The Influence Of TP53TG1 On Migration And Invasion Of Diffuse Large B-cell Lymphoma Cells By Regulating PLK1

[Objective] To investigate the expression patterns of long non-coding RNA(lncRNA)TP53TG1 in diffuse large B-cell lymphoma(DLBCL)cell lines,study the influence on proliferation,migration,invasion,cell cycle and apoptosis of DLBCL cells.To perform a preliminary investigation on the regulatory relationship between lncRNA TP53TG1 and PLK1 gene.[Methods] 1.The expression patterns of PLK1 was detected in GCB-DLBCL cell line(OCI-ly1)and ABC-DLBCL(OCI-ly3)cell line by real-time quantitative PCR(RT-qPCR).2.Lentiviral overexpression vector was constructed and lncRNA inhibitor of lncRNA-TP53TG1 was synthesized,then transiently transfected into OCI-ly3 and OCI-ly1 cells of DLBCL,respectively.3.The expression of PLKl was detected in DLBCL cell lines after up-regulation and down-regulation of lncRNA-TP53TG1 by RT-qPCR and Western blot.4.The effects were respectively detected on migration,invasion,cellcycle and apoptosis of OCI-ly1 and OCI-ly3 cells after up-regulation and down-regulation of lncRNA-TP53TG1 by Transwell migration and invasion assay and flow cytometry.[Results] 1.PLK1 was highly expressed in OCI-ly1 and OCI-ly3 cells.Total RNA was respectively extracted from LCL7,OCI-ly1,andOCI-ly3 cells.The results of RT-qPCR showed that PLK1 mRNA washighly expressed in OCI-ly1 and OCI-ly3 groups,compared with thecontrol group LCL7(P<0.05).2.LncRNA TP53TG1 was low expressed in OCI-ly1 cell and highly expressed in OCI-ly3 cell.The expression of lncRNATP53TG1 was detected by RT-qPCR.The results showed that lncRNA TP53TG1 was low expressed in OCI-ly1 cell and highly expressed in OCI-ly3 cell,compared with LCL7 cell(P<0.05).It suggested that lncRNA TP53TG1 could be a differential expressed gene for follow-up experiment.3.The up-regulation of lncRNA TP53TG1 decreased migration and invasion of OCI-ly1 cell,and induced G2 phase arrest in the cell cycle.The results of enzyme digestion and electrophoresis of recombinant plasmid were consistent with prospective.In BLAST comparison online,plasmid sequencing results showed that the over-expressed plasmid pHS-AVC-LW1020 of the target gene was 100% identical with the designed over-expressed sequence,proving that the lentiviral vector was successfullyconstructed.Lentiviral packaging was performed by using overexpressing lentiviral plasmids,HEK 293 T cell,and lentiviral packaging kits.After lentivirus was transfected in OCI-ly1 cell,the results showed that the cells number carrying green fluorescence in the transfected group was 70% under the fluorescence microscopy.The efficiency of transfection was detected by RT-qPCR,and the results showed that the expression level of lncRNA TP53TG1 in the transfected group was significantly higher than the NC group and the blank group(P<0.05).It provided that the transfection was effective for follow-up experiment.The results of Transwell migration and invasion assay showed that the migrative and invasive rate of the over-expression group was significantly lower than that the NC group and the blank group(P<0.05),indicating that over-expression of lncRNA TP53TG1 could inhibit the migration and invasion of OCI-ly1 cell.The apoptosis and cell cycle of OCI-ly1 cell was detected by flow cytometry,and the results showed that there was no significant difference among the over-expression group,the NC group and the blank group(P>0.05).However,obvious cell necrosis was presented in the over-expression group.The proportion of G2 phase cells in the overexpression group was significantly higher than the NC group and the blank group(P<0.05),providing that the expression of lncRNA TP53TG1 could induce G2 phase arrest in OCI-ly1 cell.4.The down-regulation of lncRNA TP53TG1 decreased migration and invasion of OCI-ly3 cell,increased apoptosis,and induce G2 phase arrest in the cell cycle.The expression of lncRNA TP53TG1 was interfered by transient transfection of RiboTM h-lncRNA TP53TG1 Smart Silencer in OCI-ly3 cell.The total RNA was extracted after transfection 48 h,the transfection efficiency was detected by RT-qPCR.The results showed that the expression level of lncRNA TP53TG1 in the interference group was significantly lower than the NC group and blank group(P<0.05),indicating that transfection was effective for follow-up experiment.The results of Transwell migration and invasion assay showed that the migrative and invasive rate of the interference group was significantly lower than that the NC group and the blank group(P<0.05),indicating that interference of lncRNA TP53TG1 could inhibit the migration and invasion of OCI-ly3 cell.The apoptosis and cell cycle of OCI-ly3 cell was detected by flow cytometry,and the results showed that that the apoptosis rate of the interference group was significantly higher than the NC group and the blank group(P<0.05).The proportion of G2 phase cells in the interference group was significantly higher than the NC group and the blank group(P<0.05),providing that the interference of lncRNA TP53TG1 could induce apoptosis and G2 phase arrest in OCI-ly3 cell.5.The expression of PLK1 decreased after up-regulation of lncRNA TP53TG1 in OCI-ly1 cell.The expression of PLK1 mRNA was detected by RT-qPCR.The results showed that the relative expression of PLK1 mRNA in the overexpresion group was significantly lower than the NC group and the blank group(P<0.05).The expression of PLK1 protein was detected by Western blot.The results showed that the expression of PLK1 protein in the overexpression group was significantly lower than the NC group and the blank group(P<0.05),indicating that lncRNA TP53TG1 could regulate PLK1.6.The expression of PLK1 increased after down-regulation of lncRNA TP53TG1 in OCI-ly3 cell.The expression of PLK1 mRNA was detected by RT-qPCR.The results showed that the relative expression of PLK1 mRNA in the interference group was significantly higher than the NC group and the blank group(P<0.05).The expression of PLK1 protein was detected by Western blot.The results showed that the expression of PLK1 protein in the interference group was significantly higher than the NC group and the blank group(P<0.05),indicating that lncRNA TP53TG1 could regulate PLK1.[Conclusion] It could induce G2 phase arrest and decrease cell migration and invasion ability as up-regulating lncRNA TP53TG1 in OCI-ly1 and down-regulating in OCI-ly3,which mechanism may play a role inregulating PLK1.