Expression of miR-10b gene and methylation level in schwannomas / 西安交通大学学报(医学版)

Objective To analyze miR-10b expression level and the gene upstream methylation level in schwannomas so as to explore and identify the potential target genes for miR-10b in schwannomas .Methods The miR-10b with its potential target genes including HOXB 3 ,HOXD10 ,PTEN ,PIK3CA ,MAPRE1 and HADC4 were quantitatively analyzed by PCR in 13 cases of schwannomas and 6 cases of human vestibulocochlear nerves . We studied the correlation between the differentially expressed genes and the clinical characteristics of schwannomas . Finally ,the differences in miR-10b gene upstream methylation levels were measured and analyzed by pyrosequencing between schwannomas and normal vestibulocochlear nerves .Results Compared with that of normal nerves ,the expression level of miR-10b was significantly higher (P=0 .0003) while the level of PTEN was lower (P=0 .0047) in schwannomas .Negative correlation existed between the levels of miR-10b and PTEN (P=0 .001 , r= -0 .689) . Moreover ,the methylation level of the miR-10b gene promoter was downregulated in schwannomas ;it had negative correlation with the expression level of miR-10b (P= 0 .011 , r= -0 .571) .There was a significant difference in tumor mass diameter between miR-10b higher expression group and lower group (P=0 .016);however ,there was no difference in age or recurrence rate (P>0 .05) .Conclusion The downregulation of methylation level of the promoter leads to higher expression of miR-10b gene ,and it may targetedly inhibit the expression of PTEN .

Construction of IK6 recombinant lentiviral vector and its expression and biologic feature in THP1 cells / 中国实验血液学杂志

The purpose of this study was to construct a lentiviral vector carrying IK6 gene and to observe the expression of IK6 as well as related biologic feature in THP1 cells, so as to provide an effective method to further investigate the role of this gene in leukemia. The IK6 gene was obtained by using reverse transcription polymerase chain reaction (RT-PCR). Then IK6 was recombined with the pGC-FU vector to construct a recombinant lentiviral vector named pGC-FU-IK6 gene-GFP,which was confirmed by PCR and sequencing. The 293T cells were transfected with pGC-FU- IK6-GFP by using Lipofectamine 2000. After examining the titer of the virus, pGC-FU- IK6-GFP was used to transfect THP1 cells. The transfection efficiency was detected by flow cytometry, and the expression level of mRNA and IK6-GFP fusion protein were confirmed by RT-PCR and Western blot respectively. Then the impact of IK6 on apoptosis and cell cycle was analyzed. The results showed that the IK6 gene was obtained by RT-PCR and connected into the linearized lentiviral vector to successfully constructed target plasmid named pGC-FU-IK6-GFP with Amp resistant. The target plasmid was transfected into 293T cells and the virus titer was 2.0×10(9)TU/ml. Next, THP1 cells were transfected with pGC-FU-IK6-GFP and the efficiency was up to 90%. The detection of the IK6 mRNA and IK6-GFP fusion protein in target cells showed that IK6 could promote target cell clone formation and inhibit apoptosis, but had no significant effect on the cell cycle. It is concluded that virus vector carrying IK6 gene had been successfully constructed and expressed in THP1 stably. Biology studies of target THP1 cell shows that the IK6 is likely to interfere with the function of normal Ikaros protein as tumor suppressor, and it exerts a potential anti-apoptotic effect. Thus, IK6 can promote leukemia cell growth. However, there is no significant effect on the cell cycle. It provides an effective method for exploring the function of IK6 in acute myeloid leukemia.

Mutation of isocitrate dehydrogenase 2 (IDH2) gene in Chinese AML patients and its clinical significance / 中国实验血液学杂志

