LncRNA15691 Promotes T-ALL Infiltration By Upregulating CCR9

BackgroundT-cell acute lymphoblastic leukemia（T-ALL）is a malignant and invasive hematologic tumor caused by the abnormal clonal proliferation of T-lineage progenitor cells.It accounts for approximately 20%～25%of adult acute lymphoblastic leukemia and10～15%of childhood acute lymphoblastic leukemia,respectively.Although treatment optimizations have improved remission rates and disease-free survival for T-ALL patients,primary drug resistance and long-term recurrence remain the unresolved issues.Our previous studies have identified that CCR9 selectively increased expressed on CD4⁺T cells from T-ALL patients,and that IL-2 and IL-4,together,internalized CCR9 on T-ALL CD4⁺T cells and subsequently inhibited chemotaxis and adhesion of these cells.Our later studies showed that CCR9 and its sole ligand C-C motif chemokine 25（CCL25）are involved in the infiltration and multidrug resistance of T-ALL through various signaling pathways.However,the mechanisms modulating CCR9 expression have not been well characterized.This study aims to explore the underlying mechanisms involved in CCR9 expression regulation from the perspective that long non-coding RNA（Lnc RNA）regulates gene expression,and the potential application of novel Lnc RNA in T-ALL.The present research will expand our understanding of CCR9 expression regulation that might provide new clues for the treatment of CCR9^high T-ALL.Main ContentPart I:Lnc RNA15691 promotes leukemia cell migration and invasion via upregulation of CCR9 expressionMethods:In this study,we first analyzed the expression of Lnc RNAs that had been positively correlated with CCR9 expression in dataset from the GEO database.To confirm the expression of the three most potent Lnc RNAs in T-ALL,13 patients with T-ALL,20 healthy individuals and T-ALL cell lines（MOLT-4 and JURKAT）were enrolled and included in the analysis.Real-time quantitative PCR（RT-q PCR）was employed to detect the expression of the three Lnc RNAs in T-ALL peripheral blood mononuclear cells（PBMC）and cell lines（JURKAT and MOLT-4）.To explore the information of the previously uncharacterized Lnc RNA,we first investigated the Lnc RNA chromosome localization,protein-encoding ability,secondary structure and subcellular localization through a series of biological function prediction analysis and fluorescence in situ hybridization（FISH）.To analyze the biological functions of Lnc RNA15691 in T-ALL,we designed sh RNAs specifically targeting the Lnc RNA15691 transcript for silencing,which were confirmed to efficiently knock down Lnc RNA in MOLT-4 cells via RT-q PCR.Next,the proliferation capacity of T-ALL cells with Lnc RNA15691 knocked down or overexpression CCR9 was assessed through the CCK-8,and the invasive and metastatic ability was assessed by transwell migration/invasion assay,while RT-q PCR and western blot（WB）were utilized to vertify the CCR9 expression.Finally,single-gene rescue experiments comfirmed that CCR9 as a key gene mediating the migration and invasion of T-ALL cells induced by Lnc RNA.Results:In this study,bioinformatics analysis and validation in clinical samples revealed the Lnc RNA15691 to be positively correlated with CCR9 m RNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9^high T-cell acute lymphoblastic leukemia cell lines（p<0.001）.Lnc RNA15691,a previously uncharacterized Lnc RNA,was found to be located on the long arm of human chromosome 9 and distributed in both cytoplasm and nucleus,without the ability to encode proteins and possesses a secondary structure that interacts with macromolecules via biological function prediction analysis and FISH experiment.The proliferation capacity of MOLT-4 cells with Lnc RNA15691 knocked down was not significantly different from that of cells expressing the empty vector control,as assessed through the CCK-8 assay.Interestingly,knocking down the expression of Lnc RNA15691 dramatically inhibited the migration and invasion of MOLT-4 cells.Moreover,we used CCR9^low JURKAT cells to establish stable transfectants with Lnc RNA15691 or empty control vector,and the overexpression of Lnc RNA15691significantly triggered an increase in the migration and invasion of JURKAT cells but did not affect cell proliferation.We observed a significant decrease in the expression of CCR9 m RNA after knocking down Lnc RNA15691 expression in MOLT-4 cells.In addition,this finding was corroborated by a significant decrease in the protein level of CCR9 after Lnc RNA15691 knockdown.Moreover,overexpression of Lnc RNA15691in the CCR9^low T-ALL cell line（JURKAT cells）increased the expression of CCR9 at both the m RNA and protein levels.To assess the significance of CCR9 in Lnc RNA15691-regulated leukemia cell invasion,exogenous CCR9 was expressed in MOLT-4 cells（vector and lnc RNAsh）.The results illustrated that the decreased invasiveness caused by Lnc RNA15691 depletion was rescued by exogenous expression of CCR9.Conclusion:Lnc RNA15691,a 361-nt-long RNA located on chromosome 9,is a cytoplasmic and nuclear localized Lnc RNA,which is highly expressed in T-ALL and promotes the migration and invasion of T-ALL cells by up-regulating the expression of CCR9.Part II:Lnc RNA15691 promotes T-ALL infiltration,and CCR9 was the dominant mediator of Lnc RNA15691-dependent migrating leukemia cell extramedullary infiltrationMethods:We established the cell-derived xenograft（CDX）using the stable cell lines,including Lnc RNA15691 or empty control vector,to constitutively express the luciferase gene under a ubiquitous promoter and injected into the NOD-Prkdcscid IL2rgdnull（NTG）immunocompromised mice.At the same time,the PBS was injected as the control group.Mice were treated with PBS or CCL25（30 ng per mouse）after 3 d.Engraftment and disease progression were analyzed by body weight,peripheral blood smear,bioluminescence imaging,and flow cytometry.Results:JURKAT cells expressing vector-luc or Lnc RNA15691-luc