| non-small cell lung cancer(NSCLC)is the cancer with the highest morbidity and mortality worldwide.Despite the continuous improvement in the diagnosis and treatment of NSCLC in recent years,the 5-year survival rate of patients is about 17.4%,especially the 5-year survival rate of patients with advanced stage is lower.Platinum-assisted chemotherapy is the most important treatment for NSCLC,but chemotherapy resistance affects the therapeutic effect.The mechanism of cisplatin chemotherapy resistance in NSCLC has not been fully elucidated.There is an urgent need to find new regulatory factors to inhibit cisplatin resistance in NSCLC.CircRNA is a kind of circular-closed non-codingRNA with biological characteristics of tissue specificity,stability and conservation.Studies have confirmed that circRNA is involved in tumor cisplatin chemotherapy resistance,which has broad application prospects in chemotherapy sensitivity evaluation and treatment.However,the study of circRNA in NSCLC cisplatin chemotherapy resistance is still in the initial stage,so it is necessary to continue to find circRNA differentially expressed in NSCLC cisplatin chemotherapy resistance,and explore its function and mechanism,so as to improve the mechanism of NSCLC cisplatin chemotherapy resistance,and achieve personalized treatment for NSCLC patients,thus improving the therapeutic effect,prognosis and quality of life of patients.Part Ⅰ High-throughput sequencing and bioinformatics analysis of human non-small cell lung cancer A549 and A549/DDP cell linesObjective To analyze circRNAs differentially expressed in Cisplatin sensitive A549 cells and drug-resistant A549/DDP cells of NSCLC by highthroughput sequencing and bioinformatics,and screen out circRNAs that may be involved in cisplatin resistance of NSCLC.Methods The IC50 of A549 and A549/DDP cells to cisplatin was determined by CCK-8 method.The circRNA expression profiles of A549 and A549/DDP cells were analyzed by high-throughput sequencing,and the differentially expressed circRNA were analyzed.The genes were analyzed by GO function and KEGG pathway analysis,and the most significantly differentially expressed circRNA was verified by q RT-PCR,and the target circRNA was screened out.The circRNA basic information was analyzed by circ Base database,the circRNA loop formation was detected by Sanger sequencing,and the circr Na-miRNA-mRNA regulatory network was constructed for the circRNA to analyze the function.Results The IC50 of A549 and A549/DDP cells to cisplatin were respectively;By high-throughput sequencing,7137 circRNAs were detected in A549 cells and 3700 circRNAs were detected in A549/DDP cells,and 2286 circRNAs were co-expressed,accounting for 26.7% of all circRNAs.At the same time satisfy the | log2(FC)| 1 or higher and FDR < 0.05 circRNA altogether 730,of which the A549 / DDP cells raised in 689,down 87;There were 859 mRNA significantly up-regulated and 1028 mRNA significantly down-regulated in A549/DDP cells.GO functional analysis showed that different circRNAs were mainly concentrated in biological adhesion,locomotion,cellular process,single-organism process and metabolic process.The main enrichment pathways include: Metabolic pathways,pathways in cancer,MAPK signaling,Ubiquitin mediated proteolysis,Focal adhesion,actin cytoskeleton regulation and apoptosis.There was 1887 mRNA with significant difference between the two groups,among which 859 mRNA were significantly upregulated and 1028 mRNA were significantly down-regulated in A549/DDP cells.GO analysis showed that the differential mRNA were mainly related to cellular response to stimulus,cell communication cell differentiation,cell surface receptor signaling pathways,cell proliferation,cell death,cell adhesion immune response,cell migration,cell cycle,response to drug and ERK1 and ERK2 cascade.The involved signaling pathways mainly include PI3K-Akt signaling