Laptm5 3'UTR Inhibits Mouse B Cell Lymphoma By Function As CeRNA

ObjectiveLysosomal-associated protein transmembrane5 is mainly expressed in lymphatic and myeloid cells.Previously transcriptome chip detection found that the expression of Laptm5 in B-cell lymphoma was significantly decreased when the oncogene c-myc was activated.The effects of Laptm5 on mouse B lymphoma is not clear.In this study,we want to explore the mechanism of Laptm5 3 'untranslated region function as ceRNAs(Competitive endogenous RNA,ceRNA)to affect the growth of mouse B-cell lymphoma by influencing the effect of miR-17-3p on the target protein.Method1.The expression levels of Laptm5 and miR-17-3p in mouse B-cell lymphoma cells and Clinical lymphoma tissue were detected by qRT-PCR and Western Blot.2.Laptm5 3'UTR and mutant Laptm5 3'UTR(Laptm5 3'UTRmut)overexpression plasmids were constructed and packaged to obtain virus,respectively,to infect mouse B cell lymphoma cell line 38B9.Cell count was used to detect the proliferation of cells in each group.Cell apoptosis and cell cycle changes were detected by flow cytometry.3.38B9 cells overexpressing Laptm5 3'UTR and Laptm5 3'UTRmut and empty vector(pMSCV-PIG)were injected subcutaneously into the mouse to produce tumor-bearing mouse model.The tumor growth was observed and the proliferation of tumor cells were detected by immunohistochemistry.4.38B9 cells overexpressing Laptm5 3'UTR and empty vector(pMSCV-PIG)were detected by transcriptome microarray.We correlated the mRNAs that were up-regulated by Laptm5 3'UTR and were potential target genes of miR-17-3p predicted by bioinformatics.Then the Mlkl(Ablation of mixed lineage kinase 1,Mlk1)was selected for further study.5.The miR-17-3p overexpression plasmid and luciferase-Laptm5 3'UTR fusion gene plasmid were constructed into cells.The targeting effect of miR-17-3p on Laptm5 was observed by dual luciferase reporter assay system.Western blot was used to observe the effect of miR-17-3p on Laptm5 and Mlkl protein and the effect of Laptm5 3'UTR on Laptm5 protein and Mlkl protein.6.The proliferation and cell cycle of 38B9 cells overexpressing miR-17-3p were detected by cell counting and flow cytometry.The effect of miR-17-3p on tumor growth was observed by tumor-bearing mice.Result1.The expression of Laptm5 mRNA and protein in mouse B cell lymphoma cell was significantly lower than that in normal B cells(P<0.01).The level of Latptm5 mRNA in clinical lymphoma tissue was significantly lower than that in inflammatory lymph nodes(P<0.01)The miR-17-3p was highly expressed in mouse B cell lymphoma cells(P<0.01).2.The proliferation capacity of cells overexpressing Laptm5 3'UTR was significantly lower than that in cells overexpressing Laptm5 3'UTRmut and empty vector(P<0.01).Cell cycle experiments showed that the proportion of mouse B-cell lymphoma cells expressing Laptm53'UTR in G1 phase was significantly higher than that in cells that overexpressing Laptm53'UTRmut and empty vector(P<0.01)The tumor growth of the overexpressing Laptm53'UTR was significantly slower than that of the Laptm5 3'UTRmut and empty vector groups(P<0.01).3.Dual-luciferase reporter assay confirmed that miR-17-3p could negatively regulate Laptm53'UTR and inhibit Laptm5 protein expression,but did not affect its mRNA level(P>0.05).4.By Gene chip detection of cells overexpressing Laptm53'UTR and bioinformatics retrieval,we choosed Mlkl,which was up-regulated by Laptm53'UTR and miR-17-3p predicted target gene,was selected and verified.The results showed that Mlkl protein level was also regulated by miR-17-3p.In mouse B-cell lymphoma,the expression level of Mlkl was positively correlated with the expression level of Laptm5 3'UTR.When Laptm5 3'UTR overexpressed,the expression levels of Mlkl and Laptm5 proteins were increased,while Laptm5 3'UTRmut had no significant effect on the expression of both.5.Cell counting showed that miR-17-3p significantly promoted the growth of mouse B-cell lymphoma cells.The tumor-bearing test also showed that miR-17-3p promoted the growth of B lymphoma.Conclusion1.The expression of Laptm5 in B cell lymphoma was significantly lower,and Laptm53'UTR inhibited the growth of mouse B-cell lymphoma;2.miR-17-3p can target the expression of Laptm5 negatively,Laptm5 3'UTR can inhibit the formation of mouse B-cell lymphoma by acting as the ceRNA of Mlkl and Laptm5 by competitive binding miR-17-3p and decrease the downregulation of miR-17-3p on Mlkl and Laptm5.