Study On Differential Expression Profile Of LncRNA Between Concurrent Chemoradioresistant Nasopharyngeal Carcinoma Cell Lines And Parental Cell Lines

[Objective]:By constructing concurrent chemoradioresistance model of nasopharyngeal carcinoma cells in vitro,inducing concurrent chemoradiotherapy resistance cell lines,and high-throughput sequencing method was used to compare the differential expression of lncRNAs between the concurrent chemoradioresistance cell lines and the parental cell lines,screening lncRNAs involved in the concurrent chemoradioresistance of nasopharyngeal carcinoma cells,further more,applicated bioinformatics to analyze the GO terms and signal pathways involved in target genes of differentially expressed lncRNAs,which establish the foundation for further exploration of the mechanism of concurrent chemoradioresistance of nasopharyngeal carcinoma.[Methods]:1.Construction of a concurrent chemoradioresistant cell lines for nasopharyngeal carcinoma in vitro.Nasopharyngeal carcinoma cells CNE1 and CNE2 were selected,exposed to a complete medium containing 0.05ug/ml cisplatin,and irradiated with 6Gy 6Mv X-ray.The cells were exposed to cisplatin for 24 hours and then replaced with fresh medium.After the residual cells resume growth,the above treatment is repeated to a total of 10 times,then obtained chemoradioresistance cell lines,named CNE1 CRR and CNE2CRR;2.Use clone formation was used to confirm the resistance to concurrent chemoradiotherapy of CNE1CRR and CNE2CRR.Cells obtained in step 1 and their parental cell lines were counted 300,400,800,1000,2000,and 4000 cells,and then inoculated in 6-well plates,respectively,exposed to a complete medium containing 0.05ug/ml cisplatin,gradient doses(0Gy,2Gy,4Gy,6Gy,8Gy,10Gy)of X-rays at room temperature.After the cells were exposed to cisplatin for 24 hours,the fresh medium was replaced.After 2 weeks of culture,the cells were fixed with methanol,stained with crystal violet,counting colonies which more than 50 cells under microscope,calculate the cell survival fraction,fit the survival curve by the one-hit multi-target model,and calculate D0,Dq,SF2;3.Screening for lncRNAs involved in concurrent chemoradioresistance of nasopharyngeal carcinoma.High-throughput sequencing method was used to detect the differential expressions of lncRNAs in chemoradiotherapy resistant cell lines and parental cell lines;4.Functional enrichment analysis on the target genes of differentially expressed lncRNAs.Using bioinformatics software to predict target genes of differentially expressed lncRNAs,based on the annotation of Gene Ontology database,explored GO entries for lncRNAs target genes;base on the annotation of Kyoto Encyclopedia of Gene and Genomes database,explored the signaling pathways related to target genes of differentially expressed lncRNAs;5.Verification in sequence results of lncRNAs by qRT-PCR.Randomly selected 4 lncRNAs that differentially expressed in two groups of cells,qRT-PCR was applicated to detect their expression in concurrent chemoradioresistant cell lines and parental cell lines,then compared the qRT-PCR results with the sequence results;6.Statistical analysis of data.All the data were analyzed by SPSS25.0,statistics were expressed as mean ± standard deviation(x±s),paired t test was used to obtain the statistical significance.[Results]:1.After 10 concurrent chemoradiotherapy treatments,CNE1 and CNE2 concurrent chemoradioresistant nasopharyngeal carcinoma cell lines were established;2.The clone formation method was used to confirm the resistance of CNE1CRR and CNE2CRR to concurrent chemoradiotherapy;3.Through high-throughput sequencing,2746 lncRNAs were differentially expressed in CNE1CRR and CNE1(358 lncRNAs were significantly up-regulated and 2063 lncRNAs were significantly down-regulated in CNE1CRR);3475 lncRNAs were differentially expressed in CNE2CRR and CNE2(265 and 520 lncRNAs were respectively up-regulated and down-regulated in CNE2CRR);in addition,387 lncRNAs were simultaneously down-regulated in CNE1CRR and CNE2CRR,and 49 lncRNAs were simultaneously up-regulated in CNE1CRR and CNE2CRR;4.Enrichment analysis on target genes of differentially expressed lncRNAs.In GO analysis,the target genes of differentially expressed lncRNAs was classified from three aspects:biological process,cellular component,and molecular function.It was found that target genes participate in multiple biological functions;KEGG analysis found that target genes of differentially expressed lncRNAs participate in multiple signaling pathways,different target genes can participate in one signal pathway;one target gene can participate in different signal pathways;5.Verification sequencing results of lncRNAs differential expression profile by qRT-PCR.qRT-PCR results showed that the three lncRNAs LOC102724699,LOC102724872 and AC002116.7 were simultaneously up-regulated in CNE1CRR and CNE2CRR;HNRNPU-AS1 was simultaneously down-regulated in CNE1CRR and CNE2CRR;the expression trends of qRT-PCR were consistent with the sequencing results,indicating that this sequencing results was reliable[Conclusions]:1.This experiment successfully established concurrent chemoradioresistence cell lines CNE1CRR and CNE2CRR of human nasopharyngeal carcinoma,laying a foundation for the study of chemoradioresistance mechanisms of nasopharyngeal carcinoma.2.The expression profiles of lncRNAs involved in concurrent chemoradioresistence to nasopharyngeal carcinoma cells were obtained.Diverse biological functions and signal pathways on target genes of differentially expressed lncRNAs were obtained by bioinformatics analysis,which is instructive for further study.