DNA methylation-mediated silencing of microRNA-204 enhances T cell acute lymphoblastic leukemia by up-regulating MMP-2 and MMP-9 via NF-κB.

T cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive haematological cancer of the bone marrow. The abnormal expression of microRNAs (miRNAs) is reportedly involved in T-ALL development and progression. Thus, we aimed to decipher the involvement of miR-204 silencing mediated by DNA methylation in the occurrence of T cell acute lymphoblastic leukaemia (T-ALL). miR-204 expression was determined in bone marrow and peripheral blood samples from T-ALL patients by real-time quantitative PCR (RT-qPCR) with its effect on cell proliferation evaluated by functional assays. In addition, bisulphite sequencing PCR was employed to detect the DNA methylation level of the miR-204 promoter region, and the binding site between miR-204 and IRAK1 was detected by luciferase assay. We found that miR-204 was down-regulated in T cells of T-ALL patients, which was caused by the increased DNA methylation in the promoter region of miR-204. Moreover, overexpression of miR-204 inhibited T-ALL cell proliferation while enhancing their apoptosis through interleukin receptor-associated kinase 1 (IRAK1), which enhanced the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 through activation of p-p65. Thus, miR-204 modulated MMP-2 and MMP-9 through IRAK1/NF-κB signalling pathway, which was confirmed by in vivo assay. Taken together, DNA methylation-mediated miR-204 silencing increased the transcription of IRAK1, thus activating the NF-κB signalling pathway and up-regulating the downstream targets MMP-2/MMP-9.

5-Aza-dC promotes T-cell acute lymphoblastic leukemia cell invasion via downregulation of DNMT1 and upregulation of MMP-2 and MMP-9.

5-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells, and can restore the expression of their target genes in Jurkat cells. The goal of this study was to discern the potential effect of 5-Aza-dC on the invasion of T-ALL cells in acute lymphoblastic leukemia (ALL). The role of matrix metallopeptidase (MMP)-2, MMP-9, and DNMT1 in cell invasion was determined using loss- and gain-of-function investigations in Jurkat- and Sup-T1-R cells. A nude mouse model of ALL was established for further exploration of their roles in vivo. MMP-2 and MMP-9 exhibited high expression and low DNA methylation levels in 5-Aza-dC-resistant T-ALL cells. DNMT1 was poorly expressed in 5-Aza-dC-resistant T-ALL cells and exhibited decreased enrichment in the promoter region of MMP-2 and MMP-9. Silencing of MMP-2 and MMP-9 or DNMT1 overexpression reduced T-ALL cell invasion. After treatment of Sup-T1 cells with 5-Aza-dC, MMP-2 and MMP-9 presented with reduced DNA methylation levels but increased expression, and DNMT1 expression was identified to be suppressed. Further, in vivo assays revealed that DNMT1 alleviated T-ALL by reducing the expression of MMP-2 and MMP-9 in vivo. All in all, 5-Aza-dC activates MMP-2 and MMP-9 expression by reducing DNMT1-dependent DNA methylation levels and, hence, promotes the invasion of T-ALL cells.

miR-100 inhibits cell proliferation in mantle cell lymphoma by targeting mTOR.

BACKGROUND: miR-100 is reported to be associated with cell proliferation and apoptosis. However, the function of miR-100 in mantle cell lymphoma (MCL) is unknown. The purpose of this study is to analyze the abnormal expression of miR-100 and mTOR in MCL together with their potential biological function and pathogenesis. METHOD: Eighteen MCL tissue samples and 3 cell lines (Jeko-1, Mino, Granta-519) were investigated in this research study, while eighteen samples of proliferative lymphadenitis from patients and peripheral lymphocyte cells from healthy volunteers served as controls. The expression and alteration of miR-100 and mTOR mRNA were detected by RT-PCR. The expression and alteration of mTOR protein were explored by Western blot. LV-miR-100-up and LV-mTOR-RNAi were constructed and transfected by lentivirus transfection. Cell proliferation, cell apoptosis and the cell cycle were detected using CCK-8 and flow cytometry. Bioinformatics prediction software was used to predict the miR-100 target gene of mTOR. A double luciferase experiment was used to verify miR-100 targeting at the mTOR-3'-UTR. The interaction between miR-100 and mTOR was further studied using recovery experiments. GraphPad Prism 7 software (version 7.2) was used for statistical analysis, and a P value < 0.05 was considered statistically significant. RESULTS: We found that the expression of miR-100 mRNA in MCL tissues and cell lines was lower, while that of the mTOR protein was higher. There was a negative correlation between miR-100 and mTOR in both MCL tissues and cell lines. Promoting miR-100 and inhibiting mTOR could inhibit cell proliferation, induce cell apoptosis and block the cell cycle in the G1 phase. A double luciferase reporter assay showed that mTOR was one of the target genes of miR-100. The recovery experiment demonstrated that PV-mTOR-up partially set off the effect of LV-miR-100-up on decreasing mTOR expression, inhibiting proliferation, inducing apoptosis and blocking the cell cycle in G1 phase in both Jeko-1 and Mino cells. CONCLUSIONS: Abnormal expression of miR-100 and mTOR was found in MCL, which included downregulation of miR-100 and upregulation of mTOR. The expression of mTOR is negatively correlated with miR-100. It may play an important role in MCL pathogenesis. miR-100 up-regulation can inhibit cell proliferation, promote cell apoptosis, and inhibit cell cycle in G1 phase by targeting the mTOR gene. miR-100 may potentially be an anti-mantle cell lymphoma gene.

miR­101 regulates the cell proliferation and apoptosis in diffuse large B­cell lymphoma by targeting MEK1 via regulation of the ERK/MAPK signaling pathway.

