The Biological Function Role And Molecular Mechanisms Of Circular RNA Circ＿0001165 In Esophageal Squamous Cell Carcinoma

Esophageal cancer is a highly aggressive and poorly prognostic malignant tumor of the digestive system,ranking seventh in incidence and sixth in mortality among malignant tumor-related diseases worldwide.China has a high incidence of esophageal cancer,with esophageal squamous cell carcinoma(ESCC)being the primary pathological subtype.Smoking,excessive alcohol consumption,Barrett’s esophagus,and unbalanced dietary habits are high-risk factors for esophageal cancer.According to 2022 statistics,there were253,000 new cases and 194,000 deaths from esophageal cancer in China,ranking sixth and fifth among malignant tumor-related diseases,respectively,indicating a significant disease burden.Due to the insidious onset of esophageal cancer,atypical early symptoms,and lack of specific early diagnostic markers,most patients exhibit symptoms at the middle or late stages.These factors lead to the loss of optimal surgical window,posing significant challenges for the prevention and treatment of ESCC.Surgery combined with chemoradiotherapy and targeted immunotherapy have improved the clinical efficacy of esophageal cancer,but for locally advanced and late-stage ESCC,lymph node metastasis,postoperative recurrence,and treatment resistance remain significant factors for poor prognosis.The prognosis of early-stage ESCC is markedly different from that of advanced ESCC;therefore,exploring diagnostic markers and therapeutic intervention targets for ESCC is significant for improving prevention,control,and precisely targeted therapy.Noncoding RNAs play critical regulatory roles in tumorigenesis,among which circular RNAs have become star molecules in recent research.Circular RNA(circ RNA)is an endogenous non-coding RNA with a covalently closed circular structure exhibiting specific expression in various tissues and cell types.Current research shows that circular RNAs have relatively higher stability than linear RNAs and are detectable in bodily fluids such as plasma,saliva,urine,and exosomes due to their circular structure.They are involved in various diseases’ physiological and pathological processes,making them promising biomarkers and therapeutic targets with considerable translational and application potential.Functionally,circular RNAs can serve as molecular sponges for micro RNAs(miRNAs)and proteins,or they can exert their roles by regulating protein functions and translating them into peptides.In this study,we conducted high-throughput sequencing of circular RNAs in four pairs of ESCC and adjacent tissues,identifying the highly expressed circular RNA hsa＿circ＿0001165(circ＿0001165)and investigating its clinical relevance and biological functions.We elucidate the specific molecular mechanisms that promote ESCC development,providing strategies for diagnostic markers and therapeutic targets.Part One Expression Profile of Circular RNAs and Molecular Characteristics and Clinical Relevance of circ＿0001165 in esophageal squamous cell carcinomaObjective: To identify the differential expression profiles of circular RNAs in ESCC tissues using high-throughput sequencing and to analyze the expression,molecular characteristics,and clinicopathological relevance of circ＿0001165 in ESCC tissues and cells.Methods:1.Using high-throughput sequencing of circular RNAs in four pairs of ESCC cancerous and adjacent non-cancerous tissues to map and analyze the differential expression profiles of circ RNAs in ESCC.2.Screening the highly expressed circ RNA has＿circ＿0001165 in ESCC tissues and cells through literature review and preliminary experiments and verifying its molecular characteristics using Sanger sequencing,agarose gel electrophoresis,RNase R resistance assay,nucleocytoplasmic separation assay,and FISH assay.3.Investigating the correlation between circ＿0001165 expression and clinical characteristics of patients with ESCC.Results:1.High-throughput sequencing identified 1017 upregulated circ RNA molecules and1216 downregulated circ RNA molecules.According to the composition types,circ RNAs consist of exonic,intronic,and intergenic types,with most circ RNAs derived from the backsplicing of host gene exons.2.Identification of circ＿0001165 expression and circular characteristics in ESCC.Divergent and convergent primers were designed based on the splicing sites of circ＿0001165,and Sanger sequencing was used to verify the splicing sites of circ＿0001165.Agarose gel electrophoresis was used to confirm its presence in c DNA but not in g DNA,excluding the possibility of genomic rearrangement.RNase R resistance assay demonstrated that circ＿0001165 was less susceptible to exonuclease RNase R degradation than its host gene NCOA3.q RT-PCR was used to detect the higher expression of circ＿0001165 in ESCC tissues and cells relative to normal tissues and cells.The correlation between circ＿0001165and clinicopathological characteristics was analyzed using Fisher’s exact test.Nucleocytoplasmic separation assay and RNA fluorescence in situ hybridization(FISH)assay identified that circ＿0001165 is predominantly located in the cytoplasm,with a small portion in the nucleus.3.Fisher’s exact test was used to analyze the correlation between high circ＿0001165expression and clinical staging of ESCC,and the results showed that its high expression is positively correlated with TNM staging.Conclusion:1.There are many differentially expressed circ RNAs in ESCC tissues;most circ RNAs are formed by back-splicing of exonic types.2.Circ RNA circ＿0001165 is highly expressed in ESCC tissues and is formed by backsplicing exons 4-9 of the parental gene NCOA3.It has circular characteristics and is predominantly located in the cytoplasm to