This study was purpose to analyze the frequency and of isocitrate dehydrogenase 2 (IDH2) gene mutation in acute myeloid leukemia (AML) and its clinic significance. The multiplex polymerase chain reaction (PCR) and sequencing were performed to screen 192 AML patients for exon 4 of the IDH2 gene. FLT3, NPM1, CEBPA, c-kit and WT1 mutations were also included in analysis. The results showed that IDH2 mutation was found in 14 (7.29%) of 192 patients. There were 9 AML patients with R140Q mutation, 1 patient with R140W mutation, and 1 patient with R172K mutation. IDH2 aberrations significantly more were detected in French-American-British (FAB) M5 (P < 0.005) than other types. There was no statistical difference in age, sex, WBC, platelet count, bone marrow blasts count, hemoglobin as compared with IDH2 wild-type. For immunotype analysis, IDH2 mutation patients were more likely to express CD34 and CD13, less CD36. IDH2 mutation combined with FLT3/ITD mutation was found in 7 cases, with CEBPA mutation in 4 cases, with NPM1 mutation in 4 cases, with Dnmt3a mutation in 5 cases, neither with c-kit, IDH1 or WT1 mutation for no one, which revealed a significant interaction between IDH2 mutation and the FLT3/ITD positive genotype, Dnmt3a mutated, and IDH1 wild-type. IDH2 mutation was detected in 5 (8.47%) of 59 CN-AML. There was no significant difference of IDH2 mutation incidence between the normal and abnormal karyotype. The CR rate was higher in IDH2 R140 mutated patients than wild-type ones, but there was no significant in the two group. It is concluded that the rate of IDH2 mutation is 7.29% in Chinese AML patients and 7.81% in CN-AML. IDH2 mutation is significantly associated with AML-M5, FLT3/ITD, Dnmt3a, IDH1 wild-type and fusion gene wild-type, but not with age, leucocyte and platelet counts in peripheral blood, karyotype, NPM1, CEBPA, c-kit or WT1 mutation. And IDH2 R140 mutation has no impact on CR rate.

Effect of gambogic acid on proliferation of SKM-1 cells and its mechanism / 中国实验血液学杂志

The aim of this study was to explore the effect of gambogic acid (GA) on MDS SKM-1 cell proliferation, apoptosis and their possible mechanism. Cell proliferation was determined by MTT method. The apoptosis percentage and cell cycle regulation of SKM-1 cells were analyzed by flow cytometry. Morphological features were observed by light microscopy. The mRNA expression of bcl-2 and bax were detected by RT-PCR. The results showed that GA could inhibit the proliferation of SKM-1 cells in a dose- and time-dependent manner (IC50 was 0.37 µg/ml at 48 h), increase the apoptotic percentage of SKM-1 cells, and arrest cell cycle at the G0/G1. The expression of bax mRNA was up-regulated while that of bcl-2 mRNA was down-regulated in SKM-1 cells treated with GA for 48 h. It is concluded that GA can induce apoptosis, which may be related to its effect of arresting cells at phase of G0/G1 and down-regulating bcl-2/bax ratio.

Establishment of xenotransplated mouse model using primary multiple myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish xenotransplated mouse model by non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice with primary myeloma cells.</p><p><b>METHODS</b>The model of xenograft was established in NOD/SCID mice by tail vein injection of mononuclear cells from two end stage multiple myeloma patients, three mice were inoculated for each patient. Mice were monitored weekly for body weight. Two weeks later, the human CD45(+) cells from peripheral blood of mice were evaluated by flow cytometry (FCM). The experiment endpoint was body weight loss up to 20% or had pale, vertical hair and listlessness, then spleen and liver were studied by histologic analysis, the human CD45(+)CD38(+) cells from spleen, lymph node, peripheral blood and bone marrow were evaluated by FCM.</p><p><b>RESULTS</b>Body weight of mice in group patient 1 and group patient 2 decreased seven and five weeks after inoculation respectively; the human CD45(+)CD38(+) cells appeared in the peripheral blood (26 ± 4) and (16 ± 4) days after inoculation in group patient 1 and group patient 2 respectively, and increased by time, reaching (16.2 ± 3.0)% and (31.3 ± 3.5)%, respectively at the endpoint; the spleen, liver and lymph node of both groups enlarged, the typical malignant plasma cells were observed in them. The human CD45(+)CD38(+) cells were detected in spleen, lymph node and bone marrow by FCM.</p><p><b>CONCLUSION</b>Our study successfully established a NOD/SCID mouse model xenotransplated with human primary myeloma cells.</p>

Detection of isocitrate dehydrogenase 1 gene mutation in 205 AML patients and its clinical significance / 中国实验血液学杂志

