Circular RNA CircRILPL1 Promotes Nasopharyngeal Carcinoma Malignant Progression By Activating The Hippo-YAP Signaling Pathway

ObjectiveNasopharyngeal carcinoma(NPC)is a malignant tumor originating from nasopharyngeal epithelium and has obvious regional and ethnic distribution,mostly in Southeast Asia and Southern China.Genetic components and environmental factors such as Epstein-Barr virus infection are considered to be the synergic pathogenic contributors to NPC,which exhibits strong tendency of local invasion and lymph node metastasis.However,during the pathogenesis of NPC,the oncogenic genomic changes and the relevant signaling processes remain far from clear.Circular RNAs(circRNAs)are covalently closed non-coding RNAs formed by back-splicing at the 3’ and 5’ ends.They have tissue-specific expression patterns,conserved and stable structures.In recent years,using high-throughput sequencing technology and bioinformatics techniques,several circRNAs have been found to play important regulatory roles during tumorigenesis,including NPC.However,their specific roles and pathological mechanisms remain largely unknown.CircRNA circRILPL1,which was highly expressed in NPC tissues,was identified through RNA-seq analysis.The aim of this study was to investigate the signalling pathways and action mechanisms regulated by circRILPL1 in NPC,and its important role in the malignant progression of NPC.MethodsRNA-seq dataset for NPC in the GEO database was downloaded and performed differential expression analysis using SAM.Basic circRNA information such as full-length sequences and transcripts were obtained from the circ Base website,and sequence validation of circRILPL1 was performed using Sanger sequencing.The expression of circRILPL1 in NPC clinical samples was examined by q RT-PCR and in situ hybridization(ISH).RNase R digestion and actinomycin D treatment were performed to verify the stability of circRILPL1.We performed functional assays and mechanistic research on circRILPL1 by constructing an overexpression vector and an antisense oligonucleotide probe specifically designed based on splice site.The effects of circRILPL1 on the migration and invasion of NPC cells in vitro were examined by wound healing and Transwell assays.The effect of circRILPL1 on the proliferation ability of NPC cells was investigated by MTT and colony formation assays.The effect of circRILPL1 on mechanical properties of NPC cells was determined by atomic force microscopy(AFM).A subcutaneous tumor model,a tail vein-lung metastasis model and a footpad-lymph node metastasis model were constructed to determine the tumor-promoting effects of circRILPL1 in vivo.Proteomic analysis was performed by liquid chromatography-tandem mass spectrometry to screen the signalling pathways and binding proteins regulated by circRILPL1.The binding of circRILPL1 to ROCK1 and IPO7 was examined by RNA pull down,RIP,and IF-FISH assays.The specific sequence of circRILPL1 interacting with ROCK1 and IPO7 was determined by constructing deletion-mutants of circRILPL1.The regulatory relationship between circRILPL1 and ROCK1 on key proteins of the Hippo pathway was analysed by western blotting and q RT-PCR.The effect of circRILPL1 and IPO7 on the nucleoplasmic distribution of YAP proteins was analysed by immunofluorescence and cytosolic/nuclear protein fractionation assays.Dual luciferase reporter gene assays were performed to determine the transcriptional activity of YAP.The effect of circRILPL1 on the enrichment of YAP on CAPN2 and PXN promoters was examined by ChIP assays.Finally,we also examined the expression of the key downstream target genes of circRILPL1,YAP,ROCK1 and IPO7,using immunohistochemistry(IHC)in NPC tissue samples.Results【 circRILPL1 is highly expressed in NPC and associated with poor prognosis of NPC patients】By SAM analysis of the NPC RNA-seq dataset(GSE68799),30 circRNAs with high expression in NPC tissues were identified,among which circRILPL1(hsa＿circ＿0007552)was not reported in NPC before.Results of q RT-PCR and Sanger sequencing confirmed that circRILPL1 was formed by circular splicing of the exons 3 and 4 of RILPL1pre-m RNA,with a total length of 341 nt.The q RT-PCR results showed that circRILPL1 was significantly highly expressed in NPC tissues.The results of ISH revealed that the expression of circRILPL1 in 99 NPCs was significantly higher than that in 46 non-cancer nasopharyngeal epithelial(NPE)tissues,and the overall survival rate of patients with high circRILPL1 expression was lower than that of patients with low circRILPL1 expression.RNase R digestion assays and Actinomycin D treatment showed that the stability of circRILPL1 was much higher than that of RILPL1 linear m RNA.Results of RNA cytosolic/nuclear fraction assay and FISH showed that circRILPL1 was distributed both in the cytoplasm and the nucleus.