pathway,MAPK signaling pathway,Focal adhesion,TNF signaling pathway,Ras signaling pathway,Wnt signaling pathway,Rapl signaling pathway,NF-κB signaling pathway,Hippo signaling pathway,HIF-1 Signaling pathways,apoptosis and Toll-like receptor signaling pathways.RT-q PCR results showed that the expression of hsa＿circ＿0096157 in A549/DDP cells increased by about2.5 times compared with A549 cells.hsa＿circ＿0096157 is located on chromosome 11(chr11 : 65271376-65272624)and is formed by reverse splicing of MALAT1 gene with a length of 1248 bp.The ring structure of hsa＿circ＿0096157 was confirmed by Sanger’s experiment.Bioinformatics analysis showed that hsa＿circ＿0096157 could regulate apoptosis and cell cycle related proteins.Conclusion hsa＿circ＿0096157 is abnormally high expression in A549/DDP cells,located on human chromosome 11 with a length of 1248 bp,which may be an important regulatory factor of cisplatin resistance in NSCLC.Part Ⅱ Effects of hsa＿circ＿0096157 on biological behavior and cisplatin sensitivity of A549/DDP and A549/ cellsObjective To investigate the effects of silencing and overexpression of hsa＿circ＿0096157 on proliferation,migration and invasion ability,cell cycle,apoptosis level and cisplatin sensitivity of A549/DDP cells and A549 cells,respectively.Methods hsa＿circ＿0096157 lentivirus and overexpressed plasmid were transfected into A549/DDP and A549 cells,respectively,and the silencing and overexpression effects of hsa＿circ＿0096157 were detected by q RT-PCR.Transfected A549/DDP cells and A549 cells were treated with cisplatin,and the group without cisplatin was set up.Cell proliferation activity was detected by CCK-8 method,cell apoptosis rate was detected by flow cytometry and TUNEL staining,cell cycle was detected by flow cytometry,and cell migration and invasion ability were detected by Transwell chamber assay.Results A549/DDP cells silenced by hsa＿circ＿0096157 and A549 cells overexpressed by hsa＿circ＿0096157 were successfully constructed.Functional experiments confirmed that hsa＿circ＿0096157 silencing could inhibit the proliferation,migration and invasion of A549/DDP cells,induce cell apoptosis,block cell G1 phase,and improve the sensitivity of A549/DDP cells to cisplatin.hsa＿circ＿0096157 silencing can improve the proliferation,migration and invasion ability of A549/DDP cells,inhibit apoptosis,increase the proportion of cell G2 phase,and reduce the sensitivity of A549 cells to cisplatin.Conclusion hsa＿circ＿0096157 silences can inhibit the malignant behavior of drug-resistant NSCLC cells and improve the sensitivity to cisplatin,while hsa＿circ＿0096157 overexpression can increase the malignant phenotype of NSCLC cells and reduce the sensitivity to cisplatin.Part Ⅲ Effects of hsa＿circ＿0096157 on cyclin and epithelial mesenchymal transformation of A549 and A549/DDP cellsObjective To investigate the effects of silence and overexpression of hsa＿circ＿0096157 on cycle-regulated proteins and pithelial-mesenchymal transitions(EMT)of A549/DDP and A549 cells.In order to reveal the possible mechanism of hsa＿circ＿0096157 participating in the regulation of NSCLC cell cycle and migration invasion.Methods Western blot was used to detect the effect of hsa＿circ＿0096157silencing on cell cycle regulatory proteins(p21,Bcl-2,CDK4 and Cyclin D1)and EMT marker proteins(E-cadherin,N-cadherin,Vimentin)of A549/DDP cells.Results hsa＿circ＿0096157 silencing increased the protein expression of P21,Bcl-2 and E-cadherin,and decreased the protein expression of CDK4,Cyclin D1,N-cadherin and Vimentin in A549/DDP cells.Overexpression of hsa＿circ＿0096157 decreased the expression of P21,Bcl-2 and E-cadherin proteins,and increased the expression of CDK4 and Cyclin D1 proteins.Conclusion The mechanism of cisplatin resistance of hsa＿circ＿0096157to NSCLC cells is related to the regulation of cell cycle and EMT process. |
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