The Mechanism Of LncRNA TCONS₀0015691/NFATC2/CCR9 Promoting The Infiltration Of T-ALL

Background and objectivesLeukemia is a group of hematopoietic malignancies caused by the accumulation and infiltration of abnormal hematopoietic stem cells or early progenitor cells.In children and adults with malignancies,under 35 years of age,leukemia has the highest mortality rate thereby threating the human health.T-lineage acute lymphocytic leukemia(T-ALL)is characterized by a large number of immature lymphoblastic cells invading the bone marrow accounting for 10-15%and 25%of acute lymphoblastic leukemia in children and adults,respectively.Invasion and infiltration of T-ALL commonly associated with treatment complications therefore,underscoring the molecular mechanism exploration of T-ALL invasion and infiltration to find new drug candidate targets.In our previous studies,we have found that chemokine receptor 9(CCR9)is highly expressed in T-ALL leukemia cells,which is closely related to the invasion and infiltration of T-ALL,but the regulation mechanism of CCR9 expression remains unclear.LncRNA mainly regulates the gene expression on transcriptional and post-transcriptional levels.In this study,we analyzed GEO datasets to screen lnc RNAs positively correlated with CCR9 expression.Next,the regulation CCR9 expression and its effect on T-ALL invasion and infiltration was investigated.Activated T cell nuclear factor(NFAT)and other transcription factors coordinately regulate the expression of genes that in turn participate in the development of normal T cells and the formation of a variety of tumors.Research found that NFATC2 in CD4~+T cells regulate the expression of CCR9.This study aims to investigate the molecular mechanism of lnc RNA TCONS＿00015691/NFATC2/CCR9 axis in promoting T-ALL invasion and infiltration,and to provide a new idea for the diagnosis and treatment of T-ALL.Research contents and methods1.To explore the role of LncRNA TCONS＿00015691 in regulating CCR9expression and participating in T-ALLmigration and invasion.LncRNA is positively correlated with CCR9 expression in T-ALL revealed by bioinformatics.The expression levels of lnc RNA and CCR9 were detected by Real-time PCR and Western Blotting,and LncRNA TCONS＿00015691was found to be positively correlated with CCR9.Lentiviral technology was used to construct lnc RNA stable knockdown transgenic cell lines.Transwell and Matrigel-Transwell assays were used to detect the migration and invasion ability of MOLT-4 T-ALL cell line with CCR9high expression.Animal experiments confirmed lnc RNA TCONS＿00015691 in T-ALL infiltration.2.To explore the mechanism of NFATC2 regulating CCR9 expression and participating in T-ALL invasion and infiltrationAfter knocking down the expression of NFATC1/C2/C3 in MOLT-4 cells,Real-time PCR and Western Blotting were performed to detect the expression levels of NFATC1/C2/C3 and CCR9.We found that NFATC2 can promote the expression of CCR9.NFATC2 stable knockdown MOLT-4 cell line was constructed and the migration and invasion abilities were detected by Transwell and Matrigel-Transwell experiments.The role of NFATC2 in T-ALL infiltration was verified by animal experiments.3.To explore the mechanism of LncRNA TCONS＿00015691/NFATC2/CCR9 axis promoting T-ALL invasion and infiltrationThe expression of CCR9 was upregulated by the knockdown of lnc RNA TCONS＿00015691 in MOLT-4 cells.The expression of CCR9 was detected by Real-time PCR and Western Blotting.The migration and invasion ability of MOLT-4 cells were detected by Transwell and Matrigel-Transwell assay.The animal experiment verified lnc RNA TCONS＿00015691/NFATC2/CCR9 axis in T-ALL infiltration.Results1.Bioinformatics analysis revealed three lnc RNAs including lnc RNA TCONS＿00024295,lnc RNA TCONS＿00020560 and lnc RNA TCONS＿00015691positively correlated with CCR9.Real-time PCR and western blotting showed that expression level of CCR9 was down-regulated after the silencing of lnc RNA TCONS＿00015691.Moreover,the knockdown of LncRNA TCONS＿00015691decreased the migration and invasion ability of MOLT-4 cells revealed by Matrigel-Transwell assay.After the rescue of lnc RNA TCONS＿00015691,the migration and invasion ability of MOLT-4 cells increased significantly.In vivo experiments in nude mice showed that the level of MOLT-4 cell infiltration in the lnc RNA TCONS＿00015691 knockdown MOLT-4 cells was significantly lower than that in the control group.The MOLT-4 cell organ infiltration lnc RNA TCONS＿00015691 rescue group showed a significant increase.2.CCR9 expression was down-regulated after knockdown of NFATC2 in MOLT-4 cells.Transwell and Matrigel-Transwell experiments showed that MOLT-4 cells migrated and invaded decreased after knockdown of NFATC2.In vivo experiments in nude mice showed that the NFATC2 knockdown MOLT-4 cells displayed a significantly low infiltration ability compared to the control group.The level of MOLT-4 cell infiltration in the organs of the NFATC2 rescue group was significantly increased.The expression level of NFATC2 and CCR9 decreased with the silencing of lnc RNA TCONS＿00015691 in MOLT-4 cells.The expression of CCR9 was restored after the overexpression of NFATC2.After the knockdown of LncRNA TCONS＿00015691 in MOLT-4 cells,the expression levels of NFATC2 and CCR9 were down-regulated,and the expression level of CCR9 was restored after overexpression of NFATC2 on LncRNA TCONS＿00015691 knockdown strain.Animal experiments showed that lnc RNA TCONS＿00015691 knockdown strain with overexpressed NFATC2,compared with LncRNA TCONS＿00015691 knockdown group,MOLT-4 cell infiltration level was significantly up-regulated in this group of mice,suggesting that LncRNA TCONS＿00015691 promotes CCR9 by up-regulating NFATC2 expression,which in turn mediates the organ infiltration of MOLT-4 cells in mice.Conclusion:1.LncRNA TCONS＿00015691 up-regulates the expression of CCR9 and promotes the invasion and migration of MOLT-4 cells;2.NFATC2 up-regulates the expression of CCR9,which in turn promotes the migration and invasion of MOLT-4 cells;3.LncRNA TCONS＿00015691/NFATC2/CCR9 axis plays an important role in T-ALL invasion and infiltration.