The Biological Role And Mechanism Of CircTMC5 In Gastric Cancer

Background:With the changes in human living standards and eating habits in the last years,the incidence of gastric malignant tumor has been decreasing year by year,but gastric cancer(GC)is still the fifth most common cancer in the world and the fourth leading cause of cancer death in the world.In 2018,gastric cancer mortality accounted for more than 8% of global cancer deaths,and the five-year overall survival rate for gastric cancer was only 20-30%.In China,due to the control of risk factors related to gastric cancer and the continuous work of gastric cancer screening,the overall morbidity and mortality of gastric cancer showed a downward trend,but gastric cancer is still a highly malignant tumor of digestive system,and early gastric cancer has no specific symptoms.Metastasis to distant organs is often found,such as peritoneum,lung,liver,lymph node metastasis and other organs.Therefore,searching for specific early diagnosis markers for gastric cancer and exploring the molecular mechanism of gastric cancer is still a hot spot in gastric cancer.At present,endoscopy plus biopsy pathological diagnosis is still the gold standard for diagnosing gastric cancer,but it is an invasive procedure.Recent studies have found that in order to improve the level of early diagnosis of malignant tumors,the current diagnostic methods for malignant tumors include blood detection,genetic detection,and tumor marker detection.A variety of biomarkers have been identified,such as extracellular vesicles and particles,Urine DNA methylation determination,plasma hsa_circ₀000745 level and CEA combined.Circulating(circ)RNAs have become research hotspots.Circ RNA is a non-coding RNA,as a competitive endogenous RNA(ce RNA)and plays an important role in the transcription process.It has become a key gene for tumor cell proliferation,differentiation,apoptosis and invasion,as well as cancer diagnosis,survival and metastasis.However,the role and mechanism of Circ RNAs in gastric malignant tumors have not yet been fully elucidated.In view of the above research background,we found that CircTMC5 was highly expressed in gastric cancer tissue by transcriptome sequencing.Our study intends to explore the biological role and possible mechanism of CircTMC5 in gastric cancer.Objective:1.To explore the expression of CircTMC5 in gastric cancer tissues and gastric cancer cell lines,and analyze the clinical significance,diagnostic and prognostic value of CircTMC5 expression in gastric cancer patients.2.To explore the biological effects of CircTMC5 on gastric cancer cell lines.3.To explore the relationship between CircTMC5/mi R-365-3p/RABL6 regulatory axis and the biological effects on gastric cancer.4.To explore the biological role of RABL6 in the immune regulation of gastric cancer.Method:1.The differential expression of CircTMC5 was found and identified by the 2th generation sequencing of Circ RNA transcription.2.q RT-PCR detected the expression level of CircTMC5 in gastric cancer tissues and adjacent normal tissues.According to the median expression level of CircTMC5,it was divided into high expression group and low expression group,analyzing the relationship between CircTMC5 and the clinical significance or prognosis of gastric cancer patients,and constructed a prognostic risk proportional model to analyze the prognostic factors of patients with gastric cancer by Univariate and multivariate Cox regression analysis.3.Detecting the expression of CircTMC5 in the blood of gastric cancer patients and healthy volunteers by q RT-PCR,and analyzing the diagnostic value of CircTMC5 in gastric cancer patients by ROC curve.4.Constructing a low-expression CircTMC5 stably transfected gastric cancer cell line,and detecting the biological effects of low-expression CircTMC5 on the proliferation,apoptosis,migration and invasion of gastric cancer cell lines by CCK-8,EDU,flow cytometry,and Transwell.5.Detecting the biological effects of low expression CircTMC5 in gastric cancer cell lines on apoptosis,metastasis,epithelial-mesenchymal transition(EMT)related protein expressions by Western blotting.6.Predicting the targeted binding site of CircTMC5 and mi R-361-3p by RNAhybrid 2.2 software and the targeted binding site of mi R-361-3p and RABL6 m RNA by DIANA-Tar Base v8 database.The FISH experiment proved that CircTMC5 and mi R-361-3p were expressed in gastric cancer cells.The Dual luciferase Reporter experiment verified the targeted binding relationship of CircTMC5 with mi R-361-3p or mi R-361-3p with RABL6 m RNA.q RT-PCR verified the expression of CircTMC5,mi R-365-3p and RABL6 in gastric cancer tissues and gastric cancer cell lines.7.Co-transfecting over-expressing CircTMC5 or mi R-361-3p mimic or over-expressing RABL6 vectors