| Background and purpose:Nasopharyngeal carcinoma(NPC)is a malignant head and neck tumor originating from nasopharyngeal epithelial cells.It has obvious regional aggregation,high degree of malignancy and strong ability of invasion and metastasis.Although great progress has been made in the pathogenesis,early diagnosis and clinical treatment of NPC in recent years,the efficacy and prognosis of NPC patients have not been greatly improved,and local recurrence and distant metastasis are still the main reasons for the low survival rate of advanced NPC patients.In addition,due to the hidden location and atypical early symptoms of nasopharyngeal cancer patients,many patients with nasopharyngeal cancer are initially diagnosed as advanced and miss the best clinical treatment stage,so it is easy to develop resistance to radiotherapy and chemotherapy and distant metastasis.Therefore,the development mechanism of nasopharyngeal carcinoma and the screening of clinical diagnosis and treatment targets are still the frontier and hotspot of current research in the field of nasopharyngeal carcinoma.BRD7 is a new full-length gene with low expression in nasopharyngeal carcinoma,which was independently isolated and cloned by our research group through c DNA representative difference analysis and library screening.It was confirmed that the expression of BRD7 was downregulated in nasopharyngeal carcinoma tissues and cell lines,and was negatively correlated with the clinical progress and poor prognosis of nasopharyngeal carcinoma patients.Through the negative regulation of ERK,Rb/E2 F and PI3K/AKT signaling pathways,the proliferation,migration and invasion of nasopharyngeal carcinoma cells as well as the growth and metastasis of tumors in vivo are inhibited,thus playing the role of tumor suppressor genes in nasopharyngeal carcinoma.However,it has not been reported whether BRD7 can play a role in nasopharyngeal carcinoma in the form of non-coding RNA in addition to its protein coding function.Circular RNAs(circRNAs)are a class of new non-coding RNAs with regulatory ability,which are closed circular structures formed by reverse splicing of precursor m RNA.They have been confirmed to be abnormally expressed in various tumors such as nasopharyngeal carcinoma and participate in the occurrence and development of tumors in the form of tumor genes or tumor suppressor genes.Therefore,this study intends to screen and confirm the circRNAs encoded by BRD7,reveal its expression characteristics,biological functions and mechanisms in nasopharyngeal carcinoma,and provide new molecular targets and strategies for clinical diagnosis and treatment of nasopharyngeal carcinoma.Methods:Five circRNAs encoded by BRD7 were retrieved through bioinformatics analysis,and one circRNA(circ＿0039345)splicing site was identified by agarose gel electrophoresis.The circRNA sequence was sequenced and verified by Sanger sequencing method and named circBRD7.The localization of circBRD7 in nasopharyngeal carcinoma cells was detected by FISH and cytoplasmic separation assay,and its stability was verified by actinomycin D treatment.The expression of circBRD7 in nasopharyngeal carcinoma cells and tissues was detected by qPCR assay.Subsequently,nasopharyngeal carcinoma cell lines with overexpression and silencing of circBRD7 were constructed,and various cell biological experiments including CCK-8,colony formation assay,flow cytometry,scratch healing and matrix gel invasion were performed.To investigate the effects of circBRD7 overexpression and silencing on proliferation,colony formation,cell cycle progression,apoptosis,migration and invasion of nasopharyngeal carcinoma cells.The effects of circBRD7 on BRD7 expression level and BRD7 on circBRD7 expression level were investigated by qPCR and Western Blot.The regulation mechanism of circBRD7 on BRD7 expression was investigated by Ch IPqPCR.The important role of BRD7 in tumor suppressive function of circBRD7 was discussed through a series of recovery experiments.The expressions of circBRD7 and BRD7 in nasopharyngeal epithelial tissues and nasopharyngeal carcinoma biopsies at different stages were detected by qPCR,in situ hybridization and immunohistochemistry,and the correlation between circBRD7 and BRD7 was analyzed by Pearson method.Graph Pad Prism software was used to analyze the correlation between circBRD7,BRD7 and their combined expressions and clinicopathological features.Results:1.CircBRD7 was confirmed to be a circRNA encoded by its host gene BRD7In order to confirm whether BRD7 can play a role as circRNAs in nasopharyngeal carcinoma,we predicted the circRNAs encoded by BRD7 gene through bioinformatics analysis,and found that BRD7 may encode 5potential circRNAs,including circ＿0039343,circ＿0039344,circ＿0039345,circ＿19158 and circ＿0105514.Furthermore,by designing divergent primers for splicing sites,RT-qPCR was used to verify the existence of circRNAs.Among the five potential circRNAs encoded by BRD7,only circ＿0039345 had reverse splicing site sequences.Sanger sequencing further confirmed that circ＿0039345 was