Preliminary Screening And Expression Verification Of CircRNAs For Lung Cancer In Xuanwei,Yunnan

ObjectiveLung cancer,as the most common malignant tomours in the world,especially in Asia,has a high incidence and mortalit.Lung cancer in Xuanwei?LCXW?is a kind of lung cancer which is occured in the northeast of Yunnan province.The incidence and mortality of lung cancer in this area are increasing and higher than the national average.Because of the lack of sensitive biomarkers and effective treatment methods,most of the lung cancer patients are in the advanced stage of cancer when they come to the hospital for medical treatment.Therefore,they lost the best treatment opportunities which makes severely damaged of patients' physiology and psychology.Therefore,finding effective biomarkers for LCXW is one of the greatest significant measures for the early diagnosis LCXW.CircRNA is more capable for reflecting the development stage of disease,because of its stability,time specificity and tissues specificity.We use circRNAs array to screen the differentially expressed circRNAs inLCXW,and applied real-time quantitative PCR?real-time quantitative PCR,RT-qPCR?to further verify the differentially expressed circRNA in the array,hope to screen out the potential biomarker for diagnosis of LCXW.Method1.After extracting 26 pairs of LCXW samples,the RNA was divided into 6 groups according to the stages?phase I:10/26;stage II:9/26;stage III:7/26?and tissue sources?tumour tissues and adjacent nontumorous tissues?.Each group was mixed with Arraystar Human circRNA Array V2 to analyze circRNAs expression.Using the box plot and scatter plot,the differential expression profiles of LCXW related circRNAs were established,and Fold Change>2 was thought difference significantly.And exploring the downstream target miRNA of circRNA through bioinformatics analysis.2.Analyzing and screening the differentially expressed circRNAs:?Fold Change is the greater the better;?the Raw Intensity of each circRNAs is more than 200;?the length of circRNAs is range 200bp from 2000bp;?P-value is the smaller the better;?circRNAs whose predicted target microRNAs or the parent gene has been confirmed associated with the cancers.3.Designing primers,using real-time fluorescent quantitative PCR?real-time quantitative PCR,RT-qPCR?to verify the value of selected circRNAs in 50 paired LCXW tissue.4.The clinicopathological parameters of patients were analyzed by statistics.Analysing if candidate circRNAs is differentially expressed in LCXW patients.Kaplan-Meier survival analysis was used to evaluate the relationship between the expression of circRNA and the prognosis of LCXW.And analysing the correlation between differentially expressed circRNAs and sex,age,smoking history,pathological stage,lymph node metastasis,tumor size.Result1.Through the technology of circRNAs microarray,we discovered 717 upregulated and 1055 downregulated differentially expressed circRNAs in I stage,612 upregulated and 806 downregulated differentially expressed circRNAs in II stage,183 upregulated and 455 downregulated differentially expressed circRNAs in III stage.There are 537 cicrRNAs differentially expressed in every stage,143 upregulated and 394 downregulated.Each circRNA predicts 5 possible combinations of miRNA.2.We screened the differentially expressed circRNAs,and combined the literature to investigate the predicted miRNAs of circRNAs,we selected 8 valuable circRNAs for further study at last.3.Primers were designed according to the structure of circRNAs,4 circRNAs primers were designed successfully?hsa_circRNA₁04600,hsa_circRNA₀69397,hsa_circRNA₁05013,hsa_circRNA₄06010?,and RT-qPCR was used to verify the expression of the 4 circRNAs at the level of tissue.It was found that hsa_circRNA₁04600?significantly upregulated?,hsa_circRNA₁05013?significantly upregulated?and hsa_circRNA₄06010?significantly downregulated?were differentially expressed in LCXW tissues,and hsa_circRNA₁04600 and hsa_circRNA₄06010 were consistent with the results of the chip,but hsa_circRNA₁05013 was not consistent.4.Statistics was used to analyze the correlation between the expression of circRNAs and clinicopathological parameters.It was found the upregulated of hsa_circRNA₁04600 and hsa_circRNA₁05013 were associated with tumor size.The downregulated of hsa_circRNA₄06010 was associated with the prognosis of LCXW patients.Conclusion1.Through the technology of circRNAs microarray,we discovered there were a great number of differentially expressed circRNAs in LCXW patients.2.Using RT-qPCR technology to verify candidate circRNAs at the tissue level,hsa_circma₁04600,hsa_circma₁05013 and hsa_circma₄06010 were significantly differential expression.The upregulated of hsa_circrna₁04600 and hsa_circma₁05013 was associated with tumour size,and the downregulated of hsa_circma₄06010 was associated with prognosis of LCXW.It illustrated hsa_circrna₁04600?hsa_circma₁05013 and hsa_circrna₄06010 may serve as potential biomarkers for the diagnosis of LCXW.3.This study provides new data for the study,diagnosis and treatment of LCXW.