Effect Of Paclitaxel On The Biological Function Of T Cell Lymphoma Jurkat Cell Line And Its Mechanism

Objective: In this study,the gene expression profile of the T-cell lymphoma Jurkat cell line treated with paclitaxel was analyzed by biological function experiment,second-generation sequencing technology and public database,and the results were preliminarily verified,aiming to explore the possible molecular mechanism of paclitaxel against T-cell lymphoma at the gene and protein levels.To provide more theoretical basis for the study of the mechanism of paclitaxel against T cell lymphoma.Methods: 1.CCK-8 method was used to determine the concentration of paclitaxel in human T cell lymphoma Jurkat cell line,and then cell viability and cell morphology were detected.2.The inhibitory effect of paclitaxel on proliferation of Jurkat cell line was observed by flow cytometry to detect cell cycle and apoptosis.3.Transcriptome sequencing was performed on Jurkat cell line after paclitaxel was treated with high-throughput sequencing technology,and bioinformatics analysis was performed on the sequencing results.4.Gene chips containing T-cell lymphoma were retrieved from the GEO database to obtain m RNA expression data,and core genes and key regulatory pathways were analyzed after intersection with transcriptional sequencing data.5.The transcriptome and protein levels of the core genes were verified by fluorescence quantitative PCR and Western blot assay.Results: 1.The results of CCK8 experiment showed that compared with untreated cells,the inhibition rate of cells treated with paclitaxel was significantly increased,and the increase of the inhibition rate of cells treated with paclitaxel for 48 h and 72 h was more obvious than that of 24 h,in a concentration-dependent manner;The IC₅₀ values of paclitaxel and adriamycin for Jurkat cell line were 0.004±0.0001μmol/L and 0.0591±0.0015μmol/L respectively.Under inverted microscope,it was found that with the increase of paclitaxel concentration,the cell density decreased significantly,the cell morphology was irregular,the cell shrinkage was obvious,the distribution was sparse,the cell membrane was incomplete,and the nucleus fragmentation was common.The cells in the control group were regular in morphology,vigorous in growth and uniform in size.2.Flow cytometry was used to detect cycle and apoptosis of Jurkat cells.IC₅₀ concentration paclitaxel induced significant G2/M phase arrest in Jurkat cells after 24 h and 48 h,and the early phase arrest was more significant.Compared with the control group,the apoptosis rate of Jurkat cells after treatment with 0.004 and 0.006μmol/L paclitaxel for 48 h was significantly increased,and in the early apoptosis was also higher than that in the control group and the low concentration group,but the effect on the late apoptosis was not significant.3.Jurkat cells treated with paclitaxel-seq and expressed genes of RNA analysis（P< 0.05,| log2（FC）|）1 or higher),found 351 different genes,genetic variations in 323 raised genes,28 genes.Gene functional annotation and pathway enrichment analysis were performed on DEG.GO analysis showed that the differentially expressed genes were mainly involved in signal transduction,negative regulation of cell proliferation,nucleosome assembly and other biological processes（BP）.In the cell component（CC）,plasma membrane and nucleosome were mainly constituted.It is enriched in molecular functions（MF）such as protein heterodimerization and ligand-gated ion channel active cell protein metabolism.KEGG analysis showed that differential genes were mainly involved in cancer pathways,transcriptional dysregulation in cancer and p53 signaling pathways.4.On the platform of GEO database mining from seven 22 new chip,including tumor in 720 cases,153 cases of normal sample,using 22 all differences in gene chip（| log2（FC）| ≥1 and P < 0.05）and taxol sequencing results in intersection,140 intersection genes.The GO analysis of the intersection genes showed that the intersection genes were mainly involved in signal transduction,nucleosome assembly,information signaling,cell protein metabolism and other biological processes（BP）.In the cell component（CC）,plasma membrane and nucleosome were mainly constituted.It plays a role in molecular functions（MF）such as protein heterodimerization and transcriptional activator activity.KEGG analysis showed that the intersection genes were mainly involved in the transcriptional dysregulation signal pathway in cancer.Core genes were selected by screening strategy: HIST2H2AA3,JUN,NR4A2,NR4A3,CDKN1 A,and CCNE1.5.The results of real-time quantitative PCR showed that the expression levels of Hist2H2AA3,Jun,NR4A2,NR4A3 and CDKN1 A were up-regulated and the expression levels of CCNE1 were down-regulated in Jurkat cells treated with paclitaxel IC₅₀ concentration for 48 h,and the change trend was completely consistent with the results of high-throughput sequencing.6.Western Blotting results showed that CCNE1 protein expression level decreased and Jun protein expression level showed no difference after Jurkat cells were treated with different concentrations of Paclitaxel for 48 h.Conclusion: 1.After treated with paclitaxel for 24 h and 48 h,Jurkat cells developed significant G2/M phase arrest,and the early phase arrest was more significant.At the same time,the apoptosis rate of paclitaxel group increased compared with the control group,and in Jurkat’s early apoptosis was also significantly higher than that of the control group,in a concentration-dependent manner,but had no effect on the late apoptosis.2.The mechanism of action involved in paclitaxel against T-cell lymphoma Jurkat cells may be related to negative regulation of cell proliferation,protein heterodimer activity,transcriptional activators,cell signal transduction,and transcriptional dysregulation signaling pathways;Jun,HIST2H2AA3,NR4A2,NR4A3,CDKN1 A and CCNE1 may play an important role in inhibiting Jurkat cells.3.Paclitaxel may inhibit the growth of Jurkat cells by down-regulating CCNE1 gene and causing cell cycle arrest.