The Mechanism Of CircZNF608 Inhibiting Proliferation,Migration And Invasion Of Non-small Cell Lung Cancer

Lung cancer is the second most common malignant tumor in the world with the highest mortality,accounting for 11.4% of the total cancer incidence and 18.0% of the total death.In recent years,with the improvement of diagnostic techniques and the use of thoracoscopic,targeted and immunotherapy,the survival rate of lung cancer patients has been significantly improved.The three-year survival rate has increased from 19% in 2001 to 21% in 2004,and then to 31% in 2015 through 2017.The median survival time has also increased from 8months to 13 months.However,in China,the incidence rate of lung cancer is still the highest among malignant tumors.The number of new lung cancer cases in China is estimated to be870982 in 2022,accounting for 18% of the total estimated new cancer cases.The number of deaths due to lung cancer is estimated to be 23.9% of the total deaths.Among lung cancer,non-small cell lung cancer(NSCLC)accounts for the vast majority,so the mechanism of its occurrence and development remains to be further studied.Circular RNA is a kind of circular non-coding RNA formed by backsplicing and Lariatdriven pathway.Due to its special circular structure,it is more tolerant to the digestion of exonuclease RNase R and more stable in expression than m RNA.Recent studies have shown that a large number of differentially expressed circular RNAs have also been found in tumors.Their expressions are significantly correlated with TNM stage of tumors,and have a significant impact on tumor proliferation,migration and invasion.With the increase of research,the roles of a large number of circular RNAs have been clarified in lung cancer,but their mechanism research mainly focuses on the role as a mi RNA sponge,and there is still a lack of relevant research on the mechanisms of interaction with protein or translation into protein.In order to further clarify the role of circular RNA in lung cancer,we firstly used three pairs of NSCLC and adjacent tissues for high-throughput sequencing of circ RNA.A total of9442 circular RNAs were found,of which 1114 were differentially expressed(|log FC| ≥0.585;P < 0.05).After intersection with the results of GSE112214 and q PCR verification,hsa＿circ＿0001523(circ ZNF608)was finally selected to conduct the following study to explore its effects on the proliferation,migration and invasion of NSCLC,so as to provide a possible target for the therapy of lung cancer.This topic is divided into the following four parts:Part I: Identification and clinical correlation analysis of circ ZNF608Methods:1.High throughput sequencing of circular RNA was performed in 3 pairs of NSCLC and adjacent samples,and the expression of circ RNA were analyzed by R language.2.q RT-PCR was performed to verify the expression of circ RNA in 35 pairs of NSCLC samples.3.Divergent primers and convergent primers were designed,RT-PCR was carried out in g DNA and c DNA,and the products were collected by agarose gel electrophoresis and Sanger sequencing was performed to prove the existence of circ ZNF608.4.NSCLC cell lines were treated with actinomycin D and RNase R respectively to detect the half-life and stability of circ ZNF608;The subcellular localization of circ ZNF608 was demonstrated by fluorescence in situ hybridization(FISH).5.The clinical information of 35 patients was collected to analyze the relationship between the expression of circznf608 and clinicopathological features.Results:1.A total of 9442 circ RNAs were found in NSCLC samples.Among them,86.6% of the circular RNAs are formed by backsplicing of exons and 1114 circ RNAs were differentially expressed(620 up-regulated and 494 down-regulated).2.After the intersection with results of GSE112214,three downregulated circular RNAs were obtained.It was found that circ ZNF608 was most significantly downregulated in 10 pairs of NSCLC samples.3.CircZNF608 was formed by backsplicing of exon 2 in ZNF608,with a total length of256 nt.4.The half-life of circ ZNF608 was more than 12 hours.It could tolerate the digestion of RNase R enzyme and was mainly distributed in the cytoplasm.5.The expression of circ ZNF608 was significantly correlated with the T-stage of the patients.Conclusion:CircZNF608 was significantly down regulated in NSCLC samples,and its expression was significantly correlated with tumor size.CircZNF608 was mainly localized in the cytoplasm.Part Ⅱ: The biological function of circ ZNF608 on NSCLC cellsMethods:1.The expression levels of circ ZNF608 in BEAS-2B and NSCLC cell lines were detected by q RT-PCR.The overexpression or knockdown lentivirus was used to generate circ ZNF608 overexpressed or down-regulated