| Objective:Ameloblastoma(AB),as one of the most common odontogenic tumors in the jaw,has a biological behavior of local invasiveness and recurrence.The early symptoms of AB are not obvious.Curettage or cyst fenestration is commonly used.It is easy to relapse.After long-term progression,it severely invades the jaw tissue.At this time,enlarged lesions and jaw segment resection are often used,which greatly affects the patients’ postoperative quality of life.AB,as a kind of odontogenic tumor of epithelial origin,its recurrence rate is as high as 70%,and the tumor metastasis rate is as high as 2%.In 2017,WHO’s new classification of head and neck tumors and odontogenic cysts divided AB into traditional type,single cystic type,extraosseous/peripheral type and metastatic AB,of which solid/polycystic type and connective tissue proliferation type AB collectively referred to as traditional AB(conventional ameloblastoma),and a new classification of metastatic AB has been added.Circular RNAs(circular RNAs,circ RNAs)are a type of non-coding RNAs(nc RNAs)that are different from ordinary 5’-3’ end linear RNA structures and have a closed circular structure end to end.It exists in the eukaryotic transcriptome and is not easily degraded by RNase.It mainly regulates genes related to tumorigenesis and development at the transcription and post-transcriptional levels,and plays an important regulatory role in tumor physiology and pathology.Circular RNA has a high degree of conservation and stability,expression of space and timeliness and tissue specificity,so it can be used as a potential biomarker and therapeutic target for tumor diagnosis.miRNA is a non-coding RNA molecule containing 19-25 bases.It usually regulates gene expression at the post-transcriptional level by promoting m RNA degradation,cutting specific sites,or inhibiting translation.The function of miRNA is mainly to negatively regulate target genes at the post-transcriptional level,in particular to regulate m RNA by specifically binding to 3′UTR sites or to promote the cleavage of specific sites of m RNA by binding to m RNA coding sequences,etc.,thereby regulating cell differentiation,migration,and apoptosis.Physiological processes such as death and maintaining cell homeostasis.In the occurrence and development of tumors,miRNAs play an important role.They can be used as tumor suppressors and tumor promoters to regulate the pathological process of tumors at different stages.CTNND1,also known as p120-catenin(p120),is identified as a proto-oncogene or as a tumor suppressor gene to play different physiological and pathological roles in a variety of diseases,and is mainly combined with classic and type II cadherin The cytoplasmic tail interacts to regulate cell adhesion.CTNND1 affects tumor cell adhesion and polarity in a variety of tumors,thereby affecting tumor proliferation,invasion,migration,and other features.Studies have shown that CTNND1 knockout mice found precancerous lesions and tumor lesions in the oral cavity,esophagus and stomach.The purpose of this study is to screen the data of circ RNA microarrays and conduct quantitative tissue verification to further verify its downstream miRNA and m RNA that may have binding sites with it.After the ceRNA network is further constructed,the progress of the ceRNA network axis to AB The impact of the disease provides potential new directions for the occurrence and development of AB and diagnosis and treatment.Methods:1.Screening circ RNA microarray data,potential circ RNA targets with differential expression and specific regulation based on their expression and function prediction,and tissues quantitative verification was utilized.2.Further verification of downstream miRNAs and m RNAs and potential binding sites was done.Prediction of further functional enrichment and pathway enrichment with the help of bioinformatics methods was accomplished,in order to further construct a ceRNA network.3.Immunohistochemical staining technique was used to detect the expression of CTNND1 in AB tissues.4.Nuclear and cytoplasmic isolation experiments was used to identify the expression of hsa＿circ＿0000517 in the nucleus and cytoplasm.5.Evaluation of the effect of silence hsa＿circ＿0000517 on the expression of hsa-miR-1296,and the effect of silence hsa＿circ＿0000517 on the features of AB invasion and migration through wound-healing experiment and transwell invasion experiment.6.Overexpression/inhibition of hsa-miR-1296 to observe the changes in CTNND1 transcription level and protein expression level.Wound healing experiment and transwell invasion experiment were utilized to observe the effect of overexpression/inhibition of hsa-miR-1296 on AB invasion and migration characteristics.The luciferase reporter experiment was used to verify the binding of target genes.7.Detection the expression levels of hsa-miR-1296 and CTNND1 by constructing a rescue experimental model that silences hsa＿circ＿0000517/NC and hsa-miR-1296/NC at the same time;with the aid of the wound healing experiment,the transwell invasion experiment is used to observe the response of the experimental model to AB invasion and migration.Results:1.Screen the