in gastric cancer cell lines,and detecting expression CircTMC5,mi R-361-3p and RABL6 by q RT-PCR or Western blotting.The biological effects of CircTMC5/mi R-365-3p/RABL6 regulatory axis on the proliferation,apoptosis,migration and invasion of gastric cancer cell lines by CCK-8,EDU,flow cytometry and Transwell.The expression and correlation of CircTMC5/mi R-365-3p/RABL6 regulatory axis in gastric cancer tissues and gastric cancer cell lines were detected by q RT-PCR or Western blotting.8.Analyzing the RABL6 expression based on Oncomine and UALCAN biological information database,verifing the expression of RABL6 protein in gastric cancer tissues by immunohistochemistry,and exploring the possible biological functions of RABL6 by Linked Omics database.9.Based on the Linked Omics database,RABL6 may be involved in the immune regulation of gastric cancer,and the relationship between RABL6 and chemokines or immune cells in the immune microenvironment of gastric cancer was analyzed by TISIDB and TIMER databases.The relationship of RABL6 with chemokines or immune cells in the gastric cancer microenvironment was verified by immunohistochemistry.Result:1.CircTMC5 is highly expressed in gastric cancer tissues,plasma of gastric cancer patients and gastric cancer cell lines.The expression of CircTMC5 was related to the differentiation,pathological staging,and T classification(P<0.05),but it was no related to the age,gender,drinking,smoking,CEA expression level,lymph node metastasis,helicobacter pylori infection and tumor size(P >0.05).2.CircTMC5 had a diagnostic effect in gastric cancer.ROC curve analysis showed that CEA was as a diagnostic marker,AUC was 0.910(sensitivity,0.863;specificity,0.793;P <0.001),while the AUC of CircTMC5 was 0.821(sensitivity,0.619;specificity,0.922;P <0.001).However,when CEA and CircTMC5 were combined,the AUC was 0.941(sensitivity,0.791;specificity,0.948;P <0.001).CEA and CircTMC5 can be a combined diagnostic marker for gastric cancer.3.Kaplan-Meier survival analysis showed that the overall survival rate of gastric cancer patients with high expression of CircTMC5 was low(P=0.002),while univariate or multivariate Cox regression analysis confirmed that CircTMC5 expression,differentiation and pathological stage were independent prognosis of gastric cancer patients factors(P <0.05).4.Down-regulating the expression of CircTMC5 can promote the apoptosis of gastric cancer cell lines and inhibit proliferation,migration and invasion of gastric cancer cells.5.Biological information prediction,FISH experiment,dual luciferase report experiment proved that CircTMC5 has a targeted binding with mi R-361-3p,and mi R-361-3p has a targeted binding with RABL6.In gastric cancer tissues,it was found that CircTMC5 was negatively correlated with mi R-361-3p expression(r=-0.4158,P<0.0001),and mi R-361-3p was negatively correlated with RABL6 m RNA expression(r=-0.3356,P=0.0004).There was a positive correlation between CircTMC5 and RABL6 m RNA expression(r=0.247,P=0.015).Compared with normal gastric epithelial cells,CircTMC5 was highly expressed,mi R-361-3p was low,and RABL6 m RNA was highly expressed in gastric cancer cell lines.6.In vitro studies had shown that overexpression of CircTMC5 inhibited the expression of mi R-361-3p by sponge-like adsorption,there by up-regulating the expression of RABL6 to inhibit apoptosis and promote the proliferation,migration and invasion of gastric cancer cells,while up-regulation of mi R-361-3p had the opposite effect.In vivo experiments verified relationship among CircTMC5/mi R-365-3p/RABL6 regulatory axis.7.RABL6 was highly expressed in gastric cancer.Based on bioinformatics analysis and immunohistochemistry,RABL6 was involved in the immune regulation of chemokines and immune cells in the immune microenvironment of gastric cancer.Conclusion:CircTMC5 was highly expressed in gastric cancer tissues and had the effect on promoting the malignancy of gastric cancer.The combined detection of CircTMC5 and CEA can be used as a molecular marker for the diagnosis of gastric cancer.CircTMC5 was an independent risk factor for the prognosis of gastric cancer patients.Down-regulating expression of CircTMC5 can promote the apoptosis of gastric cancer cells and inhibit proliferation,migration,invasion and EMT.The possible mechanism of CircTMC5 promoted the malignancy of gastric cancer may be through sponge-like adsorption to inhibit the expression of mi R-361-3p,and then up-regulate the expression of RABL6 to affect the biological behavior of gastric cancer.RABL6 had a regulatory effect on chemokines and immune cells in microenvironment of gastric cancer,and RABL6 also showed the prospect of targeted therapy for gastric cancer.These results provided new research directions for the mechanism of gastric cancer.