The Function And Molecular Mechanism Of FLI1circular RNA In The Development Of Small Cell Lung Cancer

Small cell lung cancer(SCLC)is the most devastative type of human lung malignancies,accounting for approximately 15%-20% of all lung cancer cases recorded.The rapid and disseminated growth and recurrence pattern remains the primary cause of poor clinical prognosis in patients with SCLC.The five-year survival of SCLC is 10% for extended stage and 2% for limited stage because many people are diagnosed at extended stage,limited stage were found only in about one-third of patients.In the last two decades,despite a high initial response rate to first-line chemotherapy,recurrence arises rapidly in the majority of cases,usually killing the patient within only a few months.Molecular targeting therapies,although being successful in the treatment of various tumors,often fail in SCLC,primarily because mechanisms of the disease remain unclear.Therefore,Clearly,identification of the molecular pathogenesis of SCLC is urgently needed to bring breakthroughs to the prognosis assessment and treatment of SCLC.Research background:FLI1(Friend leukemia virus integration 1)is an Ets(E26 transformation-specific)transcription factor family member,was first identified as a target of proviral integration in F-Mu LV-induced mouse erythroleukemia by Ben-david.Generally,FLI1 is preferentially expressed in hematopoietic cells and tissues,endothelial cells and fibroblasts,and it has been previously reported to act as a major driver of hematological malignancies.The oncogene FLI1 may regulate the biological processes of target cells via activation/repression of multiple target genes,such as Bcl-2,to inhibit apoptosis and result in tumorigenesis.With the deepening research on FLI1,recent studies have documented that FLI1 is also aberrantly expressed in several solid tumors,such as Ewing sarcoma(EWS),metastatic melanomas,nasopharyngeal carcinoma(NPC),metastatic breast cancer(MBC),endometrial cancer(EC).Currently,our research team has been confirmed that FLI1 was aberrantly expressed in SCLC tissues compared with non-small cell lung cancer(NSCLC),and knockdown of FLI1 expression significantly inhibited cellular proliferation,promoted apoptosis and repressed colony formation of SCLC cells,suggesting that FLI1 may serve as an attractive target for therapeutic intervention of SCLC.With the development of RNA-seq,circular RNAs(circRNAs)were found to be covalently closed RNA molecules,in which the 3’-and 5’-ends are linked in a non-collinear way by a process called back-splicing.Unlike in linear RNA splicing,a splice donor site is joined to a splice acceptor site upstream in the primary transcript,yielding a circRNA.Circ RNA is stable and can not be degraded by Rnase-R enzyme.Currently,it has been reported that circRNAs contain multiple miRNA binding sites and can bind to miRNAs,defined as“miRNA sponges”,leading to inhibit the activity of miRNAs and regulate the expression of their downstream target genes.Through the research of the circRNA databases(circbase,http://www.circbase.org/),we found that FLI1 gene transcript possess six kinds of circRNAs.By screening the expression level of circFLI1 s in lung cancer cell lines and lung cancer tissues,we found that FLI1 circular RNA consist of exon 2,exon 3 and exon 4(also called circFLI1E2-4)and FLI1 circular RNA consist of exon 2-exon 5(also called circFLI1E2-5)were aberrantly expressed in SCLC cell lines and SCLC tissues,compared with NSCLC.However,whether these circFLI1 s could regulate the biological processes of SCLC remains further research.Research objectives: 1.Screen the expression level of circFLI1 s in lung cancer cell lines and lung cancer tissues.2.Analyze the correlation between the expression level of circFLI1 s and clinical data of SCLC.3.Completely assess the function of circFLI1 s in tumorigenesis and development of SCLC.4.Investigate the possible mechanism of circFLI1 promoting SCLC,clarify the signal pathway and target genes regulated by circFLI1.Research method: Screen the expression level of circFLI1 s in lung cancer cell lines and lung cancer tissues using Real-time Quantitative PCR(RT-PCR)and fluorescence in situ hybridization(FISH),especially in SCLC.Analyze the correlation between the expression level of circFLI1 s and clinical data of SCLC by statistical methods.Assess whether down-regulation of circFLI1 s by short hairpin RNA(sh RNA)could affect cellular proliferation,apoptosis,cell cycle,migration and ability of colony formation by MTT assay,flow cytometry,Annexin V/7-AAD staining,Transwell assay and so on.Luciferase reporter assay,western blot,RT-PCR,FISH and reverse transcription associated trap assay(RAT)methods were used to explore the possible mechanism of circFLI1 s.Research result: 1.Circ FLI1 E2-4 and circFLI1 E2-5 were aberrantly expressed in SCLC cell lines and SCLC tissues.The expression level of circFLI1 E2-4 and circFLI1 E2-5 was positively correlated with lymph node metastisis.2.The expression level of circFLI1 E2-4 in patients with SCLC was significantly higher than that in healthy people.And When patients with SCLC got completed remission(CR)or partial remission(PR),the expression level of circFLI1 E2-4 was significantly lower than before.3.Circ FLI1 E2-4 and circFLI1 E2-5 could promote the migration of SCLC cells,but not affected cell growth,cell cycle,colony formation and apoptosis.4.Circ FLI1 E2-4 knockdown enhanced the sensitivity of SCLC cells to etoposide(VP-16),while circFLI1 E2-5 did not.5.Circ FLI1 E2-4 and circFLI1 E2-5 interacted with miR-584-3p,and could significantly inhibited activity of miR-584-3p.6.Circ FLI1 E2-4 and circFLI1 E2-5 regulated the transcriptional activity of miR-584-3p target genes and the Rho A/ROCK1 signal pathway.Research conclusions: This study investigated the function of circFLI1 s at the cellular level and molecular level,revealing that Circ FLI1 E2-4 and circFLI1 E2-5 could promote the migration of SCLC cells,but not affected cell growth,cell cycle,colony formation and apoptosis.Ulteriorly,through the exploration of molecular mechanism on circFLI1 s,we demonstrated that circFLI1 E2-4 and circFLI1 E2-5 interacted with miR-584-3p,to inhibit its activity and the transcriptional activity of downstream target genes,and then involved regulation of Rho A/ROCK1 signal pathway.Therefore,this research contributed to further understand the molecular mechanism of tumor migration and chemotherapy resistance,and provided a novel powerful clue for thoroughly research of Rho A/ROCK1 signal pathway.