MAPK kinase 1 (MEK1) is an upstream protein kinase of extracellular signal regulated kinase (ERK), which activates the ERK/MAPK (mitogen activated protein kinase) pathway. Importantly, bioinformatic analysis has shown that there is a target complementary binding site between miR­101 and MEK1. The present study aimed to ascertain whether or not miR­101 plays a role in regulating MEK1 expression, ERK/MAPK pathway activity, and the proliferation and apoptosis in a diffuse large B cell lymphoma (DLBCL) cell line. DLBCL tumor samples were collected from patients in our hospital, and lymphatic tissues with reactive lymphoid hyperplasia were selected as controls. The patients were divided into high and low expression groups, and then the survival rate of the two groups was compared using Kaplan­Meier method, as well the effect of miR­101 and MEK1 mRNA expression on survival and prognosis was analyzed. The expression of miR­101, MEK1 and p­MEK1 between normal lymphoblastic cell lines (HCC1954 BL and NCI­BL2009) and lymphoma cell lines (SU­DHL­4 and Farage) was compared. Lymphoma SU­DHL­4 and Farage cells were cultured in vitro, and then divided into the following groups: miR­NC group; miR­101 mimic group; siRNA­NC group; and siRNA­MEK1 group. The expression of miR­101, MEK1, p­MEK1, p­ERK1/2 and Bcl­2 was compared. Cell apoptosis was detected by flow cytometry, and cell proliferation was detected by EdU staining. The results showed that targeted regulation existed between miR­101 and MEK1, and the decreased expression of miR­101 was related to the pathogenesis and prognosis of DLBCL. Upregulation of miR­101 inhibited DLBCL cell proliferation and facilitated apoptosis by inhibiting the expression of MEK1.

miR-101 regulates cell proliferation and apoptosis by targeting KDM1A in diffuse large B cell lymphoma.

BACKGROUND: miR-101 is reported to be associated with cell proliferation and apoptosis. However, it is unknown whether miR-101 expression affects cell proliferation and apoptosis in diffuse large B cell lymphoma (DLBCL). The aim of the present study was to investigate the expression of miR-101 and its effect on cell proliferation and apoptosis in DLBCL. METHODS: miR-101 expression was detected in 30 cases of patients with DLBCL and normal lymph node by qRT-PCR. Then, miR-101 expression was up-regulated and down-regulated in Originated Cell Line-Large Lymphoma 8 (OCL-LY8) cell line, respectively. MTT and flow cytometry assay were used to evaluate the effect of miR-101 on cell proliferation and apoptosis, respectively. As KDM1A was confirmed to be as a specific target of miR-101 by TargetScanHuman, the relationship between MiR-101 and KDM1A was further investigated. RESULTS: miR-101 expression in patients with DLBCL was significantly reduced compared those in normal lymph node (P<0.05). miR-101 expression was significantly associated with tumor size, clinical stage and International Prognostic Index (IPI) scores (P<0.05). In OCL-LY8 cell line, miR-101 down-regulation significantly promoted cell proliferation and suppressed cell apoptosis. Meanwhile, miR-101 up-regulation reversed this effect. In addition, miR-101 negatively regulated the expression of KDM1A. KDM1A down-regulation was oberved in normal tissues compared with those in DLBCL tissues, which inhibited cell proliferation and promoted cell apoptosis. CONCLUSION: These data indicate that miR-101 regulates cell proliferation and apoptosis by targeting KDM1A, which provides a potential therapeutic for DLBCL patients.

Identification of serum miR-34a as a potential biomarker in acute myeloid leukemia.

BACKGROUND: MicroRNA-34a (miR-34a), as a tumor-suppressive miRNA, has been found to induce cell apoptosis in acute myeloid leukemia (AML). However, the diagnostic and prognostic significance of miR-34a in AML remains largely unknown. OBJECTIVE: We aimed to explore its associations with clinical characteristics and prognosis of AML patients. METHODS: This study detected serum miR-34a level in 117 diagnosed AML patients and 60 control subjects by using qRT-PCR, and results were compared to clinical features and patient outcome. Since cytogenetically-normal AML (CN-AML) has a good uniformity of cytogenetics and provides a perfect platform for detection of AML biomarkers, we further analyzed miR-34a expression in 56 CN-AML subjects. RESULTS: We found that miR-34a was significantly downregulated in AML and CN-AML patients. MiR-34a underexpression was commonly observed in AML patients with intermediate/poor risk cytogenetic, and M5 subtype. ROC analysis demonstrated that serum miR-34a could well identify AML/CN-AML patients from healthy individuals. More importantly, miR-34a expression was found negatively correlated with aggressive clinical variable, and served as an independent prognostic indicator. In addition, AML/CN-AML patients with low miR-34a expression displayed shorter overall and recurrence free survival. CONCLUSIONS: Altogether, miR-34a might have an application as a diagnostic and prognostic indicator for AML patients.

