Circular RNAs Signature Predicts The Early Recurrence Of Stage ? Gastric Cancer After Surgery And CircRNA₁00269 Inhibits Gastric Cancer Cell Growth

Research background and purposeGastric Cancer?GC?is one of the most common malignant tumors in digestive system.At present,most of the patients with gastric cancer have been in the advanced stage.About 30%-80%of patients were diagnosed with advanced gastric cancer.Even received surgical treatment,lots of patients would receive recurrence,and the prognosis of this part of patients is often poor.Among patients who received radical surgery,stage? patients had the highest recurrence rate.To predict the recurrence of the problem,we often use the the clinical predictors such as age,tumor size,depth of tumor invasion,Borrmann classification,lymph node dissection,metastatic lymph nodes and so on in gastric cancer.However,it is difficult to obtain accurate prediction of early recurrence of gastric cancer only with these indicators.Circular RNA?circRNA?has been found to be one of the widely existing forms of RNA in recent years.CircRNA is different from the traditional linear RNA,which does not have a 5 'end cap structure and a' 3 'end poly A tail structure.The expression of circRNA has been found to be abnormal in breast cancer,colorectal cancer,liver cancer and other cancers.CircRNA can be used as a biomarker for cancer.CircRNA microarray technology is an ideal and effective method to study the expression profile of circRNA,which can quickly and accurately find out the related circRNA.In the current study,we used circRNA chip to analysis the cancer tissues,trying to find the gastric cancer related circRNA and gastric cancer early recurrence associated circRNA,and further to over expressed circRNA₁00269 in tumor cells to preform preliminary analysis function,and try to find its downstream target and its specific molecular pathways and the role of the.Research methodThe first step of this study is to extract RNA from the tumor tissue and adjacent normal mucosa of patients with gastric carcinoma after surgical resection.Then conducted circRNA gene chip to understand circRNA expression in gastric carcinoma,screening out the circRNA related to gastric cancer and circRNA related to early recurrence of gastric cancer.Then verified the results of microarray applications by qRT-PCR,and constructed the early recurrence prediction model of gastric cancer.What,s more,we used the construction technology of plasmid to overexpress the low expressed circRNA:hsa circRNA₁00269 in gastric cancer cells,to observe its effect on proliferation of gastric cancer cell lines,and further explore its downstream mechanism.Research resultsIn this study,circRNA microarray was used to determine the 46 stage III gastric cancer associated circRNA and the 8 early recurrence related circRNA.Furthermore,qRT-PCR was used to detect the expression of circRNA in early stage of gastric cancer which was identified by microarray.It was found that circRNA₁01308,circRNA₁04423,circRNA₁04916 and circRNA₁00269 were independent risk factors for early recurrence of stage ? gastric cancer by Cox regression model.A risk score model was further established to predict early recurrence.The risk score =?1.434× circRNA₁01308?-?0.917 × circRNA₁04423?-?1.042 × circRNA₁04916?-?1.201 × circRNA₁00269?.The model is verified through the training and testing groups,the HR of two groups can respectively reach 6.248?95%CI 2.534-15.403,p=0.000069?and 4.886?95%CI 1.375-17.359,p=0.014?,the area under the ROC curve can reach 0.763?95%CI 0.632-0.894,p=0.001?and 0.711?95%CI 0.558-0.863,p=0.018?.Then we combined the above model and the traditional TNM staging to get a new prediction model,the new risk score-2.709*CircRNAmodel risk score +1.432*T stage + 0.678*N stage.The HR in the two groups of the training group and the test group were 22.156?95%CI 5.143-95.150,p = 0.000032?and 21.601?95%CI =2.830-164.902,p = 0.003?,ROC analysis showed that the area under the curve of the two groups was 0.866?95%CI 0.769-0.962?and 0.818?95%CI 0.698-0.938?respectively.The reliability of the two predictive equations is good,which can be used to predict the risk of early postoperative recurrence.In this study,the expression of circRNA₁00269 in gastric carcinoma was detected by qRT-PCR.The expression of its isomer LPHN2 in gastric cancer was also significantly decreased.In this study,the downstream target miRNA,miR-605-3p and miR-630 of circRNA 100269 were predicted by bioinformatics method.The expression of miR-630 was negatively correlated with circRNA 100269 by qRT-PCR.Furthermore,the direct interaction between circRNA 100269 and miR-630 in gastric cancer cells was demonstrated by dual luciferase reporter assay.CircRNA₁00269 transfection experiments showed that circRNA₁00269 can inhibit the proliferation of gastric cancer cells,and this function can be inhibited by its downstream target miR-630.Research conclusionThe expression of circRNA in gastric cancer was significantly abnormal,we identified a large number of genes related to stage ? gastric cancer and early recurrence of gastric cancer by microarray.Furthermore,a model for predicting the early recurrence of stage ? gastric cancer was established by qRT-PCR.circRNA₁00269 is a low expression of circRNA in gastric cancer,which can directly bind to miR-630 and regulate the proliferation of gastric cancer cells.