differential expression among the 60 circ RNAs with the highest/lowest differential expression multiples.The source of exons,circ RNAs that may exist in the cytoplasm and whose function may be related to tumorigenesis and development,were finally screened out of 6 circ RNAs.RT-PCR verified that 6 circ RNAs were differentially expressed in AB tissues.2.Using bioinformatics methods to predict downstream miRNA and m RNA,their function enrichment and pathway enrichment,and further construction of ceRNA network,it is found that hsa-miR-1296 and hsa＿circ＿0000517 are more likely to bind.The miRDB database,miRWalk database,and Target Scan database were used to predict m RNAs that may have potential binding sites with hsa-miR-1296.The intersection of the three databases was used to screen out 124 m RNAs,and it was found that CTNND1 is closely related to the occurrence and development of ameloblastoma.So far,the research object is preliminarily determined as hsa＿circ＿0000517/hsa-miR-1296/CTNND1 axis.3.The expression of CTNND1 was detected on AB tissue samples by immunohistochemical staining,and statistical analysis was performed by semi-quantitative methods.The results showed that CTNND1 expression was related to the age of AB patients and the pathological type of AB(P<0.05),and the location of the tumor was mainly related to plexiform AB(P<0.05).CTNND1 is mainly expressed in the cytoplasm in AB.4.The results of nuclear and cytoplasmic separation experiments show that hsa＿circ＿0000517 is expressed in the nucleus and cytoplasm.5.The hsa＿circ＿0000517 transfection silencing model was constructed,and the expression level of hsa-miR-1296 was significantly increased(P<0.05).Construction of hsa＿circ＿0000517 transfection silence model,human ameloblastoma cell migration and invasion ability was significantly enhanced(P<0.0001).6.Construct the hsa-miR-1296 mimics/inhibitor/NC transient transfection model.The expression of CTNND1 in the hsa-miR-1296 mimics group was significantly reduced(P<0.0001),and the expression of CTNND1 was significantly increased in the hsa-miR-1296 inhibitor group(P<0.01),and there was a significant difference in CTNND1 expression between the hsa-miR-1296 mimics group and the inhibitor group(P<0.0001).CTNND1 protein expression level: the expression of CTNND1 in the miR-1296-mimics group was significantly lower than that in the NC group(P<0.05),the expression of CTNND1 in the miR-1296-inhibitor group was significantly higher than that in the NC group(P<0.01),in miR-1296-inhibitor group the expression of CTNND1 in the miR-1296-mimics group was significantly increased(P<0.05).In the hsa-miR-1296 mimics/inhibitor/NC transfection model,hsa-miR-1296 mimics significantly promoted the migration rate of HAM1(P<0.001),and the migration ability of HAM1 in the hsa-miR-1296 mimics group was obvious Stronger than hsa-miR-1296 inhibitor group(P<0.001);hsa-miR-1296 mimics significantly enhanced the invasion ability of HAM1(P<0.0001),and the invasion ability of hsa-miR-1296 mimics group was significantly stronger than hsa-miR-1296 inhibitor group(P<0.0001).The results of the dual luciferase report experiment show that there is a binding between hsa-miR-1296/CTNND1.7.Construct a response experiment of silencing hsa＿circ＿0000517/NC while silencing hsa-miR-1296/NC.After silencing hsa＿circ＿0000517,the expression of hsa-miR-1296 was significantly increased(P<0.05),and the expression of CTNND1 was significantly reduced(P<0.01);After miR-1296,the expression of hsa-miR-1296 was significantly reduced(P<0.0001),while the expression of CTNND1 was significantly increased(P<0.01);reply to the experimental group(si-circ＿0000517+ miR-1296-inhibitor)hsa-miR-1296 and CTNND1 There is no significant difference in the level of expression.Constructed a rescue experimental model of silence hsa＿circ＿0000517/NC while silence hsa-miR-1296/NC,the results showed that silence hsa＿circ＿0000517 significantly enhanced the migration ability of HAM1(P<0.0001),and replied to the experimental group(si-circ＿0000517+ miR-1296-inhibitor)Was also observed to promote the migration ability of HAM1(P<0.01).Silencing hsa＿circ＿0000517 significantly enhanced the invasion ability of HAM1(P<0.0001).Conclusions:1.Hsa＿circ＿0089153,hsa＿circ＿0001955,hsa＿circ＿0000517,hsa＿circ＿0080425,hsa＿circ＿0043278,hsa＿circ＿0001667 are differentially expressed in AB;CTNND1 is mainly It is expressed in AB cytoplasm,and is related to age and AB pathological type,and the tumor location is mainly related to plexiform AB;Hsa＿circ＿0000517/hsa-miR-1296/CTNND Axis may regulate the pathological process of human ameloblastoma;2.After silencing hsa＿circ＿0000517,the expression of hsa-miR-1296 was significantly increased,the expression of CTNND1 was significantly reduced,and the ability of AB invasion and migration was significantly enhanced;After inhibiting hsa-miR-1296,the expression of CTNND1 was significantly increased;as hsa-miR-1296 was overexpressed,the ability of invasion and migration in AB is obviously enhanced;3.Hsa＿circ＿0000517/hsa-miR-1296/CTNND1 axis has a regulatory effect on AB invasion and migration. |
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