were delivered intravenously into immunodeficient mice.The successful construction of a leukemia xenograft mouse model was confirmed by the morphology of the bone marrow and peripheral blood cells of the mice.In this analysis,we observed significant body weight loss of the mice in the OE-Lnc RNA/PBS group compared with those in the vector/PBS group.In the bioluminescence imaging analysis,whole-animal imaging data showed that the bioluminescent signal intensity increased in the OE-Lnc RNA/PBS group compared with the vector/PBS group.Dissemination of JURKAT cells in several murine organs was detected with flow cytometry by gating human CD45,a human T-cell marker.Compared with those expressing the empty vector control,more JURKAT cells overexpressing Lnc RNA15691 were detected in bone marrow,peripheral blood,and spleen,indicating that leukemia cells overexpressing Lnc RNA15691 exhibited relatively higher infiltration rates.The mice were treated with the CCR9 ligand,CCL25.Notably,CCL25 treatment augmented the weight loss of mice triggered by overexpression of Lnc RNA15691 and the bioluminescent signals in the OE-Lnc RNA/CCL25 group.Moreover,after CCL25treatment,the CCR9^low leukemia cells with overexpressing Lnc RNA15691 exhibited higher infiltration rates in blood,spleen,and liver but not in bone marrow.Conclusion:Taken together,these in vivo findings showed that Lnc RNA15691promoted T-ALL cell infiltration and implied that CCR9 was the dominant mediator of Lnc RNA15691-dependent migrating leukemia cell extramedullary infiltration.Part III:Lnc RNA15691 upregulates CCR9 via increased MATR3 stabilityMethod:To specifically identify Lnc RNA15691-interacting proteins in T-ALL cells,RNA protein pull-down assay was subjected to qualitative mass spectrometry for protein identification.RNA immunoprecipitation（RIP）was employed to determine the protein that directly binds to Lnc RNA15691 in T-ALL.Cell lines with stable knockdown or overexpression of the counterpart protein was generated using lentiviral-based transduction strategy,and RT-q PCR and WB was used to assess the m RNA and protein expression of CCR9,respectively.The role of key genes was determined by single gene rescue experiments.Protein stability after Lnc RNA and protein binding was also investigated through protein degradation experiments.Results:RNA pull-down assays combined with the specific antibody showed direct binding of MATR3 to Lnc RNA15691.To test this outcome,we performed a RIP assay using specific antibodies against MATR3 in MOLT-4 cells,followed by RT-q PCR analysis.We first verified that MATR3 was efficiently pulled down by this antibody.Importantly,compared with a negative control transcript（GAPDH）,Lnc RNA15691transcripts were enriched in RIP eluates of MATR3.These data suggested that Lnc RNA15691 directly interacted with MATR3.We analyzed a microarray data set containing 174 human T-ALL bone marrow samples and 73 normal bone marrows,and found that the expression of MATR3 was significantly higher in the T-ALL samples than in normal control samples.In addition,in PBMC from T-ALL patients,the expression of MATR3 was higher than that in normal controls（p<0.001）.To test whether MATR3 played a consistent role in the upregulation of CCR9 expression,sh RNAs specifically targeting the MATR3 transcript and empty vector control were stably transfected into MOLT-4 cells.Notably,the m RNA and protein levels of CCR9were significantly decreased after MATR3 knockdown.Moreover,overexpression of MATR3 in CCR9^low JURKAT cells led to upregulated CCR9 expression at the m RNA and protein levels.Next,we sought to investigate whether the Lnc RNA15691-MATR3 complex played a role in the regulation of CCR9.The effects of Lnc RNA15691 knockdown,including the depletion of CCR9 and decreased cell migration and invasion,were abolished by MATR3 overexpression.We observed that knocking down Lnc RNA15691 in MOLT-4 cells resulted in decreased MATR3 expression at the protein level without any effect on the m RNA level.Similar results were observed in JURKAT cells,in which exogenous expression of Lnc RNA15691 upregulated the expression of MATR3 at the protein level but not at the m RNA level.By performing a CHX-based stability assay,we investigated whether the upregulation of MATR3 expression by Lnc RNA15691 may be due to increased MATR3 protein stability.In empty vector control-expressing cells,the half-life of MATR3 proteins was decreased compared to that in cells transduced with Lnc RNA15691.In addition,knocking down Lnc RNA15691 by sh RNA promoted the degradation of MATR3,suggesting that Lnc RNA15691 played a role in MATR3protein stability.PKA-mediated degradation of MATR3 and ubiquitination-based MATR3 mislocalization and accumulation in amyotrophic lateral sclerosis have been reported previously.To assess whether Lnc RNA15691 inhibited the degradation of MATR3 mediated by PKA or ubiquitination,we applied H-89,an inhibitor of PKA,and MG-132,an inhibitor of the proteasome,to a degradation assay.After knocking down Lnc RNA15691,the MATR3 content was reduced to 38.81%±1%of the basal level at 60 min.In the presence of H-89,the MATR3 content remained at 76.11%±1.67%of the basal level,which was not significantly different from that in the empty vector control.After MG-132 treatment,the MATR3 content was reduced to 46.11%±1.67%of the basal level,which was significantly decreased compared with that in the empty vector control at 60 min.To further examine whether Lnc RNA15691affects the nuclear accumulation of MATR3,we detected the presence of nuclear MATR3 in MOLT-4（vector and sh Lnc RNA）and JURKAT（vector and OE-Lnc RNA）cells.The nuclear enrichment of MATR3 was significantly increased in Lnc RNA15691-overexpressing JURKAT cells and decreased in MOLT-4 cells with the knockdown of Lnc RNA15691.Conclusion:In summary,these mechanistic studies revealed that Lnc RNA15691upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA.