[Clinical Characteristics of Acute Myeloid Leukemia Patients with RUNX1 Gene Mutation].

OBJECTIVE: To investigate the incidence of Runt-related transcription factor 1 (RUNX1) gene and its associated gene mutations in patients with acute myeloid leukemia (AML), and analyze its clinical characteristics and prognosis. METHODS: The genomic DNA-PCR method was used to detect the exon of RUNX1 gene, and the gene mutations were analyzed by genetic sequencing. NPM1, DNMT3A, FLT3-ITD, IDH1/2, K/N-RAS, CEPBA, TET2, and WT1 co-mutations were also detected. Patients were followed up to determine efficacy and prognosis. RESULTS: Among 171 patients, the RUNX1 gene mutation was detected in 17 cases, and the mutation rate was 9.9%. The type of RUNX1 gene mutation was 9 missense mutations, 4 frameshift mutations, and 4 nonsense mutations. The peripheral blood leukocyte count of the patients in mutation group was 3 (1-101) ×109/L, which was significantly lower than those in the non-mutation group [26 (1-298)×109/L] (P=0.002), while the platelet count was 79 (22-166)×109/L, which was higher than 50 (8-351)×109/L in the non-mutation group (P=0.010), and the proportion of bone marrow blasts of the patients in the mutation group was 37 (0-72)%, which was lower than 53 (0-98)% in the non-mutation group (P=0.020). The RUNX1 mutation rate in M0 type was 55.6%, and in M4 type was 13.6%, which was significantly higher than other FAB subtypes (P=0.003). There was no significant difference in age, sex, hemoglobin concentration, and counts of peripheral blood mononuclear cells (P>0.05). The prognosis of cytogenetics in the patients in the middle and high-risk groups was 88.1% and 89.7%, which were significantly higher than that in the low-risk group, and the difference showed statistically significant (P=0.018). There was no significantly relationship between RUNX1 and specific karyotype abnormalities, including Trisomy 8, Del (7q), t (8; 21), and Inv (16) (P>0.05). There was a significant relationship between RUNX1 gene mutation and IDH1/2, N/K-RAS gene mutation (P<0.01). The complete response rate (CR) of the patients with chemotherapy in the RUNX1 mutation group(37.5%) was significantly lower than 79.4% in the non-mutated group (P=0.001). The overall survival (OS) of the patients in RUNX1 gene mutation group was lower than that in non-mutation group (P<0.05). CONCLUSION: AML patients with RUNX1 gene mutation shows unique clinical and biological characteristics, RUNX1 mutation can be regarded as a molecular marker of poor prognosis in AML patients.

In vitro facilitating role of polygonatum sibiricum polysaccharide in osteogenic differentiation of bone marrow mesenchymal stem cells from patients with multiple myeloma.

BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) were proved to play a vital role in multiple myeloma (MM). Polygonatum sibiricum polysaccharide (PSP) was found to have anti-tumor pharmacological effects, yet its interaction with BMMSCs remained poorly understood. Therefore, we explore the effect of PSP on osteogenic differentiation of BMMSCs. METHODS: BMMSCs were isolated by density gradient centrifugation. CD90 and CD34 were detected by flow cytometry (FCM). Osteogenic marks were detected by quantitative real-time PCR (qRT-PCR) and Western blotting (WB). The vitality of cells treated with different concentrations of PSP was observed by Cell Counting Kit-8 (CCK-8). ALP staining kit was used to detect the activity of alkaline phosphatase (ALP). Alizarin red staining detected the formation of mineralized nodules. Osteoblast-associated genes were evaluated by qRT-PCR and WB. The phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) signaling pathways were tested by WB. RESULTS: The BMMSCs showed good growth under an inverted microscope. FCM showed that CD34 and CD45 was low-expressed, whereas CD44, CD90 and CD105 was highly expressed. Compared with the Control group, the expressions of Runx2 and ALP in cells were significantly increased. CCK-8 showed that different concentrations of PSP had no significant effect on the viability of BMMSCs. BMMSCs treated with 25 mg/l PSP were stained the most deeply by ALP. Mineralized nodules in PSP groups dramatically increased, and hit a peak under the action of 25 mg/l PSP. PSP up-regulated p-PI3K, p-AKT, and p-mTOR, but had no significant effect on PI3K, AKT, and mTOR. CONCLUSION: PSP induced osteogenic differentiation of BMMSCs from MM patients.

[Correlation of the Expression Alteration of Tim-3 with the T Cell and B Cell Dysfunction in Peripheral Blood of Multiple Myeloma Patients].