This study was purposed to detect the mutation of isocitrate dehydrogenase 1 (IDH-1) gene in patients with acute myeloid leukemia (AML) and to explore its clinical significance. The genomic DNA was extracted from mononuclear cells (MNC) of bone marrow or peripheral blood in 205 adult AML patients, the exon 4 of IDH1 gene was amplified by PCR, then the sequencing and comparison were performed. The results showed that IDH1 mutation was detected in 9 (4.39%) of 205 AML patients. There were 6 cases of R132H mutation, 1 of R132L mutation, 1 of R132G mutation and 1 of R132S mutation. Significantly more IDH1 aberrations were detected in AML-M2 (P = 0.002) than other types. And the 9 patients with IDH1 mutation were characterized by low platelet count which was lower than patients with wild type IDH1 (P = 0.003). IDH1 mutation combined with FLT3/ITD mutation was found in 5 cases, c-kit mutation in 1, NPM1 mutation in 2, and IDH1 mutation with CEBPA or WT1 mutation was not found, which revealed a significant interaction between IDH1 mutation and the FLT3/ITD positive genotype or the CEBPA wild-type. IDH1 mutation were detected in 4 of 71 (5.63%) CN-AML. There was no significant difference of IDH1 mutation incidence between the normal and abnormal karyotypes. It is concluded that the rate of IDH1 mutation was 4.39% in Chinese AML patients. IDH1 mutation is significantly associated with AML-M2, lower platelet counts in peripheral blood, FLT3/ITD mutation and CEBPA wild-type, but not with age, white blood cell count in peripheral blood, karyotype, NPM1, c-kit or WT1 mutation.

Application of FICTION technique to the detection of genetic aberrations in multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the diagnostic value of FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms) technique, combining immunofluorescence and fluorescence in situ hybridization (FISH), to detect genetic aberrations in multiple myeloma (MM).</p><p><b>METHODS</b>Bone marrow samples were collected from 18 MM and 2 plasma cell leukemia (PCL) patients. Probes targeting IgH and MMSET were prepared using a Nick Translation Kit from Bacterial artificial chromosome (BAC) clones. The immunophenotyping was achieved via the CD138 tyramide signal amplification (TSA)-mediated immunofluorescence, followed by FISH with the prepared probes \[t(4;14), t(11;14), t(14;16)\] and the commercial deletion probes (13q and p53) to detect common genetic aberrations in MM.</p><p><b>RESULTS</b>All the 20 samples were assayed with the probes mentioned above, and revealed 4 cases with t(4;14), 6 with t(11;14), 1 with t(14;16), 3 with p53 deletion; and 8 with 13q deletion. The remaining 4 cases had none of the 5 aberrations.</p><p><b>CONCLUSION</b>FICTION technique facilitates the detection of genetic abnormalities of MM in situ; enhances both efficiency and sensitivity of positive detection, thus, could be used as the screening test of molecular diagnosis of MM to guide coming-up risk-adapted therapy and evaluate prognosis.</p>

CD96 expression on bone marrow mononuclear cells in 91 patients with acute leukemia / 中国实验血液学杂志

This study was aimed to investigate the expression of CD96 on bone marrow mononuclear cells (BMMNC) from 91 patients with acute leukemia, and the results were analyzed with clinical pathological data. Flow cytometry was used to detect CD96 molecule on the bone marrow mononuclear cell surface of 91 newly diagnosed patients with acute leukemia, and 15 healthy adults were served as normal controls. The results showed that the average rate of CD96(+) expression on BMMNC (CD45(+) CD34(+) CD19(+)) of 21 patients with B-ALL was (17.41 ± 27.97)%, the average rate of CD96(+) expression on stem cells (CD45(+)CD34(+)CD7(+)) of 11 patients with T-ALL was (46.98 ± 45.55)%, the average rate of CD96(+) expression on BMMNC (CD45(+)CD34(+)CD38(-)) of 59 patients with AML was (16.69 ± 25.08)%, while the average rate of CD96(+) on BMMNC of healthy adult controls was (0.52 ± 1.84)%, there was significant difference in average rate of CD96(+) expression between above-mentioned patients and healthy adult controls (p < 0.05). Otherwise the average rate of CD96(+) on BMMNC after treatment showed no statistical difference between patient group with CR (1.68 ± 2.31) and healthy controls, but demonstrated statistical difference between patients without CR and healthy controls (p > 0.05). The leukocyte count, hemoglobin level and platelet count in CD96(+) group had no obvious difference from CD96(-) ones (p > 0.05). No change found in the field of molecular biology and cytogenetic between these 2 groups. It is concluded that CD96 expression is different in different types of leukemia. The positive expression of CD96 on bone marrow hematopoietic stem cells in patients with acute leukemia may be associated with primary drug resistance, relapse and progression. The CD96 on BMMNC of acute leukemias can be a helpful prognostic indicator in treatment response assessment.

