Screening Of Non-small Cell Lung Cancer Associated Circular RNA And Its Potential Functional Research

Backgrounds and Objectives: Lung cancer is one of the most common malignancies in the world,the current incidence and mortality of lung cancer both rank first in the world of all malignant tumors.It represents a serious threat to human life and health and brings a heavy disease burden to human beings,which is one of the important public health problems that need to be solved urgently.The proportion of non-small cell lung cancer(NSCLC)is about 80% among all the histopathological types of lung cancer,mainly composed of adenocarcinoma,squamous cell carcinoma and large cell carcinoma.NSCLC patients are often diagnosed at the advanced stage of the disease.Surgical treatment is not usually the best choice,and they are not sensitive to the main treatment methods like radio-chemotherapy.The 5-year survival rate is very low,approximately 15%.Its specific pathogenesis is not yet clear now,and lacks sensitive and specific non-invasive biomarkers as well.Abnormal expression of non-coding RNA plays an important role in the development and progression of multiple tumors.High-throughput sequencing technologies in recent years,along with the continuous maturity and development of bioinformatics,more and more circular RNAs have been identified in multiple species(H.sapiens,D.melanogaster and C.elegans,etc.).The discovery of circRNA expands the types and functions of non-coding RNA family.Some circRNAs have higher abundance than their corresponding linear mRNAs.Some of them are reported to have the function of microRNA sponges and play a role of endogenous competitive RNA(ceRNA).Owning to its closed circular structure without 3' poly-A tail,circRNA can stably exist in human tissues and plasma and blood and could not be degraded by RNase R easily.It has the characteristic of tissue-and development-specific expression pattern.Circular RNAs are closely related to the development of different diseases,thus have the potential to serve as biomarkers of diseases such as malignancies.Therefore,this study preliminarily constructed the differential expression profile of circRNAs in patients with NSCLC and provided a reference for subsequent lung cancer associated studies of circRNAs.Moreover,significantly differentially expressed circRNAs were evaluated as non-invasive diagnostic biomarkers in peripheral blood,and biological functions in NSCLC were preliminarily explored.Methods: The research design of this project consists of two parts:(1)Establishment of differential expression profile of circRNAs in tissue stage: Illumina's next-generation sequencing platform,Hiseq 1500,was used for profile construction in 10 newly diagnosed patients with non-small cell lung cancer.Bioinformatics algorithm named CIRCexplorer was used to identify circRNAs.If more than two reads of one circRNA junction site in an individual sample were identified,then we consider this circRNA as reliable.Differential expression analysis of the sequencing result was performed by using DESeq2 software of R platform to obtain differential expression profile of circRNAs in NSCLC.We also carried out pathway enrichment analysis according to the parental genes of differentially expressed circRNAs based on one online network database called Metascape.(2)Plasma circRNA identification and its preliminary functional exploration stage: Combined with one public database of serum circRNAs,we validated differentially expressed circRNAs in plasma and selected one with highest abundance.In addition,relative quantification was performed in 50 vs 50 individual matched case-control plasma to evaluate its possibility as a NSCLC biomarker.In vitro experiments(cell proliferation assay,migration and invasion)combined with bioinformatics analysis were carried out to evaluate the potential biological effects of candidate circRNA in the development and progression of lung cancer.Results: A total of 5,471 circRNAs were identified in the sequencing screening stage,including 120 up-regulated circRNAs and 65 down-regulated circRNAs(Fold change ? 2,P < 0.05).We carried out pathway enrichment analysis according to the parental genes of 185 differentially expressed circRNAs and got several tumor associated pathways like small GTPase mediated signaling pathway and DNA damage repair pathway,etc.The top 10 circRNAs that could be up-regulated in plasma were obtained by merging with one serum circRNA database.CircFARSA showed highest abundance in plasma,the results of qRT-PCR(quantitative real-time PCR)showed that the expression of circFARSA in plasma was positively correlated with the sequencing result(r=0.64,P < 0.05).The expression level of circFARSA in case group was significantly higher than that in control group(Fold change = 1.96,P < 0.001).ROC analyses revealed that circFARSA has a sensitivity of 64% and a specificity of 66%,its area under the curve(AUC)reached 0.71 which indicates that circFARSA has the diagnostic value of NSCLC to some extent.Overexpressing circFARSA via plasmid could improve the migration and invasion ability of A549 cell line in vitro.Further bioinformatics predictions showed that miRNAs(mi R-330-5p and miR-326)targeted by circFARSA could regulate the expression of oncogenes like CCND1 and other tumor associated genes,suggesting circFARSA could influence the development and progression of NSCLC.Conclusion: This study constructed the specific circRNA expression profile of NSCLC based on next generation sequencing technology for the first time.A case-control study was carried out using plasma sample and confirmed circFARSA could serve as a novel biomarker for NSCLC,in vitro experiments along with bioinformatics predictions both suggest that circFARSA might participate in the malignant phenotype of invasion and metastasis of NSCLC.