Identification And Biofunctional Study Of Circular RNA0084893 In Gastric Cancer

Background:Gastric cancer is the third world’s leading death cancer,affecting more than 400,000 people per year in China.About 80% of the patients are diagnosed in the advanced stage of gastric cancer,and the five-year survival rate is less than 20%.In contrast,patients who diagnosed at early stage have a 5-year survival rate up to 90%-95%.It is important to change the focus of anticancer from treating advanced gastric cancer to strengthening the early diagnosis and treatments of gastric cancer.As a member of the non-coding family,circular RNA(circRNA)is widely found in tissues and eukaryotic cells.Due to the lack of free 5 ’and 3’ ends and the formation of covalently closed circular structures by reverse shear,circRNAs are characterized by high stability,long half-life and so on.With the development of high-throughput sequencing technology and bioinformatics,it has been reported that circRNAs are not only abundant in tumor tissues,but also expressed in specific ways during the development of tumor.Studies have shown that circRNAs can regulate gene expression in tumor cells and change their biological characteristics,which is expected to be a new tumor marker and therapeutic target.Though the knowledge of thousands of circRNAs with known functions is expanding,there are still many circRNAs functioning in gastric cancer that remains to be detected.Objective:This study further investigated the value of early diagnosis of circRNA in GC,and conducted in vitro biofunctional experiments to explore the effects of circRNA0084893 in proliferation,migration,invasion,apoptosis and cell cycle in gastric cancer cells.Highthroughput sequencing was used to predict the possible target genes of circRNA0084893.Material & methods:The differentially expressed circRNAs in gastric cancer tissues were detected by sequencing technology.Target gene was screened by combining with the circ Base database and the results of sequencing.Quantitative real time polymerase chain reaction(q RT-PCR)was used to detect the expression level of 68 pairs of circRNA0084893 in gastric cancer tissues and adjacent tissues,and the expression levels of circRNA0084893 in normal gastric mucosal epithelial cell lines(ges-1)and gastric cancer cell lines(MKN45,mgc-803,sgc-7901,hgc-27,bgc-823 and AGS)which representing different degrees of differentiation.Correlation between the expression level of circRNA0084893 in gastric cancer tissues and the age,gender,tumor size,nerve infiltration,lymph node metastasis and TNM stage were analyzed according to pathological results.Downexpression of circRNA0084893 in gastric cancer cell lines were studied by transfection of small interfering RNA(si RNA).Biofunctional studies were carried out by the Cell Counting Kit-8(CCK-8)assay,colony formation assay,Transwell assay,scratch wound assay and flow cytometry assay.Finally,RNA sequencing was used to detect the target genes of circRNA0084893 and predict its possible mechanisms.Results:According to q RT-PCR,circRNA0084893 was downregulated in both GC tissues and cell lines(MKN45、MGC-803、 SGC-7901、 HGC-27、 BGC-823 and AGS).It was also related to the tumor size(P=0.045)、differentiation(P=0.037)、nerve invasion(P=0.023)and biomarker CA724(P=0.003).According to the receiver operating characteristic curve,the area under the curve was calculated to be 0.643,indicating that circRNA0084893 may be valuable as a molecular biomarker for gastric cancer.In vitro functional studies showed that knockdown of circRNA0084893 facilitated the proliferation,migration and invasion of GC cells,blocked the cell cycle in S phase,affected the gastric cancer cells apoptosis.Sequencing results suggested,after knockdown circRNA0084893 can cause a variety of changes of m RNAs and mi RNAs.It also had certain influence on endogenous competitive RNA network.These effects may regulate the gastric cancer related genes transcription,cause disorder of transcription,abnormal gene expression,etc.Conclusion:CircRNA0084893 was downregulated in both GC tissues and cell lines.Circ RNA0084893 is associated with tumor size,differentiation,nerve infiltration and tumor marker CA724 in gastric cancer,which also has possible value in the clinical diagnosis of gastric cancer and is expected to be used as a clinical molecular biomarker for gastric cancer.Downregulation of circRNA0084893 can promote proliferation,migration and invasion of gastric cancer cells,prevent gastric cancer cells from transitioning from S phase to G2 phase and affect cell apoptosis.Circ RNA0084893 can cause changes in endogenous competitive RNA networks that may affect the expression of gastric cancer related m RNAs and mi RNAs.