Activation,Functions And Molecular Mechanisms Of CD109 In Nasopharyngeal Carcinoma And Fundamental Investigation On Its Application In Targeted Therapy

Background: Nasopharyngeal carcinoma(NPC)is a special type of head and neck malignant tumor with obvious regional and ethnic aggregation,and is highly prevalent in East and Southeast Asia.EBV infection,genetic factors,and environmental factors are the risk factors for NPC.Early diagnosis and effective treatment provide a significantly higher survival rate for patients with NPC.The clinical cure rate of early-stage NPC patients(Ⅰ-Ⅱ stage)who are sensitive to radiotherapy is high.The clinical cure rate is high,and the 5-year overall survival(OS)rate is 95.0%.However,since the pathogenesis of NPC is concealed and the early symptoms are not obvious,60.0%-80.0% of patients have been in the advanced stage(Ⅲ-Ⅳ stage)at their first diagnosis.The treatment effect of advanced-stage patients is not ideal and the 5-year OS rate is only 41.0%-63.0%.Meanwhile,58.0% of NPC patients experience recurrence and metastasis,and the median survival time is only about 20 months.Therefore,early diagnosis and effective treatment provide a significantly higher survival rate for patients with NPC.Objectives: To clarify the targeted binding ability of aptamer S3 to NPC cells.To detect the binding between S3 and CD109.To determine the expression levels of CD109 in NPC tissues and cells,and the correlation between CD109 expression levels and the prognosis of NPC patients.To investigate the effects of S3-Protamine-Si CD109(SPS)nanoparticles on the biological functions of NPC cells in vitro and in vivo.To investigate the biological functions of CD109 in NPC and the molecular mechanisms of CD109 promoting NPC development.To clarify the up-regulation mechanism of CD109 expression in NPC.Methods: 1 Clarify the up-regulation mechanism of CD109 expression in NPC:(1)Quantitative real-time PCR(qPCR)and western blot(WB)methods were used to analyze the effects of LMP1,p50,and mi R-203a-3p on CD109 expression in NPC.(2)Chromatin immunoprecipitation assay(ChIP),and dual-luciferase assays were used to analyze the upregulation mechanisms of CD109 in NPC.2 Study on the functions and mechanisms of CD109 in NPC:(1)The CCK8,colony formation,Edu,wound healing,transwell invasion,and flow cytometry assays were used to detect the effects of CD109 on NPC cells in vitro.(2)Subcutaneous transplantation tumor and lung metastasis models were established in nude mice to study the effects of CD109 on tumorigenicity and metastasis abilities of NPC cells in vivo.(3)The whole transcriptome sequencing technology was used to explore the molecular mechanisms of CD109 promoting NPC development.Further experiments were performed to elucidate the key signal pathways downstream of CD109 in NPC.3 Study on the targeted recognition ability of aptamer S3 to NPC cells and its application in targeted therapy by combining with si CD109:(1)The targeted recognition ability of aptamer S3 to NPC cells was detected by confocal microscopy imaging,flow cytometry,and in vivo fluorescence imaging.(2)Aptamer pull-down,immunofluorescence co-localization,and flow cytometric analysis were used to examine the binding capacity of S3 to CD109.(3)The expression of CD109 in NPC tissues and cells was analyzed with the Gene Expression Omnibus(GEO)and TCGA data,immunohistochemistry(IHC),and WB methods.(4)Kaplan-Meier method and log-rank test were used to analyze the survival rate.The univariate and multivariate Cox regression was used to evaluate whether CD109 was an independent prognostic factor for patients with NPC.