formed by reverse shearing of BRD7 exon 6-8,with a total length of 420 bp.These experimental results support the existence of circ＿0039345 in nasopharyngeal carcinoma cells,and named it circBRD7.Subsequently,the expression of circBRD7 in normal nasopharyngeal epithelial cells NP69 and 9 nasopharyngeal carcinoma cell lines(CNE1,CNE2,HNE1,HNE2,HONE1,5-8F,HK1,6-10 B and C666-1)was detected by RT-qPCR.And the results showed that,expression of circBRD7 in nasopharyngeal carcinoma cell lines was significantly lower than that in normal nasopharyngeal epithelial cell line NP69.In addition,we detected the expression of circBRD7 in nasopharyngeal non-cancer tissues and nasopharyngeal biopsy tissues by RT-qPCR,and found that the expression of circBRD7 in nasopharyngeal carcinoma tissues was significantly lower than that in nasopharyngeal noncancer tissues.The results of fluorescence in situ hybridization and nucleocytoplasmic separation showed that circBRD7 was distributed in both the nucleus and cytoplasm,with the nucleus being the dominant one.To test the stability of circBRD7,we treated 5-8F and CNE2 cells with actinomycin D to inhibit the intracellular RNA transcription,and then extracted total RNA for RT-qPCR detection.It was found that circBRD7 was more stable than the linear m RNA of BRD7 gene.These results suggest that circBRD7 is a down-regulated true circRNA encoded by BRD7 gene in nasopharyngeal carcinoma.2.CircBRD7 can inhibit the proliferation,migration,invasion and epithelial-mesenchymal transformation of nasopharyngeal carcinoma cells,thus playing the role of tumor suppressor gene in nasopharyngeal carcinomaCircBRD7 was overexpressed and silenced in nasopharyngeal carcinoma cell lines 5-8F and CNE2 by using circBRD7 overexpressed plasmid and silenced si RNAs,respectively,and the overexpression and silencing efficiency of circBRD7 were verified by RT-qPCR.By CCK-8and colony formation experiments,it was found that overexpression of circBRD7 could inhibit proliferation and colony formation of nasopharyngeal carcinoma cells.Silencing circBRD7 significantly promoted proliferation and colony formation of nasopharyngeal carcinoma cells.Flow cytometry results showed that overexpression of circBRD7 significantly inhibited G1/S process and promoted apoptosis of nasopharyngeal carcinoma cells.The results of wound healing experiment and matrix gel invasion experiment showed that overexpression of circBRD7 could inhibit the migration and invasion of nasopharyngeal carcinoma cells,while silencing circBRD7 expression had the opposite results.The changes of cell cycle,apoptosis and EMT related molecules before and after circBRD7 overexpression were detected by Western Blot.It was found that overexpression of circBRD7 inhibited the expression of CDK4,Total-PARP,N-cadherin,Vimentin and Snail,and promoted the expression of P21,Cleaved-PARP,E-cadherin and other molecules.Silencing circBRD7 expression showed the opposite result.These results suggest that circBRD7 plays a role of tumor suppressor gene in nasopharyngeal carcinoma.3.CircBRD7 can promote the enrichment of H3K27 ac in the BRD7 promoter region and thus promote the transcriptional activation and expression of BRD7A large number of studies have shown that the regulation of circRNA on host genes is an important molecular mechanism for its function.BRD7 is the host gene of circBRD7.Therefore,in this study,we first discussed the influence of circBRD7 on the expression of its host gene BRD7 and its regulatory mechanism.RT-qPCR and Western Blot analysis showed that overexpression of circBRD7 could promote the m RNA and protein expression of BRD7.Silencing circBRD7 results in reduced BRD7 expression.In addition,we also found that BRD7 expression could upregulate circBRD7 expression in a positive feedback manner.RNA fluorescence in situ hybridization and nucleo-cytoplasmic separation assays have confirmed that circBRD7 is predominantly distributed in the nucleus.These results suggest that circBRD7 may regulate BRD7 expression through transcriptional mechanisms.Dualluciferase reporter assay showed that overexpression of circBRD7 could enhance luciferase activity of BRD7 promoter reporter plasmid.Through bioinformatics analysis of BRD7 transcriptional modification regions,it was found that the promoter region of BRD7 was highly enriched with H3K27 ac modification,suggesting that circBRD7 may up-regulate BRD7 expression by enhancing H3K27 ac modification in the BRD7 promoter region.Ch IP-qPCR results showed that H3K27 ac was enriched in BRD7 promoter region in both nasopharyngeal carcinoma 5-8F and CNE2 cells,and circBRD7 enhanced H3K27 ac modification in BRD7 promoter-2000～-1407 region.The micro RNAs co-binding circBRD7 and its host gene BRD7 were further studied,and it was found that mi R-498 and mi R-944 could both bind to them,but the two micro RNAs absorbed by circBRD7 could not regulate BRD7 expression.These results suggest that ce RNA is not an important mechanism for circBRD7 to regulate the expression of its host genes,and that circBRD7 mainly promotes