Profiles And Bioinformatics Analyses Of CircRNAs In Taxol-resistant NSCLC

Background and Objective:The development of acquired chemoresistance appears to be common phenomenon during the treatment for patients with lung cancer,which throws a stumbling block to the successful implementation of therapeutic strategies.At present,the researches on RNA field have gradually become hotspot.CircRNA was recently identified to be a special class of RNA molecules.They were regarded as miRNA sponges to be implicated in physiological and pathological processes.This study aimed to explore the molecular mechanisms and signaling pathways involving taxol resistance-related circ RNAs.Methods:1.We selected lung adenocarcinoma cell line A549 and resistant cell line A549/Taxol,which generated from parental A549 cells via induction by taxol.Then,we observed the difference in appearance of the two cell lines using high power lens of the inverted optical microscope.CCK-8(Cell Counting Kit-8)assay was performed to detect the viability of the two cell lines when exposed in different concentration of taxol.The absorption values of optical density in two groups was recorded at 450 nm using a automatic microplate reader.After that,IC50 s of taxol acting on the two cell lines were calculated and taken into comparison to validate the resistance to taxol in A549/Taxol cells.2.We conducted high-throughput circRNA microarray to compare expression profiles between parental and resistant cell lines and found circRNAs with differential expression.3.We utilized the miRNA prediction software based on miRanda database to predict miRNAs related to the differentially expressed circRNAs according to complementary sequence paring.MiRNAs-related signaling pathways and target genes were investigated by DIANA-miRPath database.And the circRNA-miRNA interaction network was established by Cytoscape software.4.We carried out GO analysis and pathway analysis to predict biological function and signaling pathways,in which the host genes of differentially expressed circRNAs were implicated.Results:1.Validation of taxol-resistance: Compared to A549 cells,the shape of A549/Taxol cells was elongated and cell-cell adhesion was largely decreased.Calculated by the absorption values of optical density,the IC50 of A549/Taxol cell line was about 73 times higher than A549 cells.2.Differential expression: The high-throughput circRNA microarray examined approximately 88 thousand circRNAs.When fold change(FC)> 2.0 and p ? 0.05,the results proclaimed that a total of 11281 circRNAs seemed to be differentially expressed between the two cell lines.Among which,the upregulation of 2909 circRNAs and the downregulation of 8372 circRNAs were shown.3.MiRNA prediction: Through miRNA response elements(MREs),the majority of differentially expressed circRNAs could complementarily bind to conserved seed sequence in miRNAs.Among which,several miRNAs have been documented to influence the sensitivity of cancer cells to taxol,such as miR-141-5p and mi R-34c-5p.In DIANA-miRPath database,miR-141-5p is implicated in cell cycle.And miR-34c-5p participates in apoptosis and focal adhesion.These signaling pathways are related to taxol resistance in cancers.Whereas,a small number of circRNAs in microarray data had no correlation with mi RNAs.This study only constructed the interaction network for the significantly differential circRNAs in expression and corresponding mi RNAs according to fold change.4.Function prediction: GO analysis showed that the host genes of differentially expressed circRNA mostly was involved in the biological processes including cell cycle,cell metabolism and cell-cell adhesion,which were closely linked to the capabilities of cell proliferation and migration.Pathway analysis showed that the host genes were involved in the signaling pathways incurring the resistance of cancers to chemotherapeutic drugs including taxol,such as integrin signaling pathway,adherens junction,ErbB signaling pathway and so on.Conclusions:NSCLC cell line A549/Taxol was confirmed to be resistant to taxol.The appearance of A549/Taxol presented EMT(epithelial-mesenchymal transition)-like form due to the property of drug resistance,enhancing the abilities of cells to migrate and invade.This study researched the differential expression profiles of substantial circRNAs and the notable role of these circRNAs played in taxol-resistant NSCLC,suggesting that circRNAs could promote the initiation and progression of chemotherapy resistance.Therefore,circRNAs may serve as excellent diagnostic markers and therapeutic targets in the future clinical application.