LncRNA-NEAT1 promotes proliferation of T-ALL cells via miR-146b-5p/NOTCH1 signaling pathway.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant tumor of the hematopoietic system, which can develop at any age, with the symptoms of weakness, fatigue, enlarged lymph nodes, or weight loss. Nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in the process of T-ALL, but the regulatory mechanism is still not known clearly. METHODS: The expression levels of NEAT1 and miR-146b-5p in T-ALL cells were performed by qRT-PCR and NOTCH1 protein level- wwWwas determined by western blot assay. Dual-luciferase reporter assay was used to detect the interaction between NEAT1 and miR-146b-5p, as well as miR-146b-5p and NOTCH1. The cell proliferation was measured by using MTT assay and colony formation assay. RESULTS: The expression levels of NEAT1 were markedly increased, but miR-146b-5p levels were reduced in T-ALL cells. Knockdown of NEAT1 or overexpression of miR-146b-5p decreased NOTCH1 expression, inhibited the proliferation of T-ALL cells. MiR-146b-5p bound both NEAT1 and NOTCH1 3'-UTR directly. Finally, inhibition of miR-146b-5p could abrogate the effects of NEAT1 knockdown on the proliferation of T-ALL cells. CONCLUSION: NEAT1 promotes the proliferation of T-ALL cells by sponging miR-146b-5p to upregulate the expression of NOTCH1. The results of this study provide new insight into the action mechanism of NEAT1 modulating T-ALL progression.

A TET2 rs3733609 C/T genotype is associated with predisposition to the myeloproliferative neoplasms harboring JAK2(V617F) and confers a proliferative potential on erythroid lineages.

Common germline single-nucleotide polymorphisms (SNPs) at JAK2 locus have been associated with Myeloproliferative neoplasms (MPN). And, the germline sequence variant rs2736100 C in TERT is related to risk of MPN, suggesting a complex association between SNPs and the pathogenesis of MPN. Our previous study (unpublished data) showed that there was a high frequency distribution in rs3733609 C/T genotype at Ten-Eleven Translocation 2 (TET2) locus in one Chinese familial primary myelofibrosis. In the present study, we evaluate the role and clinical significance of rs3733609 C/T genotype in JAK2V617F-positive sporadic MPN (n = 181). TET2 rs3733609 C/T genotype had a higher incidence (13.81%; 25/181) in JAK2V617F-positive sporadic MPN patients than that in normal controls (n = 236) (6.35%; 15/236), which was predisposing to MPN (odds ratio(OR) = 2.361; P = 0.01). MPN patients with rs3733609 C/T genotype had increased leukocyte and platelets counts, elevated hemoglobin concentration in comparison with T/T genotype. Thrombotic events were more common in MPN patients with rs3733609 C/T than those with T/T genotype (P < 0.01). We confirmed that rs3733609 C/T genotype downregulated TET2 mRNA transcription, and the mechanism may be involved in a disruption of the interaction between CCAAT/enhancer binding protein alpha (C/EBPA) and TET2 rs3733609 C/T locus.TET2 rs3733609 C/T genotype stimulated the erythroid hematopoiesis in MPN patients. Altogether, we found a novel hereditary susceptible factor-TET2 rs3733609 C/T variant for the development of MPN, suggesting the variant may be partially responsible for the pathogenesis and accumulation of MPN.

An intron mutation in the ACVRL1 may be associated with a transcriptional regulation defect in a Chinese family with hereditary hemorrhagic telangiectasia.

PURPOSE: To identify a novel pathogenic gene mutation present in a Chinese family with hereditary hemorrhagic telangiectasia (HHT) and to determine if an intron mutation may influence the transcriptional activity of the ACVRL1 gene. METHODS: HHT family members were ascertained following the presentation of proband and involved subjects. All family members (n = 5) and 113 healthy individuals were genotyped for the variant in intron 6 c.772+27G>C of ACVRL1 gene. The genomic structure of ACVRL1 in affected HHT patients and healthy individuals was determined by long range PCR and sequencing. The expression of ACVRL1 mRNA and protein in patients with HHT was evaluated using real-time polymerase chain reaction and immunoblot analysis. Luciferase activity assay and electrophoretic mobility shift assay (EMSA) were performed to uncover the mechanism of intron-related transcriptional regulation. RESULTS: Only one novel mutation in intron 6 (c.772+27G>C) of ACVRL1 gene, no other mutation, abnormal splice, gross genomic deletion or rearrangement was found in this HHT2 family. Compared with healthy individuals, ACVRL1 mRNA and protein were significantly decreased in affected HHT2 individuals. Luciferase activity assay demonstrated that the transcriptional activity of the mutated ACVRL1 was significantly lower than that of the wild-type of intron 6; EMSA results showed that intron 6 c.772+27G>C mutation was able to inhibit the binding of transcriptional factor Sp1. CONCLUSIONS: A novel intron mutation in ACVRL1 gene is associated with familial HHT2. The mechanisms may be involved in the down-regulation of ACVRL1 gene transcription.