perform its biological functions.3.The high expression of circ＿0001165 is positively correlated with TNM staging of ESCC tissues,indicating its potential as a clinical biomarker.Part Two Study on the Biological Function of circ＿0001165 in the Progression of Esophageal Squamous Cell CarcinomaObjective: To investigate the biological effect of circ＿0001165 related to the development of ESCCMethods: Validation of its biological function in ESCC using gain-and loss-offunction cell experiments in vitro and animal experiments in vivo.Firstly,stable cell lines with knockdown of circ＿0001165 and plasmids with overexpression of circ＿0001165 will be constructed,and their knockdown and overexpression efficiency will be verified using q RT-PCR.Verify its effect on the proliferation ability of ESCC using CCK8,Ed U,and colony formation proliferation assays.Verify its effect on ESCC migration and invasion using Transwell migration,invasion,and wound healing assays.Construct a subcutaneous tumor model in nude mice to verify the impact of circ＿0001165 knockdown cell lines on tumor formation in vivo.Results:1.Cell function experiments,including CCK8,Ed U,colony formation,scratch assays,and Transwell assays,verify that circ＿0001165 can enhance the proliferation,migration,and invasion of ESCC cells.2.Subcutaneous tumor formation experiments in nude mice confirmed that circ＿0001165knockdown inhibits ESCC proliferation in vivo.Conclusion: circ＿0001165 has biological functions that promote the development of esophageal squamous cell carcinoma;Part Three Study on the Molecular Mechanisms of circ＿0001165 in Promoting the Development of esophageal squamous cell carcinomaObjective: To investigate the upstream regulatory mechanisms controlling circ＿0001165 formation and the absorption of micro RNAs and target genes in ESCC.Methods: Using bioinformatics to predict relevant RNA binding proteins involved in circ＿0001165 circularization,q PCR to verify RNA-binding proteins promoting circularization and the correlation of circ＿0001165 expression with tissue levels and changes,and RIP assays to verify the binding sites of RNA-binding proteins binding to precursor RNA NCOA3.By bioinformatically analyzing the potentially absorbed micro RNAs of circ＿0001165and using dual-luciferase reporter assay,FISH assay,RIP assay,RNApulldown assay,and q PCR to validate interactions between circ＿0001165 and downstream micro RNAs.Functional rescue experiments validated that circ＿0001165 can adsorb micro RNAs to promote ESCC cell proliferation,migration,and invasion.Bioinformatics and transcriptome sequencing were used experimentally to validate that circ＿0001165regulates target gene expression by adsorbing micro RNA.q RT-PCR and WB assays to validate micro RNA-regulated target gene expression.Dual-luciferase reporter assays verified the interaction between micro RNAs and the 3’-UTR regions of target genes.The relative expression of target genes in ESCC and adjacent tissues was detected,and the correlation with clinical prognosis was analyzed.Correlation analysis was conducted among circ＿0001165,micro RNAs,and target genes.Functional rescue experiments validated that circ＿0001165 can promote ESCC cell proliferation,migration,and invasion by targeting micro RNAs to regulate the target gene signaling axis.Results:1.Bioinformatics predicted,and experiments verified that EIF4A3 regulates circ＿0001165 circularization.Bioinformatics predicted that circ＿0001165 could potentially adsorb micro RNAs.RNA-pulldown,RIP,and dual-luciferase reporter assays showed that circ＿0001165 can adsorb miR-381-3p to promote ESCC development.FISH experiments confirmed that circ＿0001165 and miR-381-3p are co-localized in the cytoplasm.2.qRT-PCR experiments confirmed that miR-381-3p expression levels are significantly downregulated in ESCC tissues and cells compared to normal tissues and cells.Overexpression and inhibition of miR-381-3p,along with proliferation,migration,and invasion-related functional assays(CCK8,Ed U,colony formation,and Transwell),confirmed that miR-381-3p inhibits ESCC cell proliferation,migration,and invasion.Functional rescue experiments using CCK8,Ed U,colony formation,and Transwell assays confirmed that inhibiting miR-381-3p can rescue the inhibition of ESCC cell proliferation,migration,and invasion caused by circ＿0001165 knockdown.3.Transcriptome sequencing of circ＿0001165 knockdown KYSE150 cells and joint analysis with micro RNA-predicted target gene databases confirmed that circ＿0001165 can adsorb miR-381-3p to upregulate the expression of the target gene TNS3.High expression levels of TNS3 in ESCC tissues are positively correlated with prognosis.TNS3 expression in ESCC tissues is positively correlated with circ＿0001165 expression and negatively correlated with miR-381-3p expression.Functional rescue experiments confirmed that overexpression of TNS3 can partially rescue the inhibition of ESCC cell proliferation,migration,and invasion caused by circ＿0001165 knockdown.Conclusion:1.EIF4A3 is highly expressed in ESCC tissues and is positively correlated with high circ＿0001165 expression,regulating the circularization of circ＿0001165.2.Circ＿0001165 promotes ESCC development by targeting and adsorbing miR-381-3p in ESCC.miR-381-3p is lowly expressed in ESCC tissues and cells and has biological functions inhibiting ESCC cell proliferation,migration,and invasion.3.circ＿0001165 can target and adsorb miR-381-3p to upregulate the expression of the target gene TNS3.TNS3 is highly expressed in ESCC tissues and is associated with poor prognosis.TNS3 overexpression can rescue the proliferation,migration,and invasion abilities inhibited by circ＿0001165 knockdown,indicating that circ＿0001165 promotes ESCC development through the miR-381-3p/TNS3 axis.