Analysis of IKAROS6 expression in 73 patients with chronic myelogenous leukemia / 中国实验血液学杂志

This study was aimed to evaluate IKAROS6 expression in patients with chronic myelogenous leukemia (CML) and its clinical significance. cDNAs from 73 CML patients were amplified by PCR and sequenced for IKAROS expression to elucidate clinical characteristics in IKAROS6 positive patients. The results showed that there was no IKAROS6 gene expression in 8 healthy controls and 15 CML patients in chronic phase and accelerated phase, and 15 cases (35.71%) were IKAROS6 positive in lymphoblast crisis samples among 42 newly diagnosed CML; however, none was found in myeloblast crisis of 16 newly diagnosed CML. Among 42 lymphoblast crisis of CML, the complete remission (CR) rate of IKAROS6 expression positive patients reached 40% (6/15), which was obviously lower than that in IKAROS6 negative patients (85.19%, 23/27) (p < 0.01), IKAROS6 positive patients relapsed after CR for 15 (2 - 18) months with relapse rate 66.7% (4/6), which was higher than that in expressed wild type IKAROS gene patients (21.74%, 5/23) (p < 0.05). It is concluded that abnormal expression of IKAROS gene dominated by IKAROS6 isoforms can be detected in lymphoblast crisis samples of CML patients. Abnormal expression of IKAROS gene may be an important factor in lymphoblast crisis of CML. Therefore, detection of IKAROS gene expression may be important for target therapy and evaluation of clinical prognosis of CML patients.

Effect of 5-Aza-CdR on biological activity and inhibitor of DNA binding 4 gene expression in human erythroleukemia cell line K562 / 中国实验血液学杂志

This study was aimed to investigate the effect of 5-Aza-CdR on the biological activity of human erythroleukemia cell line K562 and the expression of inhibitor of DNA binding 4 (ID4). ID4 methylation in K562 cell line was detected by methylation-specific PCR. RQ-PCR was used to analyze the expression levels of ID4 mRNA in K562 cell line treated by different concentrations of 5-Aza-CdR. Cell apoptosis rate and cell cycle were analyzed by flow cytometry. The result showed that ID4 gene methylation existed in K562 cells, ID4 mRNA expression in K562 cells treated with 5-Aza-CdR increased in a concentration-dependent manner, the difference between experimental groups was statistical significant (p < 0.01). The 5-Aza-CdR could enhance the apoptotic rate of K562 cells in time and dose-dependent manner, the apoptotic rate of K562 cells highly correlated to relative expression level of ID4 mRNA (r = 0.95). After the K562 cells were treated by 5-Aza-CdR for 48 hours, cells in G(0)/G(1) phase increased, cells in G(2)/M phase decreased along with enhancement of drug concentration. It is concluded that methyltransferase inhibitor 5-Aza-CdR can re-express the silent ID4 gene in K562 cells. The upregulation of ID4 may be a key factor to give rise to cell apoptosis, and the cell cycle of K562 cells can be arrested by 5-Aza-CdR.

Effect of sodium valproate on human myelodysplastic syndrome cell line SKM-1 and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of sodium valproate(VPA) on human myelodysplastic syndrome cell line SKM-1 and its mechanism. The cell proliferation was determined by MTT assay, cell apoptosis was analyzed by flow cytometry. The expressions of c-flipl, c-flips and dlk1 mRNA were detected by RT-PCR. The results showed that VPA could inhibited the growth of SKM-1 cells in dose- and time-dependent manners. The flow cytometric analysis indicated that VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner. The expressions of c-flipl, c-flips and dlk1 mRNA in SKM-1 cell treated with VPA decreased using of VPA. It is concluded that VPA can induce apoptosis and inhibited proliferation of SKM-1 cells. In this process, the decreasing of c-flipl, c-flips and dlk1 mRNA expression may play important roles.

ERK pathway change in the differentiation of human MDS cell lines SKM-1 induced by sodium butyrate / 中国实验血液学杂志

The study was purposed to investigate the role of extracellular signal-regulated kinase (ERK) pathway in the differentiation of human MDS cell lines SKM-1 induced by sodium butyrate (NaB), and to elucidate the molecular mechanism of differentiation in SKM-1 cells induced by NaB. The expression levels of total ERK and phosphorylated-ERK were determined by Western blot. The effect of NaB in combination with the ERK inhibitor PD98059 on the proliferation/differentiation of SKM-1 cells was studied, and then the expression levels of the P21 and HDAC protein were detected by Western blot. The results showed that the expression level of phosphorylated ERK was down-regulated by the 1 mmol/L NaB, and the level of total ERK had not changed. NaB or combination of the MEK inhibitor PD98059 with NaB could increase the differentiation of the SKM-1 cells and up-regulated the levels of the P21 and HDAC protein, but the effect of combination of NaB with PD98059 was higher than that of NaB alone. It is concluded that the inhibition of ERK may be involved in sodium butyrate inducing differentiation in SKM-1 cells.