[The frequency of JAK2 V617F mutation, expression level of phosphorylated JAK/STATs proteins and their clinical significance in myeloproliferative disorders patients].

OBJECTIVE: To investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features. METHODS: The JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation. RESULTS: 1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group. CONCLUSIONS: MPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.

[Induction of active antitumor immune response by myeloma idiotype protein-pulsed dendritic cells].

BACKGROUND & OBJECTIVE: Most multiple myeloma (MM) patients could not be cured by high-dose chemotherapy and bone marrow transplantation. This study was designed to investigate in vitro killing effect of tumor-specific cytotoxic T lymphocytes (CTLs) stimulated by idiotype protein (Id)-pulsed dendritic cells (DCs) on autologous MM cells. METHODS: DCs were generated from peripheral blood monocytes of 6 MM patients using interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). After cultured for 5 days, immature DCs were pulsed with Idû tumor necrosis factor-alpha (TNF-alpha) was added at the 7th day. Id-pulsed DCs were cocultured with autologous T cells for 3 days to induce tumor-specific CTLs. MTT assay was used to detect proliferation of autologous T cells, and evaluate killing effect of CTLs on autologous MM cells. RESULTS: Mature DCs were successfully induced. Id-pulsed DCs markedly increased proliferation of autologous T cells in a dose-dependent manner; stimulation index (SI) of Id-pulsed DCs was the highest [(39.1+/-6.0)%] when the radio of DCs to T cells was 10:1, which was significantly higher than those of unpulsed mature DCs [(19.3+/-7.7)%], Id-pulsed immature DCs [(15.9+/-6.1)%], and unpulsed immature DCs [(11.4+/-4.9)%] (P < 0.01). Id-pulsed DCs induced anti-MM activity of CTLs in a dose-dependent manner. Unpulsed mature DCs also induced cytotoxicity of CTLs against autologous MM cellsû however, when DC:T was 30:1, killing rate of MM cells was significantly higher in Id-pulsed mature DCs group than in unpulsed mature DCs group [(70.1+/-7.9)% vs. (40.8+/-7.8)%,P < 0.05]. KRN7000-pulsed mature DCs stimulated proliferation of allogeneic T cells in a dose-dependent manner; when DC:T was 1:10, SI was significantly higher in KRN7000-pulsed mature DCs group than in unpulsed mature DCs group [(38.5+/-5.7)% vs. (20.2+/-5.7)%, P < 0.05]. CONCLUSIONS: Mature DCs could be induced and Id with biological activity could be extracted from peripheral blood of MM patients. Id-pulsed DCs could induce antitumor immune response. KRN7000 could improve the immune function of in vitro cultured DCs.

[In vitro anti-myeloma effects induced by myeloma idiotype-protein pulsed dendritic cell vaccine].

OBJECTIVE: To investigate the specific antitumor immune response induced by idiotype protein (Id)-pulsed dendritic cells (DC) in vitro. METHODS: DC was generated from peripheral blood monocytes of the multiple myeloma (MM) patients using GM-CSF, IL-4, and TNF-alpha. The DCs were pulsed with idiotypic fragment, the F(ab')2 fragment of M protein from MM patient at the immature stage. The morphologic characteristics of the cells were observed with light and electron microscopes. The phenotypic features were analyzed with FACS, MTT assay was employed to evaluate the proliferation of autologous T cells and the inhibition rate of MM cells. RESULTS: DC precursors in peripheral blood could be induced to typical mature DC in medium containing GM-CSF, IL-4 and TNF-alpha. Mature DC with Id could increase the proliferation of the autologous T cells and activate naive T cells to become tumor specialized cytotoxic T lymphocytes (CTL). The CTL at different doses showed significant inhibition on or killing ability to autologous MM cells in vitro. CONCLUSIONS: In a suitable cytokine environment, the DC precursors from peripheral blood of MM patients could be induced to functional DC, and vaccination of Id-pulsed DC could induce active antitumor immune response.