【 circRILPL1 promotes the proliferation and metastasis of NPC cells,and alters the mechanical properties of NPC cells in vitro】Overexpression of circRILPL1 by constructing a circRILPL1 overexpression vector,or its expression was knocked down by using the specific ASO targeting circRILPL1 splice site.Wound healing and Transwell assays showed that circRILPL1 could significantly enhance the migration and invasion of NPC cells.MTT and colony formation assays showed that circRILPL1 promoted NPC cells proliferation.AFM assays showed that overexpression of circRILPL1 resulted in weakened adhesion and decreased stiffness of NPC cells,suggesting that cells were easier to detach from the surrounding tissues and their deformability was enhanced,which is consistent with the elevated capabilities of invasion and metastasis in NPC cells with high expression of circRILPL1.And opposite results were obtained after knockdown of circRILPL1.Immunofluorescence showed that overexpression of circRILPL1 promoted the polymerization of actin filaments,whereas knockdown of circRILPL1 resulted in reduced microfilaments and loss of cytoskeleton integrity in NPC cells.The above results indicated that circRILPL1 regulated the mechanical properties of NPC cells and promoted their proliferation,invasion,and migration in vitro.【circRILPL1 promotes the proliferation and metastasis of NPC cells in vivo】The subcutaneous tumor model showed that overexpression of circRILPL1 significantly increased the volume and weight of tumors in nude mice.IHC data revealed that circRILPL1 promotes the expression of Ki67,indicating that circRILPL1 promoted NPC cells proliferation in mice.In the tail vein-lung metastasis model,the number and area of lung metastatic nodules were significantly increased in the circRILPL1 overexpression group,while there were fewer and smaller lung metastatic nodules in the circRILPL1 knockdown group when compared with the control group,indicating that circRILPL1 promoted lung metastasis of NPC cells in mice.In the footpad-lymph node metastasis model,circRILPL1 significantly increased the weight and volume of inguinal lymph nodes.H&E staining confirmed that the area and number of metastatic tumor cells in lymph nodes were significantly increased after overexpression of circRILPL1,while there were fewer metastatic tumor cells in lymph nodes in the circRILPL1 knockdown group compared to the control group.All of these data demonstrated that circRILPL1 promoted NPC growth and metastasis in vivo.【circRILPL1 activates the Hippo-YAP signaling pathway】The liquid chromatography coupled to tandem mass spectrometry was performed to identify differentially expressed molecules regulated by circRILPL1 in HNE2 cells.DAVID analysis further revealed that some proteins were enriched in the Hippo signalling pathway.Western blotting showed that phosphorylation of YAP(Ser127 and Ser397),total YAP,and phosphorylation of LATS1 were modulated in NPC cells by overexpression or knockdown of circRILPL1,with no significant effect on MST1.Cycloheximide(CHX)treatment and ubiquitination assays revealed that circRILPL1 inhibited the ubiquitin-mediated degradation of YAP and increased its protein stability,leading to the upregulation of YAP protein level.Immunoprecipitation showed that circRILPL1 inhibited the binding of YAP to 14-3-3.Furthermore,circRILPL1 enhanced the abundance of YAP in nucleus while reduced the amount of YAP in cytoplasm.The CTGF luciferase reporter assay revealed that circRILPL1 significantly enhanced the transcriptional activity of YAP.The expression of YAP was higher in the circRILPL1 overexpression group and lower in the circRILPL1 knockdown group in nude mice subcutaneous tumors and lung metastases tumors.Overexpression of YAP partially restored the inhibitory effect of circRILPL1 knockdown on migration,invasion,and proliferation of NPC cells.AFM assays revealed that overexpression of YAP partially reversed the circRILPL1 knockdown-induced changes of cell biophysical properties.Additionally,knockdown of YAP significantly impaired the promotive effects of circRILPL1 overexpression on migration,invasion,and proliferation in NPC cells.Alterations of mechanical properties induced by circRILPL1 overexpression in NPC cells were also reversed by knockdown of YAP.These data suggested that the function of circRILPL1 in NPC cells was dependent on activation of the YAP.