cell lines;2.CCK8 and EDU were performed to detect the effect of circ ZNF608 on the proliferation of NSCLC cells;The effects of circ ZNF608 on the migration and invasion of NSCLC cells were detected by tranwell assay and wound healing.3.Xenograft tumor nude mice was established by subcutaneously injected with NSCLC cell overexpressed circ ZNF608.Lung metastasis model was used to access the effect of circ ZNF608 on metastasis of NSCLC cells in vivo.Results:1.CircZNF608 was significantly down regulated in NSCLC cell lines compared with BEAS-2B.2.CircZNF608 significantly inhibited the proliferation,migration and invasion of NSCLC cells in vitro.3.CircZNF608 significantly inhibited tumor proliferation and metastasis in vivo.Conclusion:CircZNF608 inhibited the proliferation,migration and invasion of NSCLC cells in vitro and in vivo.Part III: CircZNF608 binds to IGF2BP3 protein in NSCLC cellsMethods:1.CSCD and Circinteractome were performed to predict the mi RNAs that circ ZNF608 could interact with.CSCD and Starbase was used to predict the RNA binding proteins(RBPs)that circ ZNF608 could bind to.The coding potential of circ ZNF608 was evaluated with CircRNAdb website;2.The RBPs interacted with circ ZNF608 was confirmed by RNA pull-down and RIP assay;3.FISH and immunofluorescence were used to verify the subcellular localization of circ ZNF608 and RBP respectively;4.The transcriptome,proteome and clinical information of NSCLC were downloaded from TCGA and CPTAC databases to explore the expression of IGF2BP3 in NSCLC and its relationship with clinicopathological features.The relationship between IGF2BP3 and prognosis was analyzed by Kaplan-Meier plotter;5.TCGA pan-cancer data including 33 cancer types was downloaded from Xena browser website and used to explore the role of IGF2BP3 in multiple cancer types;6.The protein-protein interaction network(PPI)of IGF2BP3 was constructed by String database and and functional analysis was performed by Metascape database.Results:1.CircZNF608 bound to IGF2BP3 in NSCLC cells;2.CircZNF608 and IGF2BP3 were co-located in the cytoplasm;3.IGF2BP3 was significantly overexpressed in NSCLC samples and correlated with T stage,N stage and prognosis of patients;4.IGF2BP3 was significantly overexpressed in multiple cancer types and associated with poor prognosis.Conclusion:CircZNF608 binds to IGF2BP3 protein in NSCLC cells.Part IV CircZNF608 competitively interacts with IGF2BP3 and downregulates the expression of MYCMethods:1.The efficiency of IGF2BP3 overexpression and knockdown was verified by real-time quantitative PCR and Western Blot(WB).The proliferation of NSCLC cells was examined by CCK8 and EDU.Tranwell and wound healing assays were used to detect the changes of migration and invasion ability of NSCLC cells;2.WB was used to detect the effect of overexpression or knockdown of circ ZNF608 on the expression of IGF2BP3,and real-time quantitative PCR was performed to verify the change of expression of circ ZNF608 due to the overexpression or knockdown of IGF2BP3;3.Catrapid website was performed to predict the binding site of circ ZNF608 and IGF2BP3.The binding site was confirmed by constructing IGF2BP3 truncated plasmid with flag tag and RIP experiment;4.The m6 A methylation modification sites on circ ZNF608 were predicted by GEO database data,SRAMP and RMbase website,which were verified by Me RIP,RIP and luciferase assay;5.The target genes of IGF2BP3 were found by consulting the previous studies,and the effects of circ ZNF608 and IGF2BP3 on the expression of target genes were verified by q PCR and WB;6.RIP,Actinomycin D and luciferase assay were used to detect the effect of circ ZNF608 on IGF2BP3 binding MYC;7.The expression of ZNF608,DNMT1 and DNMT3 b in NSCLC and the methylation level of promoter of ZNF608 were analyzed by Ualcan database.Results:1.IGF2BP3 promoted the proliferation,migration and invasion of NSCLC cells in vitro;2.Overexpression of circ ZNF608 can inhibit the promoting effect of IGF2BP3,and knockdown of circ ZNF608 can rescue the inhibitory effect after IGF2BP3 was downregulated;3.There was no obvious mutual interference between the expression of circ ZNF608 and IGF2BP3;4.CircZNF608 bound to the KH3-4 domain of IGF2BP3;5.Circznf608 bound to IGF2BP3 in an m6 A dependent manner;6.CircZNF608 competitively interacted with IGF2BP3 and downregulated expression of MYC;7.CircZNF608 decreased the binding of IGF2BP3 to MYC and the stability of MYC m RNA;8.In NSCLC,the expression of DNMT1 and DNMT3 b increased,and the methylation level of ZNF608 gene promoter elevated significantly.Conclusion:CircZNF608 downregulated the expression of MYC in NSCLC cells by competitively interacting with IGF2BP3 in an m6 A dependent manner.