Analysis Of Long Non-coding RNA Expression In Exosomes Of Nasopharyngeal Carcinoma Cells Resistant To Concurrent Chemoradiotherapy

Objective(s):In this project,we constructed a model of nasopharyngeal carcinoma cells for concurrent radiochemotherapy in vitro,induced the establishment of nasopharyngeal carcinoma radiochemotherapy resistant cell lines,and detected and screened the key exosomal lncRNAs molecules that lead to nasopharyngeal carcinoma radiochemotherapy resistance using biochips.The research results will provide a scientific basis for revealing the role of exosomal IncRNAs in nasopharyngeal carcinoma cells' concurrent radiochemotherapy resistance,which has important theoretical significance.It will lay a solid foundation for further finding related clinical intervention measures to improve the efficacy of radiotherapy and chemotherapy for nasopharyngeal carcinoma,and it has good clinical application value.Methods:1.Select the nasopharyngeal cancer cell lines CNE-1 and CNE-2,use a large-dose continuous radiotherapy and chemotherapy continuous shock method,give 0.05?g/mL cisplatin and 6Gy 6MV X-ray,repeat treatment for a total of ten times,induce the establishment of nasopharyngeal carcinoma CNE-1 CRR,CNE-2 CRR.2.Identify the resistance of nasopharyngeal carcinoma to radiochemotherapy resistant cell lines by plate cloning experiment.3.Use differential centrifugation to extract the exosomes produced by nasopharyngeal cancer cells and identify their size,morphology,marker protein and particle concentration and particle size to meet exosome standards.4.Using biochip technology to detect the differential expression levels of exosomal IncRNAs of CNE-1 CRR and CNE-2 CRR cell lines and the exosomes of their respective mother cell lines,and according to the results of biochip detection,screen out concurrent radiochemotherapy Resistance to key exosomal IncRNAs molecules.Results:1.After a total of ten treatments during the same period of radiotherapy and chemotherapy,successfully constructed in vitro nasopharyngeal carcinoma radiochemotherapy resistant cell lines CNE-1 CRR and CNE-2 CRR models.2.The plate clone formation experiment method has confirmed that CNE-1 CRR and CNE-2 CRR cell lines are resistant to radiochemotherapy compared with their respective mother cell lines,and using GraphPad prism8.0 software,using"multi-target click mathematical model",Draw cell survival curve.3.Exosomes were extracted from nasopharyngeal carcinoma cells by the traditional differential centrifugation method,and identified by transmission electron microscopy,Western Blot and nanoparticle tracing analysis(NTA).Exosomes were successfully isolated from nasopharyngeal carcinoma cell lines.4.Extract the total RNA of exosomes from nasopharyngeal carcinoma cells,use a micro-spectrophotometer to detect the concentration and purity of total RNA of exosomes,and finally analyze the CNE-1 CRR and CNE-2 CRR exosomal IncRNAs The differential expression profiles of exosomal IncRNAs in their respective mother cell strains were screened for the key up-regulation and down-regulation of key exosomal lncRNAs.Conclusion(s):In this experiment,CNE-1 CRR and CNE-2 CRR models have been successfully established after repeated treatment of concurrent radiotherapy and chemotherapy.Using IncRNAs biochip detection technology,the differential expression profiles of exosomal IncRNAs of CNE-1 CRR and CNE-2 CRR and their respective parent cell line exosomes were constructed.The IncRNAs that were significantly up-regulated in the exosomes of CNE-1 CRR and CNE-2 CRR were selected as MALAT1,FAM212B-AS1,LINC-PINT and H19,and the significantly down-regulated lncRNAs were SNHG15,CDKN2B-AS1,ZFAS1,CCAT1,SNHG6,GAPLINC and TUG1,These differentially expressed exosomal lncRNAs may play an important role in the formation of chemoradiotherapy resistance in nasopharyngeal carcinoma.