【 circRILPL1 inhibits the LATS1-YAP kinase cascade by binding to ROCK1】Rho associated coiled-coil containing protein kinase 1(ROCK1),which may bind to circRILPL1,was screened by mass spectrometry analysis of RNA pull down products.RNA pull down,RIP and IF-FISH assays confirmed the binding of circRILPL1 to ROCK1.Western blotting results showed that circRILPL1 promoted the phosphorylation of Thr 696 on MYPT1,a well-known substrate of ROCK1,but had no effect on the expression of ROCK1 at the protein and m RNA levels.Treatment with ROCK1 inhibitor Y27632 or si RNA targeting ROCK1 in NPC cells significantly inhibited dephosphorylation of LATS1,which in turn inhibited dephosphorylation of YAP and resulted in decrease of YAP protein level.When Y27632 or ROCK1 si RNA were used in circRILPL1-overexpressing NPC cells,the dephosphorylation of LATS1 and YAP,and the stability of YAP induced by circRILPL1 were significantly reduced.These results suggested that circRILPL1-mediated ROCK1 activation was critical for the regulation of LATS1-YAP kinase cascade.Transwell and MTT assays demonstrated that knockdown of ROCK1 partially reversed the promoting effects of circRILPL1 overexpression on the invasion and proliferation capabilities of NPC cells.Together,these data demonstrated that ROCK1,as an important downstream effector of circRILPL1,is essential for the activation of YAP and promoting the proliferation and migration of NPC cells.【circRILPL1 promotes YAP protein nuclear translocation by binding to IPO7 】According to mass spectrometry analysis,we screened that importin7(IPO7),a nuclear transport receptor,was pulled down by circRILPL1.The interaction between circRILPL1 and IPO7 was further confirmed by RNA pull down,RIP assay,and IF-FISH assays.But ROCK1 did not bind to IPO7.To explore the role of IPO7 in the circRILPL1 regulated pathway,the data showed that circRILPL1 facilitated the entry of IPO7 into nucleus,but had no effect on IPO7 protein and m RNA expression.In addition,a weak interaction existed between endogenous IPO7 and YAP.But IPO7 did not regulate the expression and phosphorylation of YAP.Immunoprecipitation and immunofluorescence assay showed that circRILPL1 promoted the binding between IPO7 and YAP proteins.Knockdown of IPO7 hindered circRILPL1-induced translocation of YAP from the cytoplasm to the nucleus and inhibited YAP transcriptional activity,indicating that circRILPL1 increasing the YAP nuclear import was partially IPO7-dependent.And this inhibition was more pronounced after simultaneous knockdown of IPO7 and ROCK1.【 circRILPL1 interacts with ROCK1 and IPO7 through nucleotides136-189】Using cat RAPID and RNAfold bioinformatics tools,two well-characterized stem loop structures(136-189 nt and 251-291 nt)on circRILPL1 were predicted to bind with ROCK1 and IPO7 proteins.RNA pull-down and RIP assays showed that the deletion mutant DEL1 lack of136-189 nt failed to bind with ROCK1 and IPO7 proteins,whereas deletion mutant DEL2 lack of 251-291 nt still possessed the binding ability,suggesting that the 136-189 nt region was essential for circRILPL1 binding with ROCK1 and IPO7 proteins.The data showed that circRILPL1-DEL1 failed to activate the Hippo-YAP signaling pathway and could not stabilize and promote nuclear translocation of YAP protein,when compared with wild-type circRILPL1.Also,circRILPL1-DEL1 failed to promote the activation of ROCK1 and lost the potential to facilitate the binding between YAP and IPO7.Wound healing and MTT assays showed that the deletion mutant circRILPL1-DEL1 had no effect on the migration and proliferation of HNE2 cells.These data indicated that binding with ROCK1 and IPO7 through nucleotides 136-189 is crucial for circRILPL1 to promote malignant progression of NPC.IHC assays showed that the expression levels of YAP and ROCK1 protein were significantly higher in NPC clinical tissues than that in NPE tissues,while levels of IPO7 protein in NPC and NPE tissues had no difference.【circRILPL1-YAP signaling promotes the transcription of CAPN2 and PXN】To search for the key downstream effectors activated by the circRILPL1-YAP axis in NPC cells,those upregulated proteins identified by mass spectrometry analysis following overexpression of circRILPL1 and associated with cytoskeleton remodeling and malignant behaviors of cancers were further analyzed.The results revealed that circRILPL1 promoted the m RNA and protein expression of CAPN2 and PXN in NPC cells.Overexpression of YAP partially reversed the decrease of CAPN2 and PXN m RNA induced by circRILPL1 knockdown.ChIP experiment showed that circRILPL1 prompted the enrichment of YAP on the CAPN2 and PXN promoters in NPC cells.These results suggested that circRILPL1 promoted the transcription of CAPN2 and PXN through activation of YAP.ConclusionIn summary,circRILPL1,as a tumor activator,bound to and activated ROCK1 to inhibit the LATS1 kinase,which led to reduction of YAP phosphorylation at Ser127 and Ser397 sites.Binding and cooperating with nuclear transport receptor IPO7,circRILPL1 promoted YAP nuclear translocation by enhancing its interaction with IPO7.circRILPL1 activated the Hippo-YAP signaling pathway to promote the transcription of CAPN2 and PXN and NPC malignant progression.