutaneous tumor model were constructed to explore the effect of circCAMSAP1 on NPC cell metastasis and proliferation ability in vivo.We searched for circCAMSAP1 potential regulatory proteins by highthroughput mass spectrometry and NPC microarray data analysis after overexpression or knockdown of circCAMSAP1.The effect of circCAMSAP1 on SERPINH1 expression was explored by qRT-PCR and Western blotting,and the specific molecular mechanisms involved were explored by bioinformatics analysis,dual-luciferase reporter gene assay,circRIP assay,actinomycin D assay.The interaction between SERPINH1 and c-Myc was then explored by Co-immunoprecipitation assay and immunofluorescence co-localization assay.The effects of circCAMSAP1 and SERPINH1 on c-Myc expression and the specific mechanisms involved were explored by Western blotting,protein stability assay,protein ubiquitination assay,protein phosphorylation assay,nuclear-plasmin separation protein analysis and dual-luciferase reporter gene assay.The expression of circCAMSAP1,SERPINH1 and c-Myc was detected by in situ hybridization assay and immunohistochemistry in NPC tissues and correlation analysis was performed.The effects of c-Myc on circCAMSAP1 expression and the mechanisms involved were explored by qRT-PCR,bioinformatics analysis,dual-luciferase reporter gene assay and chromatin immunoprecipitation experiments.We explored the regulatory role of SRSF10 on circCAMSAP1production by bioinformatics analysis,qRT-PCR and RNA-binding protein immunoprecipitation assays.Finally,the regulatory role of c-Myc on SRSF10 was explored by qRT-PCR and Western blotting.Research Results[circCAMSAP1 is highly expressed in NPC tissues]Bioinformatic analysis of high-throughput RNA sequencing data(GSE68799)revealed that circCAMSAP1 expression was significantly upregulated in NPC tissues compared to NPE tissues.The high expression of circCAMSAP1 in NPC tissues and cell lines was confirmed by qRT-PCR and in situ hybridization assay,and was positively correlated with clinical stage and TNM stage of NPC patients.Sanger sequencing confirmed that circCAMSAP1 is spliced from exons 2-3 of the CAMSAP1 precursor mRNA.RNase R exonuclease and actinomycin D treatment of NPC cells confirmed that circCAMSAP1 was significantly more stable than linear CAMSA P1 mRNA in NPC cells.RNAFISH analysis and RNA nuclear and cytoplasmic isolation showed that circCAMSAP1 was distributed predominantly in the cytoplasm of NPC.[circCAMSAP1 promotes metastasis and proliferation of NPC in vitro and in vivo]After successful overexpression or knockdown of circCAMSAP1 in NPC cells,we confirmed that circCAMSAP1 significantly promoted migration and invasion of NPC cells in vitro by Wound-healing assay and Transwell assays.MTT assay and Cell cycle analysis confirmed that circCAMSAP1 promoted the proliferation of NPC cells in vitro.We constructed a nude mouse lung metastasis model by tail vein injection of NPC cells,and compared the number and size of lung metastatic nodules,the results showed that circCAMSAP1 promoted NPC cells metastasis in vivo.A nude mouse subcutaneous tumor model was constructed by subcutaneous injection,the size of subcutaneous tumors was compared,the results showed that circCAMSAP1 promoted the proliferation of NPC cells in vivo.