the transcriptional activity and expression of BRD7 by enhancing the enrichment of H3K27 ac in the BRD7 promoter region.4.Restoring BRD7 expression can partially reverse the inhibition of circBRD7 on malignant phenotype in nasopharyngeal carcinoma cellsTo investigate the role of BRD7 in circBRD7-mediated tumor suppressive phenotype of nasopharyngeal carcinoma,we co-transfected 5-8F and CNE2 cells with si BRD7 and circBRD7 to restore BRD7 expression.By CCK-8,colony formation assay,flow cytometry,wound healing and matrix gel invasion experiments,it was found that the inhibition of circBRD7 overexpression on cell proliferation,colony formation,cycle progression,migration and invasion and the promotion of apoptosis could be partially reversed after the restoration of BRD7 expression.Cell cycle,apoptosis and expression of EMT-related molecules were further examined.It was found that the restoration of BRD7 expression could reverse the inhibitory effect of circBRD7 on CDK4,Total-PARP,N-cadherin,Vimentin and Snail,and the promoting effect on P21,Cleaved-PARP,E-cadherin and other molecules.These results suggest that circBRD7 plays a role as a potential tumor suppressor gene in the development and progression of nasopharyngeal carcinoma by positively regulating the expression of its host gene BRD7 in nasopharyngeal carcinoma cells.5.In vivo experiments confirmed that circBRD7 inhibited tumor growth by enhancing BRD7 expressionThe nasopharyngeal carcinoma cell line 5-8F with circBRD7 overexpression and BRD7 restored expression was used to construct the nude mouse xenograft tumor model of nasopharyngeal carcinoma cells.By detecting the growth curve and tumor size of xenograft tumor in nude mice,it was found that overexpression of circBRD7 significantly inhibited the growth of xenograft tumor in nude mice.Restoration of BRD7 expression can partially reverse the inhibitory effect of circBRD7 overexpression on tumor growth.Furthermore,immunohistochemistry was used to detect the proliferation,cell cycle and expression of EMT-related molecules in nude mice,and it was found that circBRD7 overexpression could inhibit the expression of Ki67,CDK4 and Vimentin,and promote the expression of P21 and E-cadherin.Restoration of BRD7 expression reversed the inhibition or promotion of circBRD7 on the expression of these molecules.Therefore,in vivo results further confirmed that circBRD7 inhibited tumor growth in vivo through positive regulation of BRD7 expression.6.Expression and clinical correlation of circBRD7 and BRD7 in nasopharyngeal carcinoma biopsy tissuesIn order to better understand the expression and clinical correlation of circBRD7 and BRD7 in nasopharyngeal carcinoma clinical samples,we detected the expression of circBRD7 and BRD7 in 43 nasopharyngeal noncarcinoma patients and 78 clinical biopsy patients with different stages of nasopharyngeal carcinoma by in situ hybridization(ISH)and immunohistochemical staining(IHC),respectively.It was found that both circBRD7 and BRD7 were underexpressed in nasopharyngeal carcinoma tissues,and circBRD7 expression was positively correlated with BRD7 expression.The expression of circBRD7 and BRD7 and their combination were further analyzed in relation to the clinicopathological features of nasopharyngeal carcinoma.The results showed that circBRD7 and BRD7 had no significant correlation with the age and gender of patients with nasopharyngeal carcinoma,but had a significant negative correlation with clinical stage.The combined analysis of circBRD7 and BRD7 expression yielded similar results.These results suggest that low circBRD7 expression and low BRD7 expression can be used as important molecular markers for the evaluation of malignant progression of nasopharyngeal carcinoma,and targeting circBRD7/BRD7 signaling axis is expected to be a potential molecular target for the clinical treatment of nasopharyngeal carcinoma,with important clinical significance.Conclusion:1.CircBRD7 was confirmed to be the circRNA encoded by its host gene BRD7,which can inhibit the proliferation,migration and invasion of nasopharyngeal carcinoma cells and tumor growth in vivo,thus playing the role of tumor suppressor gene in nasopharyngeal carcinoma.2.CircBRD7 can enhance the transcriptional activity and expression of its host gene by enhancing H3K27 ac enrichment in the BRD7 promoter region.3.Restoration of BRD7 expression can reverse the inhibitory effect of circBRD7 on the proliferation,migration,invasion and tumor growth of nasopharyngeal carcinoma cells,and support the inhibition of the occurrence and development of nasopharyngeal carcinoma by promoting the expression of its host gene.4.The low expression of circBRD7 in nasopharyngeal carcinoma tissues is positively correlated with the expression of BRD7 and negatively correlated with the clinical stage of nasopharyngeal carcinoma patients.The combination of circBRD7 and BRD7 can be used as an important molecular marker for the evaluation of malignant progression of nasopharyngeal carcinoma. |
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