The Expression Profiling Characters Of CircRNAs In Non-small Cell Lung Cancer With RNA Microarray Analysis

Part ? Screening of circRNAs expression profile in non-small cell lung cancer by microarray chip techniqueResearch purposeThe differential expression of circRNAs,in non-small cell lung cancer(NSCLC)tissues and adjacent normal epithelial tissues was detected by circRNAs microarray technique.The expression profile of circRNAs in NSCLC was established.MethodsTotal RNA was extracted from lung cancer tissues and adjacent normal tissues of 3 patients with non-small cell lung cancer treated by surgery in Zhujiang Hospital of Southern Medical University,Total RNA.was extracted by Trizol(Invitrogen)method.UV spectrophotometer was used to detect the concentration and purity of RNA,and the integrity of RNA was detected by formaldehyde discoloration gel electrophoresis.After the extracted RNA passed the qualitative examination,the enriched circular RNA was amplified and transcribed into fluorescent cRNA..Purified labeled cRNA.The concentration and specific activity of labeled cRNA were measured.After labeling,prepare 50?1 hybridization solution and distribute it to gasket slide.After hybridization,the chip was washed and scanned by Axon GenePix 4000B to extract the signal value of the probe.T test was used to determine the significance of differentially expressed genes(p<0 05).Fold change and p-value were used to screen the differential expressions of circRNAs between the two groups.ResultsTotal RNA quality identification result:total RNA quality identification result:A260/A280 ratio is close to 2.0,and A260/A230 ratio is more than 1.8,indicating that total RNA quality of all tissue samples is reliable,no degradation or other impurity pollution,such as DNA,protein,etc.,A subsequent microarray chip and other experiments were performed.The circular RNA microarray chip selection showed that there were 103 circulating RNAs up-regulated by more than 2-fold in non-small cell lung cancer compared with normal lung epithelial tissue,with 81(p<0.05)down-regulation of more than 2-fold.conclusionThere is a significant difference in the expression of the circular RNAs between non-small cell lung cancer and the adjacent normal lung epithelial tissue,which may be associated with the development of non-small cell lung cancer and the development of the disease.Part ? Target Gene Prediction and Pathway Analysis of differentially expressed circRNAsResearch purposeUsing biological information software to predict circRNAs target gene,to explore the mechanism of the effect of circRNAs on the occurrence and development of non-small cell lung cancer(NSCLC).MethodsAccording to the Fold change value,P value and original signal value,eight ideal circRNAs were selected to predict the target gene.Then,the relationship between differentially expressed circRNAs and non-small cell lung cance,(NSCLC)genes was analyzed by the prediction software based on Targetscan,miRTarBase,miRDB and MicroRNA.org target gene prediction database.ResultsEight circRNAs target genes with significant expression differences were selected.Each circRNAs was based on the complementary relationship of the related genes in the prediction software.conclusionThe miRNA gene sponge is one of the most common methods known to play the function of the circular RNA,according to the predicted target genes,the analysis of the circular RNA participates in the occurrence and development of the non-small cell lung cancer by affecting the miRNA.Part ? The use of real-time fluorescence quantitative PCR to verify the expression of circulating RNAs in non-small cell lung cancer tissuesResearch purposeA real-time fluorescence quantitative PCR technique was applied to the non-small cell lung cancer specimen and the internal reference for further verification of the circulating RNA,which was obtained by the microarray chip experiment and expressed in the non-small cell lung cancer-related gene.methodsFrom November 2016 to March 2017,6 patients undergoing radical lung cancer surgery in Zhujiang Hospital of Southern Medical University were selected.According to the tissue samples provided by these patients,the samples were verified by qRT-PCR,and the samples were collected and preserved in the same way as the first chapter.According to the operation instructions,the sample RNA was used for cDNA synthesis,and fluorescence quantitative PCR.was used.ResultsQRT-PCR confirmed that the relative expression level of circRNAs-453 was lower than that of internal reference,and the relative expression level of circRNAs-60461 was significantly higher than that of internal control(p<0.03548<0.05),and the relative expression level of circRNAs-60461 was significantly higher than that of internal reference(p<0.00336<0.05).The relative expression level of circRNAs-86694 was significantly higher than that of internal control(p<0.01498<0.05).conclusionThe results of the circular RNAs chip screening and the real-time fluorescence quantitative PCR validation results confirmed that there were indeed abnormal expression of the circle RNAs between the non-small cell lung cancer and the normal lung epithelial tissue,and we screened and verified that the circle RNA-60461 and the circle RNA-86694 were likely to have a relationship with the onset of non-small cell ung cancer.The abnormal expression of the circular RNA-60461 and the circular RNA-86694 and the development of the non-small-cell lung cancer may be related,and the function of the circular RNA-60461 and the circular RNA-86694 lays a good foundation for the biological function experiment and the animal experiment of the next step,and provides a new train of thought and platform.