Molecular Mechanisms And Epidemiological Studies Of Circular RNA And Its Associated Axes Influencing The Progression Of Gastric Cancer

Background :The mortality and incidence of gastric cancer continue to rank among the top five in the global tumor incidence rate and mortality rate and,and it is one of the main malignant tumors in China.Although the incidence and mortality of gastric cancer have decreased with the improvement of diagnosis and treatment technology,the prognosis of gastric cancer patients is still poor.The main reason is that most patients with gastric cancer are already in the advanced stage of gastric cancer when they are diagnosed and treated,and the easy metastasis and drug resistance of gastric cancer make the diagnosis and treatment effect very limited.Therefore,it is urgent to identify early diagnostic indicators with high sensitivity and specificity or effective treatment targets to improve the efficiency of gastric cancer diagnosis and treatment and reduce the incidence and mortality of gastric cancer.Circular RNA（circRNA）has the innate advantages of being a target for diagnosis and treatment of diseases—high stability,high abundance,and disease expression specificity,but the mechanism of circRNA influencing the progression of gastric cancer and its value as a prognostic and therapeutic target are currently unclear,so it is necessary to clarify the circRNA associated with gastric cancer and systematically study the value of the identified target circRNA as a target for the diagnosis and treatment of gastric cancer.Objectives:1.Identify gastric cancer related circRNA;2.Explore the prognostic value of gastric cancer related circRNA;3.Elucidate the molecular mechanisms of target circRNA and its associated axes influencing the progression of gastric cancer;4.Analyze the expression of target circRNA and explore the clinical application value of target circRNA in plasma.Methods:1.The circRNA microarray method was used to identify the difference in expression of circRNA between three pairs of gastric cancer tissues and their adjacent tissues,and the different expression circRNA was selected according to the fold change（FC）value and P value between the two groups（|FC|>2 and P<0.05 is the filter criterion）.In addition,10 differentially expressed circRNA were randomly selected for clinicopathological parameter correlation analysis,survival analysis,prognosis-related model construction,and nomogram construction.ROC curve,calibration curve and decision curve were used to evaluate constructed model.2.Compare the microarray results of this study with the GEO dataset,and predicting the ceRNA network,GO and pathway of the common differentially expressed circRNA,firstly,then selecting the different expression circRNA with the greatest degree of relevance to the survival time of gastric cancer patients as the research object.The characteristics of circRNA and its localization in cells were determined by RNase R,Dactinomycin D and fluorescence in situ hybridization experiments.In vitro siRNA was used to construct circRNA knockdown cell model,and this model was used to explore the effects of circRNA on the proliferation,migration and invasion ability of gastric cancer cells.After the successful construction of a subcutaneous tumor model of nude mice with gastric cancer cells,in vivo siRNA was used to explore the potential therapeutic value of the targeted circRNA.After determining the function of this circRNA,the mechanism of it on the progression of gastric cancer was investigated by double luciferase reporter gene,real-time fluorescence quantitative polymerase chain reaction,CCK8,wound healing,transwell,western blotting,RNA-binding protein immunoprecipitation and immunofluorescence assays.Finally,it was determined by CCK8 experiment whether the sensitivity of cells to chemotherapy drug 5-FU was affected by the target circRNA.3.The highest dysregulated expression circRNA in the mocroarray results of this study was used as the second target circRNA for further mechanism study.The characteristics of the target circRNA was also determined by RNase R,radiomycin D and fluorescence in situ hybridization experiments.Then,lentiviral and siRNA were used to construct target circRNA overexpression and knockdown cell models to explore their effects on the proliferation,migration and invasion ability of gastric cancer cells.After using siRNA in vivo to determine whether the target circRNA had an effect on reducing tumor size and growth rate,a