LncRNA ANRIL promotes cell proliferation, migration and invasion during acute myeloid leukemia pathogenesis via negatively regulating miR-34a.

OBJECTIVE: LncRNA ANRIL (antisense non-coding RNA in the INK4 locus) was highly expressed in acute myeloid leukemia (AML) patients to promote AML pathogenesis. In this study, we aimed to investigate the roles and molecular events of ANRIL associated with AML progression. METHODS: Expression patterns of ANRIL and miR-34a in the bone marrow (BM) samples and cell lines were determined using qRT-PCR. Cell proliferation, apoptosis, migration and invasion of cells with ANRIL knockdown or miR-34a overexpression were assessed by CCK-8, EdU staining, flow cytometry and Transwell assays, respectively. The dual-luciferase reporter assay was employed to validate the relationship between miR-34a and Histone deacetylase 1 (HDAC1). The binding of E2 F1 (E2 F transcription factor 1) with gene promoter was analyzed by ChIP. Furthermore, the tumorigenicity of AML was determined by xenograft transplantation in nude mice. RESULTS: ANRIL was up-regulated both in the BM samples from AML patients and cell lines (HL-60 and THP-1), of which expression was negatively correlated with miR-34a expression. ANRIL knockdown inhibited cell proliferation, migration and invasion but promoted apoptosis of AML cells, while overexpression of miR-34a exerted opposite effects. miR-34a was verified as a downstream gene targeted by ANRIL. Moreover, HDAC1 was a direct target of miR-34a, and HDAC1 overexpression impaired the recruitment of E2 F1 to ASPP2 (apoptosis stimulating proteins of p53) gene promoter. ANRIL knockdown significantly inhibited the tumorigenesis of AML. CONCLUSION: ANRIL promotes AML development through HDAC1-mediated epigenetic suppression of ASPP2 via negatively regulating miR-34a, which might serve as a therapeutic target for AML treatment.

Associations of IL-10 gene polymorphisms with acute myeloid leukemia in Hunan, China.

We investigated the possible association of interleukin-10 (IL-10) single nucleotide polymorphisms (SNPs) and susceptibility to acute myeloid leukemia (AML) in 115 patients and 137 healthy controls. Genetic analysis of IL-10 SNPs at -819 and -592 was carried out with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The IL-10 mRNA expression of AML patients and controls with different genotype was detected by real-time quantitative polymerase chain reaction (RT-PCR). Genetic analysis of IL-10 revealed that the -819AA genotype frequencies and the -819A allele frequencies in the AML group were higher than in the controls (59.1% vs 40.9%; 75.6% vs 63.9%, respectively); there were remarkable differences in -819T/C and -592A/C gene distribution (P<0.05) and the TA haploid frequencies were higher in the AML group (75.6% vs 63.9%, P<0.05). IL-10 mRNA expression in incipient AML patients was obvious higher than the non- tumor group and the remission group (7.78?10-3 vs 2.43?10-3, 3.64?10-3, P<0.05).The study suggested that the haploid TA and genotype TA/TA may be associated with AML in Han people in Hunan province.The IL-10 SNPs at -819 and -592 sites were associated with AML and may affect IL-10 mRNA expression in AML patients.

[Apoptosis-inducing Effect of 8-Bromo-7-Methoxychrysin on K562 cells].

This study was purposed to investigate the apoptosis-inducing effect of 8-bromo-7-methoxychrysin (BrMChR) on leukemia K562 cells as well as the variation of caspase-3 activity and phosphorylated Akt (p-Akt) expression of K562 cells during the process of apoptosis. MTT assay was used to determine the inhibitory effect of BrMChR on proliferation of K562 cells. Cell apoptosis was assayed by AO/EB staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining. The expression level of p-Akt was measured by Western blot. The results showed that BrMChR had the inhibitory effect on proliferation of K562 cells and could induce apoptosis of these cells in dose-dependent manner, and these effects were significantly stronger than ChR. After treatment of K562 cells with 3 µmol/L ChR for 12 hours, the apoptosis rate was only 3.68%, but the apoptosis rate of K562 cells treated with 3 µmol/L BrMChR was 21.8%. In the same time, the caspase-3 activity significantly increased (p < 0.05), but the expression of p-Akt was down-regulated (p < 0.01). It is concluded that BrMChR can induce apoptosis of K562 cells and with effect stronger than chR. P-Akt may participate in the apoptosis process of K562 cells induced by BrMChR.

[Expression and SNP analysis of BRD7 gene in acute leukemia cells].

OBJECTIVE: To evaluate the expression level of BRD7 gene in bone marrow mononuclear cells (BMNCs) in patients with acute leukemia (AL) and to analyze BRD7 single nucleotide polymorphism(SNP). METHODS: RT-PCR was used to detect BRD7 expression in patients with AL and normal bone marrow subjects. Single-strand conformation polymorphism and DNA sequence analysis were also used to identify BRD7 mutation or SNP to investigate the relation between BRD7 and AL. RESULTS: BRD7 mRNA in BMNCs from 52 patients with AL and 30 control subjects was expressed. The mRNA relative expression of BRD7 in patients with AL was higher than that of the control group (P=0.001). Three SNPs (C657A,C495T and A737G) in BRD7 gene coding region (447 approximately 844 bp) were found, and A737G was coupled with C495T . The allele frequencies of SNP C657A were not significantly different between AL and the control group. The genotype and the allele frequencies of the 2 coupled SNPs were significantly different (P<0.01). But there was no significant discrepancy among the mRNA expression levels of AA, AG, and GG genotypes in the leukemia group (P>0.05). CONCLUSION: Expression of BRD7 gene is up-regulated in AL cells. The 2 coupled SNPs (C495T and A737G ) in BRD7 gene coding region (447 approximately 844 bp) are correlated with AL, indicating that SNPs may be one of the genetic susceptibility factors of AL.

[Effect of diallyl disulfide on expression and secretion of VEGF in HL-60 leukemic cells].

The study was aimed to investigate the expression of VEGF mRNA and VEGF protein in HL-60 cells treated with diallyl disulfide (DADS), and to explore the antileukemic mechanism of DADS in respect of VEGF production. Semi-quantitative RT-PCR and ELISA were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by DADS respectively. The results showed that the expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells. The expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells could be down regulated by treatment with 0.625, 1.25, and 2.5 microg/mL DADS for 48 and 72 hours and the effects had a dose dependent relationship (r > 0.9, P < 0.01). The differences between DADS treated HL-60 cell groups and the control group were statistically significant (P < 0.01), there were also statistically significant differences among three DADS-treated HL-60 cell groups (P < 0.05). It is concluded that DADS effectively inhibits the proliferation of human leukemia cell line HL-60 cells; DADS exerts its antileukemic effects by reduction of the expression of VEGF mRNA and VEGF protein secretion.