Effects of ligustrazine on expression of VCAM-1/VLA-4 in syngenic bone marrow transplantation of mice / 中国实验血液学杂志

To explore the effect of ligustrazine on the expression of adherent molecule VCAM-1/VLA-4 of bone marrow cells in syngenic bone marrow transplantation (BMT) mice, the mice were divided into 3 groups: normal group (which received no treatment), BMT control group and ligustrazine-treated groups. BMT mouse models were established. The BMT control group and the ligustrazine-treated group were orally administered 0.2 ml saline per mouse and 2 mg ligustrazine per mouse, respectively, twice a day. On the day 7, 14, 21, 28 after BMT, mice were respectively killed. Bone marrow nucleated cells were detected, and then the expression of VCAM-1/VLA-4 was assayed by immunohistochemistry, RT-PCR and flow cytometry analysis, respectively. The results showed that in ligustrazine-treated group, the accounts of bone marrow nucleated cells on the day 7, 14, 21, 28 after BMT were all higher than that in BMT control group. The expression level in the ligustrazine-treated group was significantly higher than that in the BMT control group (P < 0.05 or P < 0.01). It is concluded that ligustrazine can enhance VCAM-1/VLA-4 expression in bone marrow after syngenic bone marrow transplantation in mice, which may be related to the mechanisms underlying the ligustrazine accelerating hematopoietic reconstitution in allogenic bone marrow transplantation.

The effects of Mcl-1 gene on ATRA-resistant HL-60 cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the role of Mcl-1 gene in resistance of all-trans retinoic acid (ATRA) of leukemia cells.</p><p><b>METHODS</b>Long-term, intermittent and repetitive exposure of HL-60 cells to ATRA was used to establish a multidrug-resistance cell line (HL-60/ATRA). HL-60/ATRA cells were transfected with Mcl-1 small interference RNA (siRNA) by Lipofectamine 2000. Western blot was used to detect the expression of Mcl-1. The proliferation, apoptosis and differentiation were evaluated by MTT assay, in situ nick end-labeling (TUNEL) and NBT assay, respectively.</p><p><b>RESULTS</b>The HL-60/ATRA could keep its undifferentiated and proliferative status to a high concentration of ATRA (100 nmol/L) with highly expressed Mcl-1 protein (relative grey scale 0.624 +/- 0.127). Mcl-1 gene knockdown by siRNA (relative grey scale 0.267 +/- 0.086) could reverse the resistance of ATRA of HL-60/ATRA by inhibiting proliferation, and inducing differentiation and apoptosis [apoptosis rate (18.5 +/- 4.5)%].</p><p><b>CONCLUSION</b>Mcl-1 gene might be involved in ATRA resistance in HL-60 cells and inhibiting its expression could be a new approach to ATRA resistance reversion.</p>

Effect of sodium butyrate in combination with ATRA on the proliferation/differentiation of MDS cell line SKM-1 / 中国实验血液学杂志

The study was purposed to explore the molecular mechanisms of sodium butyrate (NaB) action on SKM-1 cell proliferation/differentiation and to study its synergistic effect with all-trans retinoic acid (ATRA). SKM-1 cells were grown in the absence or presence of NaB and/or ATRA; the percentage of viable cells was determined by trypan blue exclusion; differentiation was investigated by nitro-blue tetrazolium (NBT) reduction; adhesion molecules of cell surface were analysed by FACS; cell cycle distribution was studied after DNA staining by propidium iodide; D-type cyclins, CDK and P21 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that NaB and/or ATRA blocked cells mainly in the G0/G1 phase of the cell cycle; ATRA inhibited the mRNA expression of CDK6, CDK4, cyclin D3 and cyclin D1; NaB inhibited the mRNA expression of CDK2, cyclin D2 and cyclin D1; ATRA and NaB inhibited the mRNA expression of CDK6, CDK4, CDK2, cyclin D1, cyclin D2 and cyclin D3; ATRA and/or NaB both stimulated p21 expression at the mRNA levels. It is concluded that the NaB effect on cell proliferation/differentiation may be linked to its ability to induce expression of p21 mRNA and inhibit the cyclin D-CDK complexes. These observations support the claim that NaB has the synergistic effect with ATRA.