OBJECTIVE: To study the correlation of the expression alteration of Tim-3 with the T cell and B cell dysfunction in peripheral blood of multiple myeloma (MM) patients. METHODS: 30 patients diagnosed as MM from October 2016 to October 2018 were selected and enrolled in MM group, and 30 healthy persons whose sex and age was matched with the MM patients were selected and enrolled in healthy control group (HC). The blood samples from MM patients and HC were collected, and the peripheral blood mononuclear cells (PBMNC) were separated by density gradient centrifugation, then the serum was kept for further study. The ratios of CD3+CD4+Tim-3+T cells, CD3+CD8+Tim-3+T cells and the CD19+CD20-CD38+B cells were analysed by flow cytometry (FCM)，and the concentration of T cell-related cytokines IFN-γ, TNF-αand B cell-related antibodies IgA, IgM and IgG were measured by ELISA. At the same time, the differences of the ratios of CCD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells and plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG between the MM patient and HC were estimated, and the correlation of the ratio of CD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells with the ratio of plasmablast and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG in MM patients were analyzed. RESULTS: The ratio of CD3+CD4+Tim-3+T, CD3+CD8+Tim-3+T cells increased in MM patients, while the ratio of CD19+CD20- CD38+B cells and the concentration of IFN-γ, TNF-α, IgA, IgM and IgG decreased in MM patients. And there was a negative correlation of the ratio of CD3+CD4+Tim-3+T cells with CD19+CD20-CD38+B cells and the concentration of IFN-γ, IgA, IgM and IgG in MM patients, while the ratio of CD3+CD8+Tim-3+T cells just negatively correlated with the concentration of TNF-α. CONCLUSION: Expression of Tim-3 on CD4 and CD8 cells elevates in the peripheral blood of MM patients, which also correlates with the function suppression of T and B cells.

MiR-486-5p inhibits the proliferation of leukemia cells and induces apoptosis through targeting FOXO1.

AIM: Studies have reported that micro (miR)-486-5p plays a crucial part in the progression of leukemia, however, to the best of our knowledge, few studies have been conducted on its mechanism in leukemia. In this study, the mechanism of miR-486-5p in leukemia cells was pointed out and its possible target genes were analyzed for the purpose of providing new therapeutic strategies for treating leukemia patients. METHODS: MiRNA expression of Leukemia cells (K562, Kasumi-1, and THP-1) and primary leukocytes was detected by Real-time Quantitative polymerase chain reaction(qPCR). The activity of the cells was assessed using the Cell Counting Kit-8 (CCK-8). Apoptotic cells were analyzed by a flow cytometer (FCM). Caspase-3 activation in leukemia cells was determined by Western blot. Targetscan 7.2 was used to predict the potential targets of miR-486-5p and further confirmed by dual-luciferase reporter assay. RESULT: miR-486-5p was significantly down-regulated in leukemia cells. The over-expression of miR-486-5p notably increased the apoptosis and caspase-3 activity in leukemia cells. There was a predicted interaction site for miR-486-5p in the FOXO1 3'-UTR. Furthermore, this study showed that FOXO1 was significantly up-regulated in leukemia cells, the growth of which was depressed by the up-regulation of miR-486-5p. CONCLUSION: miR-486-5p may inhibit the proliferation of leukemia cells and induce apoptosis through targeting FOXO1.

Investigation of a novel biomarker, neuropilin-1, and its application for poor prognosis in acute myeloid leukemia patients.

According to the previous studies, numerous biomarkers impact on the prognosis of acute myeloid leukemia (AML) and the prediction for AML had been improved tremendously in the past decades. However, accurate risk-stratification at diagnosis or prognosis remained difficult. In order to further investigate the prognosis evaluation biomarker, the transcription or expression of neuropilin-1 (NRP-1) in 87 AML patients and 32 non-malignant controls were examined. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect the NRP-1 expression. Clinical data were collected and analyzed for the 87 AML patients. The results indicated that high NRP-1 expression discriminated the complete remission (CR) rate of AML patients (22.12 % vs. 68.04 % for AML, P < 0.01). De novo AML patients tended to express higher NRP-1 proteins than relapsed AML patients. The overall survival (OS) and relapse-free survival (RFS) rate of the high NRP-1 expression patients decreased significantly compared with the low NRP-1 expression patients (P < 0.001). NRP-1 was revealed to be an independent risk factor for OS in AML (P = 0.003). In conclusion, NRP-1 could predict the shorter OS and RFS rate, and also related with the CR response in AML. Therefore, NRP-1 may act as a more aggressive and promising predictor for the poor prognosis of AML.

[Influence of As(2)O(3) on proteasome beta(1)-subunit in NB4 cells].

This study was aimed to explore the correlation between effects of arsenic trioxide on NB4 cell differentiation and the change of beta(1)-subunit of 26S proteasome. NB4 cell in 0.5 micromol/L As(2)O(3) was incubated for 24 hours and 48 hours, then total protein was extracted, expressions of subunit beta(1) and PML-RARalpha fusion protein were determined by Western blot. The results indicated that the expression of 26S proteasome beta(1)-subunit increased after incubation with As(2)O(3) for 24 hours, but after culture with As(2)O(3) for 48 hours, the expression of beta-subunit decreased to the baseline. Meanwhile, the expression of PML-RARalpha fusion protein obviously decreased after 24 hours, and kept low level at 48 hours. It is concluded that the expression of 26S proteasome beta(1)-subunit increases after exposure to As(2)O(3). Increment of 26S proteasome beta(1)-subunit may be associated with the degradation of PML-RARalpha fusion protein and plays roles in the differentiation and apoptosis of NB4 cells.