(5)The CCK8,wound healing and transwell invasion assays were used to detect the effects of SPS on 5-8F cells.The effect of SPS on tumorigenicity in vivo was detected by a xenograft tumor model.Results:1 The IHC,qPCR,and WB results showed that EBV-LMP1 was positively correlated with CD109 expression.LMP1 upregulated CD109 expression by activating p50.qPCR and WB results showed that p50 positively regulated CD109 expression.ChIP assay and dual-luciferase reporter assay demonstrated that p50 bound to the CD109 promoter region.At the post-transcriptional level,LMP1 up-regulated CD109 expression by inhibiting the expression of mi R-203a-3p,which inhibited CD109 expression by directly binding to the 3’-UTR region of CD109 m RNA.2 The CCK8,colony formation,and Edu assays showed that depletion of CD109 in 5-8F cells could inhibit the proliferation ability of 5-8F cells,and over-expression of CD109 in 6-10 B cells could enhance the proliferation ability of 6-10 B cells.The wound healing assay showed that depletion of CD109 in 5-8F cells could suppress the migration ability of NPC cells,and overexpression of CD109 could strengthen the migration ability of NPC cells.Transwell invasion assay showed that depletion of CD109 could inhibit the invasion ability of NPC cells and overexpression of CD109 enhance the invasion ability of NPC cells.The flow cytometry assay showed that depletion of CD109 in 5-8F cells arrested the cell cycle at the G1/S phase;overexpression of CD109 could promote the G1/S phase conversion.The flow cytometry assay showed that depletion of CD109 induced the apoptosis of NPC cells;overexpression of CD109 inhibited the apoptosis of NPC cells.In vivo experiments showed that depletion of CD109 in 5-8F cells could decrease the tumor weight and volume compared to the 5-8F-control group,and the protein level of CD109,Ki67 was decreased remarkably.Meanwhile,overexpression of CD109 in 6-10 B cells could increase the tumor weight and volume of tumors compared to the 6-10B-control group,and the protein levels of CD109 and Ki67 increased in the 6-10B-CD109 group.In the lung metastasis model,compared to the 5-8F-control group,the number of lung metastatic nodules was significantly decreased in the 5-8F-sh CD109 group.After the depletion of CD109 in NPC cells,the protein level of TGFBR2 increased significantly.Overexpression of CD109 in NPC cells promoted the degradation of TGFBR2 by the ubiquitin-dependent protein degradation pathway,and the TGF-β signaling pathway was inactivated.Meanwhile,CD109 could also activate the Wnt-β-catenin signaling pathway by inhibiting the ubiquitination of β-catenin.3 Confocal microscopy imaging,flow cytometry,and in vivo fluorescence imaging results showed that S3 could specially bind to NPC cells in vitro and in vivo.Aptamer pull-down,immunofluorescence co-localization,and flow cytometric analysis demonstrated that CD109 was the binding target of S3.CD109 was highly expressed in both NPC tissues and cells.The high expression of CD109 was positively correlated with a poor prognosis for NPC patients.The univariate and multivariate Cox regression results showed that CD109 was an independent prognostic factor for NPC patients.The size of SPS was about 103 nm.The CCK8,wound healing and transwell invasion assays demonstrated that the SPS nanoparticles could inhibit the proliferation,migration,and invasion abilities of NPC cells.In the animal model,the SPS could suppress the tumor formation of NPC cells in vivo.Conclusions:1 LMP1 up-regulated the expression of CD109 in NPC through a transcriptional pathway mediated by p50 and a post-transcriptional pathway mediated by mi R-203a-3p.p50 is the transcriptional activator of CD109.mi R-203a-3p could suppress the expression of CD109 in NPC by binding the 3’-UTR of CD109.2 CD109 functions as an oncogene protein that promotes NPC cell proliferation,migration,invasion,and metastasis.High expression of CD109 in NPC suppresses TGF-β signaling via promoting the ubiquitin-dependent proteolysis of TGFBR2.CD109 could also activate the Wnt pathway via suppressing the ubiquitin-dependent proteolysis of β-catenin and stabilizing it.3 Aptamer S3 presents a high affinity for CD109 on NPC cells both in vitro and in vivo.We reveal that CD109 is highly expressed in NPC tissues and NPC cells and find that high CD109 expression is correlated with an unfavorable prognosis in NPC patients.The SPS nanoparticles can selectively target NPC cells and silence CD109 expression.Functional analysis results indicate that SPS nanoparticles significantly inhibit NPC cell proliferation,migration,and invasion in vitro and tumorigenicity in vivo.