The Screening Of RNA And Analyzing Of Differentially Expressed Genes In Plasma Of Lung Cancer Patients

The incidence and mortality of lung cancer are extremely high.One of the reasons is that the early diagnosis of lung cancer is very difficult.In order to improve the 5-year survival rate of lung cancer patients,a sensitive and non-invasive diagnosis method is urgently needed.In recently,with the development of sequencing technology,the researches of lung cancer biomarkers have become more and more popular,and more and more potential lung cancer biomarkers have been discovered,including various proteins,DNAs,RNAs(micro RNAs,long-non-coding RNAs,circular RNAs),etc.Circular RNAs are a series of endogenous non-coding RNAs.The most notable feature is that it has a closed-loop structure,which makes it more resistant to exonuclease and can be stably present in tissues and body fluids.Moreover,circ RNAs have tissue and developmental stages specificity,and are closely related to diseases.Some circ RNAs can act as mi RNAs sponges and participate in RNA regulatory networks,so circ RNAs can be the ideal potential disease biomarkers.Although many studies are focused on the researches of lung cancer biomarkers,only few reports of extracellular RNAs(ex RNAs)in lung cancer patients.Ex RNAs(including m RNAs,lnc RNAs,circ RNAs,etc.)in plasma contains the information of nucleic acid from various tissues of the body.In some way,it can reflect the health status of individuals.Due to the low invasiveness of obtaining plasma,plasma is also an ideal material for tumor biomarkers.Based on the background above,we tried to use RNA-seq technology to analyze the composition of ex RNAs by using a small amount of peripheral blood plasma,and compare the expressed genes in plasma of patients with non-small cell lung cancer(NSCLC)and healthy controls,after a series of bioinformatics processing,the differentially expressed genes(DEGs)and circ RNAs related to non-small cell lung cancer(NSCLC)were screened.Subsequently,quantitative PCR technology was used to validate the differentially expressed genes and circ RNAs,and finally screened out potential tumor biomarkers of NSCLC.The research and results of this article can be divided into the following three parts:1.Sequencing results of extracellular RNAs in plasma: In this article,high-throughput sequencing technology was used to characterize ex RNAs in 500 μL plasma of 6 non-small cell lung cancer patients and 6 healthy control individuals.The results showed that the expression of mitochondrial r RNAs(MT-r RNAs)and mitochondrial t RNAs(MT-t RNAs)in plasma of patients with non-small cell lung cancer increased significantly.The other types of RNAs did not change significantly in NSCLC.Among them,protein＿coding RNAs and lnc RNAs account for the major part,regardless of the number of reads or the type of genes.MT-r RNAs and MT-t RNAs may serve as potential markers to distinguish NSCLC.But the expression of circ RNAs is low and the co-expression level is low.The reason of this needs to be further explored.2.Differentially expressed genes analysis in plasma of lung cancer: There are 640 differentially expressed genes(368 up-regulated and 272 down-regulated)in NSCLC plasma compared with normal controls.After a series of functional enrichment and WGCNA analysis,we determined that 7 key DEGs are highly correlated with lung tumorigenesis,including COX1,COX2,COX3,ND1,ND2,ND4 L and ATP6.And these 7 DEGs have also been confirmed by quantitative PCR analysis.3.Preliminary exploration of hsa＿circ＿0000722 as a tumor marker of NSCLC: The circ RNAs sequencing data of 6 non-small cell lung cancer patients and 6 healthy control individuals were analyzed.Using quantitative PCR to validate the expression of hsa＿circ＿0000722 in NSCLC plasma,the results showed that the expression of hsa＿circ＿0000722 in non-small cell lung cancer patients was significantly up-regulated.The target micro RNAs prediction and the KEGG pathway analysis were performed.The results showed that multiple metabolic pathways targeting mi RNAs are closely related to the occurrence of lung cancer.Further confirmed the potential of hsa＿circ＿0000722 as a tumor marker for NSCLC.Based on the high-throughput sequencing researches of ex RNAs of a small amount plasma from patients with non-small cell lung cancer,we found that the RNAs includes mitochondrial r RNAs(MT-r RNAs)and mitochondrial t RNAs(MT-t RNAs),m RNAs(COX1,COX2,COX3,ND1,ND2,ND4 L and ATP6)and hsa＿circ＿0000722 expressions have increased significantly in plasma with non-small cell lung cancer,and these RNAs have the potential to become the tumor biomarkers for non-small cell lung cancer.