[circCAMSAP1 binds SERPINH1 mRNA and promotes its stabilization]Non-coding RNAs need to function by regulating protein function.We searched for differentially expressed proteins after overexpression or knockdown of circCAMSAP1 by high-throughput mass spectrometry and focused on SERPINH1 in combination with NPC microarray data.SERPINH1 expression was also significantly elevated in clinical samples of NPC.qRT-PCR and Western blotting confirmed that circCAMSAP1 promotes the expression of SERPINH1.Based on the cellular localization of circCAMSAP1 and bioinformatics analysis,we hypothesized that circCAMSAP1 may directly bind SERPINH1 mRNA.Dual-luciferase reporter gene assay and circRIP assay confirmed that circCAMSAP1 directly binds SERPINH1 mRNA 3’UTR region.In eukaryotes the mRNA 3’UTR region is closely related to its stability.Actinomycin D assay confirmed that circCAMSAP1 promotes the stability of SERPINH1 mRNA in NPC cells.We then confirmed that SERPINH1 promoted NPC metastasis and proliferation by Wound-healing assay,Transwell assay and MTT assay,Rescue assay confirmed circCAMSAP1 promoted NPC metastasis and proliferation by up-regulating SERPINH1 expression.[SERPINH1 binds c-Myc and inhibits its ubiquitinated degradation]We analyzed SERPINH1 potential interacting proteins by bioinformatics and confirmed the interaction between SERPINH1 and cMyc in NPC by immunoprecipitation assay and immunofluorescence colocalization assay.Western blotting and qRT-PCR confirmed that circCAMSAP1 promoted c-Myc expression at the post-translational level through SERPINH1.Protein stability assay confirmed that circCAMSAP1 and SERPINH1 promoted the stability of c-Myc.Protein ubiquitination assay and protein phosphorylation assay confirmed that circCAMSAP1 inhibited c-Myc phosphorylation via SERPINH1 and then inhibited c-Myc ubiquitination,which ultimately stabilized c-Myc in NPC.We then examined the expression of circCAMSAP1,SERPINH1 and c-Myc by in situ hybridization and immunohistochemistry in NPC tissues obtained in a nude mouse lung metastasis model and a nude mouse subcutaneous tumor model The result showed that circCAMSAP1 was successfully overexpressed or knocked down,SERPINH1 and c-Myc were highly expressed in tissues overexpressing circCAMSAP1 and low expressed in tissues knocking down circCAMSAP1,the expression profiles of the three were positively correlated.[c-Myc combined with SRSF10 promotes circCAMSAP1 production]Bioinformatics software showed that c-Myc could bind to CAMSAP1 promoter region.We have demonstrated that c-Myc is positively regulated by circCAMSAP1 in NPC.qRT-PCR identified that c-Myc promotes the expression of CAMSAP1 pre-mRNA and circCAMSAP1,dual-luciferase reporter gene assay and chromatin immunoprecipitation assay identified that c-Myc binds to the promoter region of CAMSAP1 and promotes gene transcription,suggesting that circCAMSAP1 promotes malignant progression of NPC via a SERPINH1/c-Myc positive feedback loop.SRSF10 had been predicted to bind to the introns flanking exons 2 and 3 of CAMSAP1 pre-mRNA and was also highly expressed in NPC cells.qRT-PCR and RNA immunoprecipitation assay confirmed that SRSF10 bound to the flanking intron region of circCAMSAP1 pre-mRNA and promoted the expression of circCAMSAP1.qRT-PCR and Western blotting showed that c-Myc promoting SRSF10 expression in NPC.Taken together,these results indicate that c-Myc is involved in the transcription and formation of circCAMSAP1,cooperating with SRSF10.ConclusionIn this study,we reported a new circRNA,circCAMSAP1 was highly expressed in NPC tissues and cell lines and positively correlated with the clinical stages and TNM stages of patients.circCAMSAP1 promoted metastasis and proliferation of NPC cells in vitro and in vivo.Further,we demonstrated that circCAMSAP1 promoted the growth and aggressiveness of NPC cells by stabilizing SERPINH1 expression through binding to SERPINH1 mRNA 3’UTR.Additionally,the binding of SERPINH1 to cMyc inhibited the ubiquitination-degradation of c-Myc.Meanwhile,c-Myc along with SRSF10 could promote CAMSAP1 pre-mRNA transcript and back-splicing,and form a positive feedback on circCAMSAP1 production,resulting in proliferation and metastasis of NPC.This study expands the understanding of circRNA function in NPC pathogenesis and suggests a novel circRNA as a potential biomarker and therapeutic target for NPC.