[Effects of Silencing NSD2 Gene by shRNA on Proliferation, Apoptosis and Akt /mTOR Signal Pathway in OCI-Ly3 Cells].

OBJECTIVE: To investigate the effect of silencing NSD2 gene by RNA interference on the proliferation, apoptosis and the alteration of Akt /mTOR signaling pathway in diffuse large B cell lymphoma OCI-Ly3 cells. METHODS: The shRNA targeting NSD2 gene was transfected into OCI-Ly3 cells by lentivirus infection. The NSD2 mRNA and protein were detected by real time Q-PCR and Western blot, respectively. The cell proliferation was detected by CCK-8 and apoptosis was measured by flow cytometry. The expressions of BCL-2, BAX, caspase-3, Akt, p-Akt, p-mTOR, p-P70S6K, H3K36me2 were detected by Western blot. RESULTS: After transfecting the OCI-Ly3 cells by NSD2-shRNA for 72 h, the expressions of NSD2 mRNA and protein both were down-regulated(P<0.05), the proliferation rate of cells in NSD2 shRNA group was significantly lower than that in control and Neg shRNA groups (P<0.05); the apoptosis rate of cells in NSD2 shRNA group was significantly higher than that in control and neg-shRNA group (30.37±4.22)% vs 1.36±0.52 % and 2.17±1.43)%(P<0.05); the expressions of BAX and caspase-3 were up-regulated, while the expression of BCL-2 was down-regulated; the H3K36me2 level significantly decreased as compared with control group, no obvious decrease of the total protein level of AKT was found, but the expressions of p-Akt, p-mTOR and p-70S6K were down-regulated. CONCLUSION: The silencing NSD2 gene can inhibit the proliferation and induce the apoptosis of OCI-Ly3 cells, their mechanisms may relate with regulating the H3K36me2 level, specifically inhibiting the activivty of AKT/mTOR signal pathway.

Ginsenoside Rg1 Activates Dendritic Cells and Acts as a Vaccine Adjuvant Inducing Protective Cellular Responses Against Lymphomas.

Ginsenoside, a natural triterpenoid saponin, exhibits immunomodulatory and anticancer activities. In the present study, we demonstrated that ginsenoside Rg1 induced secretion of cytokines, including interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and IL-1ß, and chemokines such as IL-8 and IP-10 in a dose-dependent manner by human peripheral blood mononuclear cell (PBMC)-derived dendritic cells. Rg1 stimulated the expression of the surface molecules CD83, CD80, and human leukocyte antigen - antigen D related (HLA-DR) and decreased the expression of CD14. In in vivo experiments, C57BL/6 mice were divided into four groups, immunized with ovalbumin (OVA), OVA plus Rg1, Rg1, and phosphate-buffered saline (PBS), respectively. Splenocytes from C57BL/6 mice immunized with OVA plus Rg1 produced more antigen-specific splenocyte proliferation activity. The level of IFN-γ and IL-4 in the splenocytes was also upregulated when in vitro stimulated with OVA257-264 or OVA. After in vivo injection of tumor-forming E.G7-OVA cells, the survival rate of mice immunized intraperitoneally in OVA plus Rg1 immunized mice was higher than that in OVA immunized mice or PBS immunized mice. Thus, Rg1 induced a potent vaccine adjuvant effect and elicited antitumor immunity that polarized a Th1 type immune response. Rg1 could have potential as a prophylactic vaccine adjuvant to control lymphomas.

Effect of BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells.

OBJECTIVE: To determine effect of G9a inhibitor BIX-01294 on proliferation, apoptosis and histone methylation of acute T lymphoblastic leukemia cells (MOLT-4 and Jurkat) and to explore the underlying mechanism. METHODS: Cell proliferation was detected by MTT assay and apoptosis and cell cycle were measured by flow cytometry. Western blot was performed to determine expression of caspase-3, Bcl-2, Bax, P21, P15 and DNMT1 as well as levels of histone H3 acetylation, histone H3K9 mono- di- and tri-methylation. RESULTS: BIX-01294 inhibits expression of Bcl-2, upregulates expression of Bax and caspase-3 and induces cell apoptosis. BIX-01294 upregulates cell cycle inhibitor P21 expression and induces cell cycle arrest in the phase G0/G1. Furthermore, BIX-01294 suppresses expression of DNA demethylase DNMT1 and promotes expression of tumor suppressor protein P15, thereby inhibiting proliferation of MOLT-4 and Jurkat cells. BIX-01294 downregulates histone H3K9 mono- and di-methylation levels and has no effect on H3K9 trimethylation and histone H3 acetylation. CONCLUSION: Taken together, our results indicate that by regulating H3K9 methylation and cell cycle, BIX-01294 inhibits the proliferation and induces apoptosis of acute T lymphoblastic leukemia cells.