subcutaneous tumor model of nude mice was constructed with target circRNA overexpressed cell line,and the effect of target circRNA on the proliferation and invasion of cancer cells in vivo was clarified by the in vivo lung metastasis model.The Ch IRP-MS experiment was used to determine the protein（RBP）bound to target circRNA,and the western blot,RNA-binding protein immunoprecipitation,immunofluorescence,and Dactinomycin D experiments were used to determine the mechanism of target circRNA in the progression of gastric cancer.In addition,the experimental methods to determine the mechanism of target circRNA as ceRNA in gastric cancer progression were: double luciferase reporter gene,real-time fluorescence quantitative polymerase chain reaction,CCK8,wound healing,transwell,Western blotting method,RNA-binding protein immunoprecipitation,immunofluorescence assays,etc.4.The target circRNA in method 2,the target circRNA in method 3,two randomly selected circRNA from published literature in pubmed and impact factors are between 10-510 during the period 2000/1/1-2021/12/31 related to gastric cancer,and circRNA in the gastric cancer plasma circRNA microarray dataset in GEO were used as the candidate circRNA.Then,built a recombinant plasmid using p MD18-T vector and used DH5α as competent cell.Real-time fluorescence quantitative polymerase chain reaction absolute quantification explored the expression of circRNA in gastric cancer patients’ plasma and healthy control plasma samples to confirm the target circRNA in this part.Chi-square tests determined clinical indicators associated with target circRNA.Univariate logistic regression analysis,multivariate logistic regression analysis,random forest,and five-fold cross validation method were used to construct the models of relevant indicators,and the models were evaluated by using ROC curve,calibration curve,decision curve,accuracy,precision,F1 score,etc.Results:1.Circ RNA microarray identified a total of 11,514 circRNAs,with a total of 25 different expression circRNA.10 circRNA were randomly screened（hsa_circ₀004662,FC=8.784;hsa_circ₀008586,FC=2.082;hsa_circ₀044301,FC=2.215;hsa_circ₀000081,FC=-2.126;hsa_circ₀043138,FC=3.495;hsa_circ₀001113,FC=-2.171;hsa_circ₀072309,FC=-2.619;hsa_circ₀001055,FC=-2.108;hsa_circ₀004796,FC=2.115;hsa_circRNA₄04289,FC=2.108;their P are all less than 0.05）.After verification with tissue samples,hsa_circ₀004662,hsa_circ₀008586,hsa_circ₀044301,hsa_circ₀043138,hsa_circ₀001113,hsa_circ₀072309,and hsa_circ₀001055 expression trends were consistent with microarray results.The survival analysis results showed that there was a significant correlation between hsa_circ₀004662,hsa_circ₀008586,hsa_circ₀044301,hsa_circ₀000081,hsa_circ₀043138,hsa_circ₀004796and hsa_circ₀001113 and the survival time of patients.Multivariate Cox regression analysis yielded three circRNA-constructed prognostic models,and the risk score calculation formula of the prognostic model was: risk Score =hsa_circ₀044301*0.9603 + hsa_circ₀043138*0.3689-hsa_circ₀001113*1.1983,the AUC predicted by the model for one-year,three-year and five-year survival time of patients,respectively: 0.770,0.895,0.903,and the model was an independent prognostic factor for patient survival.The calibration curve of the nomogram was highly consistent with the standard curve,and the decision curve indicates that some patients will bring greater net benefits if they accept the nomogram.2.Hsa_circ₀043138,hsa_circ₀044301,hsa_circ₀041481,and hsa_circ₀025135 are the shared different expression circRNA in our dataset and GSE89143.KEGG enrichment analysis showed that these circRNA were associated with m TOR and MAPK signaling pathways.Hsa_circ₀044301 was selected as the target circRNA in this chapter because it participated in prognostic model building and HR was the largest.The stability and half-life of hsa_circ₀044301were significantly higher than those of linear RNA,and it was mainly localized in the cytoplasm.When hsa_circ₀044301 in the cell was knocked down,it can inhibit the proliferation,migration and invasion of cells.After knocking down the hsa_circ₀044301 in vivo,the growth rate and size of the tumor were significantly reduced.Hsa-miR-188-5p can significantly affect the fluorescent activity of hsa_circ₀044301 wild-type luciferase,and the effect of hsa_circ₀044301 on the proliferation and invasion of gastric cancer cells was affected by hsa-miR-188-5p.The downstream target gene DAXX of hsa-miR-188-5p also had a directed association with hsa_circ₀044301,and DAXX can also affect the role of hsa_circ₀044301 on the biological characteristics of gastric cancer cells.Hsa_circ₀044301 has a positive effect on the expression of the key protein ERK1/2 in the MAPK signaling pathway and can affect the role of the ERK1/2inhibitor GDC-0994.Knocking down hsa_circ₀044301 could enhance cellular sensitivity to5-FU.3.The hsa_circ₀004662 with the highest dysregulated expression in the microarray results of this study（FC=8.784,P=0.011）.Hsa_circ₀004662 had a higher stability and half-life in cells than linear RNA,and was mainly localized in the cytoplasm.Hsa_circ₀004662 overexpression could enhance the proliferation,migration and invasion of gastric cancer cells,while knocking down could reduce the proliferation,migration and invasion of gastric cancer cells.In vivo knocking down hsa_circ₀004662 also reduces tumor growth rate and size,and vice versa.In vivo metastasis models show that hsa_circ₀004662 overexpression could increase the number of tumor metastases in the lungs of nude mice.A total of 26 RBPs were identified in the Ch IRP-MS experiment and can be specifically bound to hsa_circ₀004662.The RIP experiment performed on selected 3 RBPs showed that S100A8 had the highest abundance of hsa_circ₀004662 binding.S100A8 can significantly affect the expression of key proteins such as Ki67 and MMP2,and the correlation coefficients between the expression level of Ki67 and the change of S100A8 expression level under the influence of various time points of Dactinomycin D in the hsa_circ₀004662-treated cells were high.Bioinformatics predicted that all seed regions at the end of hsa-miR-424-5p 3’UTR can be combined with hsa_circ₀004662,and hsa-miR-424-5p can affect the fluorescent activity of hsa_circ₀004662 wild-type luciferase,which also has an impact on the role of hsa_circ₀004662 in gastric cancer cells.The downstream target gene KIF23 can also affect the effect of hsa_circ₀004662 on the biological characteristics of gastric cancer cells,and the expression of Ki67 is also significantly reduced after KIF23 knockdown,and RIP experiments show that KIF23 had a direct binding relationship with S100A8.4.Candidate circRNA were hsa_circ₀044301,hsa_circ₀004662,circ CCDC9（Hsa_circ₀000944）,circ AKT3（hsa_circ₀000199）and hsa_circRNA₁02737（the ID in circ Base is hsa_circ₀001017;Dataset GSE93541）.The Mann-Whitney test showed that the level of circ AKT3,hsa_circ₀044301 and hsa_circ₀004662 in the plasma of gastric cancer patients was higher than that of healthy control groups with statistical difference.The three circRNA correlated with the tumor longest diameter,lauren typing,and blood test indicators: HDL and LYMPH#.There was significant collinearity in the expression of circ AKT3,hsa_circ₀044301,and hsa_circ₀004662 in the patient’s plasma.The logistic regression model showed that the effect and AUC of the circRNA+CEA combination for the diagnosis of the longest diameter of the tumor were better than they single combined with blood type,age and gender.The coincidence between the calibration curve and the standard curve was acceptable,and if some patients accept the model,it will bring certain net benefits.In addition,the 3 circRNA also had an indicative effect on the patient’s lauren classification.The model of circ AKT3,hsa_circ₀044301,hsa_circ₀004662 and some clinical indicators were jointly constructed by random forest,the trained AUC of it reached0.7999 and the test set reached 0.7802,the accuracy,precision and F1 score were all high.The results of the five-fold cross validation method showed that the AUC mean of the five model validation sets was 0.620,and the mean accuracy,precision and F1 score were all higher than 0.5,which once again indicated that circ AKT3,hsa_circ₀044301 and hsa_circ₀004662 have the potential to be used as diagnostic tumor characteristic targets.Conclusion:1.Gastric cancer related circRNA could construct prognosis related models and had certain predictive value.2.Hsa_circ₀044301 could affect the biological characteristics of gastric cancer through the hsa-miR-188-5p/DAXX axis and the MAPK signaling pathway,and has the potential to be used as a target for gastric cancer treatment.3.Hsa_circ₀004662 could affect the progression of gastric cancer through the S100A8/Ki67 and hsa-miR-424-5p/KIF23 axes,and has the potential to be used as a therapeutic target for gastric cancer.4.Circ AKT3,hsa_circ₀044301 and hsa_circ₀004662 have the potential value to be used as diagnostic tumor characteristic targets.