| Background:Cervical cancer is the most common gynaecological tumor,with nearly 600,000 new cases reported each year.Despite data showing a decrease in the incidence rate and mortality of the disease in Europe,America,and other developed countries,the incidence rate of cervical cancer in China is still rising.The 2020 WHO Classification of Tumour Pathology can categorise cervical cancer into the following groups:One is squamous cell carcinoma.Adenocarcinomas and malignant lesions.3.Other epithelial tumors,such as unclassifiable cervical cancer,adenosquamous and mucoepidermoid carcinoma,and neuroendocrine cancers like NET1/2 and neuroendocrine carcinoma.Squamous cell carcinoma,which has an incidence rate of 80%to 85%,is the most common pathological subtype of cervical cancer.Patients with cervical squamous cell cancer typically present between the ages of 50 and 55,and early surgical therapy is the main treatment option.Surgery is used to remove the primary tumour focus,halt the spread of cervical squamous cell carcinoma cells,and give adjuvant therapy in line with the pathology after surgery to improve the prognosis for patients with cervical cancer.However,patients with advanced cervical squamous cell carcinoma frequently develop metastases as a result of delaying therapy.Patients with only surgical treatment have a poor prognosis;instead,most patients undergo radiotherapy,targeted therapy,or immunotherapy.Non-coding RNAs known as circular RNAs（circRNAs）have a circular structure,good stability,and are challenging for RNA enzymes to degrade.They are regarded as a novel biomarker and potential therapeutic target due to the fact that it has been shown that they greatly control the growth of tumours.CircRNAs are hardly ever mentioned in studies on biomarkers of distant metastasis of cervical squamous cell carcinoma.This study mainly examines circRNAs related to distant cervical squamous cell carcinoma metastases and their regulatory mechanisms in order to create a new potential biomarker for the treatment of cervical squamous cell carcinoma.Methods:Firstly,circular RNA sequencing was used to analyze the circRNA expression imbalance between cervical squamous cell carcinoma tissue and adjacent tissues.The circular structure of circCCDC134 was amplified using specific primers,followed by Sanger sequencing to identify its cyclization sites and determine its circular structure.Ribonuclease R（RNase R）and transcription inhibitor actinomycin D treated circCCDC 134 and linear mRNA CCDC134 to evaluate the stability of circCCDC 134.The FISH experiment was used to detect the localization of circCCDC134 in cervical squamous cell carcinoma cells,and qPCR was used to detect the expression of circCCDC 134 in tumor tissues of early cervical cancer（23 cases）,advanced cervical cancer tissues（10 cases,including 7 cases of stage ⅢC and 3 cases of stage Ⅳ）,and cervical squamous cell carcinoma cells.To explore the potential mechanism of upregulation of circCCDC 134 in cervical squamous cell carcinoma,circPrimer and SRAMP tools were used to predict and predict the m6A modification site of circCCDC 134.CircRNA immuno coprecipitation silver staining and mass spectrometry were used to analyze various binding proteins of circCCDC 134.Cell function experiments（clone formation and invasion migration experiments）were conducted to explore the relationship between the expression imbalance of circCCDC 134 and changes in the proliferation.migration,and invasion ability of cervical squamous cell carcinoma cells.In addition,a xenograft growth and lung metastasis model was established to demonstrate the effect of circCCDC 134 on the proliferation and metastasis ability of transplanted tumors.Mechanism research:1.circRNA immuno coprecipitation and RNA immuno coprecipitation were used to analyze the interaction between circCCDC 134 and p65.Chromatin immunoprecipitation combined with gene sequencing（ChIP-seq）and transcriptome sequencing after circCCDC 134 interference were used to find the downstream genes regulated by p65.2.In order to explore miRNAs that can sponge with circCCDC 134,TCGA miRNA seq data,circBank database,and circinteractome database were combined for analysis.To further verify the relationship between circCCDC134 and hsa-miR-503-5p.we carried out circRNA immune coprecipitation,FISH.RNA immune coprecipitation and Luciferase reporter gene detection experiments.Results:The sequencing results showed that there were 133 circRNAs with imbalanced expression in tumor tissue compared to adjacent tissues（64 circRNAs with increased expression and 69 circRNAs with decreased expression,P<0.05）.Based on the sequencing results.circCCDC 134（hsacirc0008806）was ultimately selected as the research object.The qRT PCR results showed that compared to adjacent tissues,the expression of circCCDC 134 was significantly increased in early cervical cancer tissues（P<0.05）;CircCCDC 134 further increased in advanced cervical cancer tissue（P<0.05）.Predictive analysis using circPrimer and SRAMP tools revealed that circCCDC 134 has an RNA methylation（m6A）modification site.TRAP Western blotting and RNA immuno coprecipitation experiments showed that ALKBH5 protein and YTHDF2 protein could bind circCCDC 134.The results of m6A RNA immunoprecipitation experiment and actinomycin D experiment showed that the ALKBH5 mediated m6A methylation of circCCDC 134 increased its RNA stability through YTHDF2,leading to the up regulation of circCCDC 134 expression.The results of cell function experiments showed that compared to the control group（Si-NC/ME180 and Si-NC/SiHA）,the knockdown group of circCCDC134（Si cicCCDC134-1/ME180 and Si cicCCDC134-2/ME180;Si cicCCDC 134-1/SiHA and Si cicCCDC134-2/SiHA）had significantly reduced cell proliferation,migration,and invasion abilities（P<0.05）.In the overexpression group of circCCDC134（EX cicCCDC134/HCC94）,cell proliferation,migration.and invasion abilities were enhanced（P<0.05）.In the experimental model of cervical squamous cell carcinoma xenograft,the shRNA circCCDC 134/ME180 group showed reduced proliferation,slower growth,and smaller tumor weight.The lung metastasis experiment results showed that the shRNA circCCDC 134/ME180 group had smaller lung metastasis tumor volume and fewer lesions.The results of TRAP Western blot and RNA immuno coprecipitation experiments showed that circCCDC 134 could interact with p65.By combining the ChiI P seq data of p65,the AnimalTFDB 3.0 prediction data of p65,the mRNA seq of Si-circCCDC134-1/ME180,and the sequencing results of the transcriptome and the ChiIP data results of cervical squamous cell carcinoma,it was found that circCCDC 134 could enhance HIF1A transcription through interaction with p65.Based on the comprehensive analysis of miRNA seq,circBank,and circRNA databases,it was found that only hsa-miR-503-5p was low expressed in cervical squamous cell carcinoma tissue and may be adsorbed by circCCDC 134.The analysis results of TRAP combined with qPCR experiment,FISH experiment,RIP experiment and Luciferase reporter gene experiment showed that circCCDC 134 could up regulate the expression of MYB in cervical squamous cell carcinoma cells through the molecular sponge adsorption of hsa-miR-503-5p.Further ChIP results indicate that MYB can promote the metastasis of cervical squamous cell carcinoma by enhancing HIF1A transcription.Conclusions:In conclusion,this study found that circCCDC 134 has m6A modification that can be mediated by ALKBH5,thereby affecting the expression of circCCDC 134.CircCCDC 134 recruits p65 in the nucleus and acts as a miR-503-5p sponge to regulate the expression of MYB in the cytoplasm,ultimately enhancing HIF1A transcription and promoting the growth and metastasis of cervical squamous cell carcinoma.Meanwhile,these research results indicate that circCCDC 134 is an important potential therapeutic target in cervical squamous cell carcinoma,and provide new regulatory insights for exploring the carcinogenic mechanism of circCCDC 134 in cervical squamous cell carcinoma. |
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