Upregulated CCAT1 Promotes Development Of Gastric Cancer By CeRNA Mechanism

Introduction:Gastric cancer is the third leading cause of cancer mortality worldwide.Recent findings in gastric cancer-causing gene expression mechanisms,however,hold promise for the development of more effective diagnostic and treatment strategies.A novel mechanism for gene expression control mediated by long non-coding RNAs(lncRNAs)has been described,which have emerged as pivotal regulatory components orchestrating extensive cell processes and connections within eukaryotic cells.Recent studies have reported that several lncRNAs are aberrantly expressed and linked to the onset and progression of this malignancy.These lncRNAs have gained value for such clinical purposes as novel biomarkers and therapeutic targets;yet despite this potential,many issues remain in this rapidly growing field.Colon cancer–associated transcript-1(CCAT1)is a newly discovered lncRNA.Previous studies have shown that CCAT1 can play a role in promoting gastric cancer development,whereas the expression and clinical significance of CCAT1 in the development of gastric cancer has not been clearly reported.In this present study,we observed the CCAT1 expression in gastric cancer,its relationship with the clinicopathologic and the prognosis of gastric cancer,and its potential role in the diagnosis of gastric cancer.Moreover,we examined the distribution of CCAT1 in gastric cancer cells.The expression of CCAT1 in the blood of GC patients and normal volunteers was examined and its diagnostic significance was analyzed.Furthermore,we examined the effect of CCAT on the proliferation and invasion of gastric cancer cells and determined CCAT1 function.Finally,we observesed the underlying mechanism of CCAT1 in GC through bioinformatic and large data analysis combined with a series of in vitro molecular biology experiments.Methods:Quantitative real time-PCR(RT-qPCR)assay was performed to detect the expression of CCAT1 in 68 cases of GC tissues,paired tumor-proximal gastric mucosa and normal gastric mucosa,19 cases of gastric intraepithelial neoplasia(GIN),and the sera of these 68 GC patients and 22 healthy volunteers.The relationship between CCAT1 and clinicopathologic features of GC patients,and the ROC curve of CCAT1 in the sera of gastric cancer were analyzed.The effect of CCAT1 on the proliferation and invasion of gastric cancer was detected by CCK-8 and Transwell assay in vitro.Bioinformatic analysis and luciferase reporter assay were used to analyze the possible binding of target miRNAs CCAT1,and protein to miRNAs.RT-qPCR and Western blotting were used to validate the target miRNA and proteins.Results:1.CCAT1 expression was significantly differed in GC(185.43 ± 21.37),GIN(121.30 ± 43.61),tumor-proximal mucosa(8.9 ± 1.21),and normal mucosa(1.5 ± 0.55,P <0.001).CCAT1 mRNA expression level showed significant differences in gastric cancer tissue with dfferent Lauren grade(P < 0.001).2.CCAT1 was Spearman analysis results showed that high CCAT1 expression in GC tissues was associated with high tumor burden including larger tumor size,more lymphatic metastasis and advanced TNM stage(all p < 0.05).3.Kaplan–Meier analysis and the log-rank test showed that the mean survival time of patients with high CCAT1 expression was 23.41 months(95% CI,17.72 ~ 59.31months),the mean survival time of patients with low CCAT1 expression was(95%CI,15.41 ~ 34.18 months),there was no significantly difference between patients whose tumors showed high CCAT1 expression with those whose tumors showed low CCAT1expression(P < 0.584).4.CCAT1 expression were significantly higher in GC cells than that of normal gastric mosca.CCAT1 expresses both in the cellular and cytoplasm,while mainly expresses in the cellular nucleus.5.CCAT1 is overexpressed in sera of patients with GC(47.40 ± 6.60)compared with healthy controls(0.62 ± 0.06;p < 0.001).The diagnostic value of CCAT1 for gastric cancer was analysised using receiver operating characteristics(ROC)curves,which showed that the area under the ROC(AUROC)of the CCAT1 signature was 0.801,suggested that the expression of CCAT1 in sera may potentially be a biomarker for diagnosis of gastric cancer.6.Overexpression of CCAT1 significantly inhibited the proliferation,invasion and migration of gastric cancer cells(P < 0.01).Knockdown of CCAT1 significantly inhibited the proliferation,invasion and migration of gastric cancer cells(P <0.01).7.By using bioinformatics analysis combined with CCAT1 intervention experiment,we found that CCAT1 can complementarily binding with miR-490-3p in gastric cancer,and over-expression of CCAT1 can significantly inhibit the expression of miR-490-3p(P<0.05);Luciferase reporter assay further confirmed that miR-490-3p binds to CCAT1(P<0.05).8.The expression of miR-490-3p was significantly decreased in 32 cases of gastric carcinoma tissue samples(P <0.001),and its expression was negatively correlated with the expression of CCAT1 in the same group(Pearson analysis,P = 0.020).9.CCAT1 may reduce the inhibitory effect of mi R-490-3p on its target gene SMARCD1 through complementarily binding with miR-490-3p,and exert oncogenic function through regulating miR-490-3p / SMARCD1 signaling.Conclusion:In conclusion,the CCAT1 is overexpressed during the tumorigenesis and development of gastric cancer,and its highly corrected with the progression of gastric cancer,CCAT1 may reduce the inhibitory effect of miR-490-3p on its target gene SMARCD1 through complementarily binding with miR-490-3p,thus participate in the regulation of gastric cancer cell malignant phenotype.Furthermore,the expression of CCAT1 in sera may potentially be a biomarker for diagnosis of gastric cancer,and CCAT1 may be a potential therapeutic target.