The Role And Mechanism Of Hsa＿circ＿0001681 In Non-small Cell Lung Cancer

Objective: We screened NSCLC tissues for the aberrantly expressed circ RNAs by RNA sequencing.We examined circ RNA expression in NSCLC tissues and cells and analyzed its clinical significance.We further explored the biological roles and mechanisms of circ RNAs in NSCLC.Our study may provide a potential biomarker for NSCLC diagnosis and treatment.Methods: The highly expressed circ RNA,hsa＿circ＿0001681(abbreviated circ1681),was identified by RNA sequencing and bioinformatics analysis.We employed q RT-PCR to detect circ1681 expression level in NSCLC cell and tissues.Circ1681 knockdown and overexpression were performed by si RNA and plasmid transfection,respectively.Cell counting assay and colony formation assay were used to evaluate the effect of circ1681 on NSCLC cell proliferation.Transwell migration and invasion experiments were used to examine the effect of circ1681 on the metastatic ability of NSCLC cells.The effects of circ1681 on cell apoptosis and cell cycle were detected by flow cytometry.A subcutaneous tumor mode in nude mice was used to identify the impact of circ1681 in vivo.The downstream target genes and signaling pathways of circ1681 were validated by transcriptome sequencing.Circ1681-interacting proteins were predicted by bioinformatic and verifyed by RIP assay.The localization of FUS and circ1681 in NSCLC cells was examined by immunofluorescence assays.The biological role of FUS in NSCLC was detected by si RNA transfection experiment,and the regulation of FUS on circ1681 was detected by co-transfection experiment.The downstream target gene of circ1681/FUS was screened by q RT-PCR and verified by RIP experiments.Results: Circ1681 was found to be highly expressed in NSCLC tissues and cells.Circ1681 is a 516-nucleotide-long reverse splicing of exons 2-6 of the RAPGEF5 gene on chromosome 7,which can resist the digestion of RNase R and is mainly distributed in the cytoplasm.Knockdown of circ1681 decreased NSCLC cell proliferation,migration,and invasion,enhanced the proportion of apoptotic cells and caused cell cycle arrest in NSCLC cells.Furthermore,knockdown of circ1681 inhibited epithelial-mesenchymal transition(EMT)in NSCLC cells.Circ1681 overexpression resulted in the opposite effects.Moreover,circ1681 knockdown inhibited the formation of subcutaneous xenograft tumor in nude mice.Transcriptome sequencing confirmed the downregulation of genes such as WEE1 and the inhibition of Wnt/β-catenin,and STAT3 signaling pathways in NSCLC cells by circ1681 knockdown.A possible binding between FUS protein and circ1681 was predicted by bioinformatics,and confirmed by RIP assay.FUS protein and circ1681 were co-localized in the cytoplasm.FUS knockdown inhibited the proliferation,migration and invasion of NSCLC cells and FUS knockdown reversed the tumor-promoting effect of circ1681.The expression level of WEE1 was downregulated by both circ1681 and FUS knockdown.RIP assay showed that FUS protein bound to WEE1 m RNA.Conclusions: Circ1681 was upregulated in NSCLC cells and tissues and had the potential to be a biomarker for NSCLC.Circ1681 regulates the expression of WEE1 gene by binding to FUS protein to promote NSCLC progression.