Objective:To explore the genes related to primary myelofibrosis (PMF) and signaling pathways as well as the possible clinical significance.Methods:A total of 3 mRNA expression datasets of PMF (GSE26049, GSE61629 and GSE53482) were downloaded from Gene Expression Omnibus (GEO) database, including the data of peripheral blood samples from 55 PMF patients and 58 controls. The differentially expressed genes (DEG) between PMF patients and the controls were identified by using online tool GEO2R. Gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the common DEG of the 3 datasets, and then protein interaction (PPI) network was constructed. The key nodes of the common DEG in PPI network were calculated by using MCC method and Degree method in cytoHubba program; finally the top 10 hub genes were selected and the hub genes shared by the 2 methods were obtained. Peripheral blood samples of 25 PMF patients and 10 controls (normal hematopoietic stem cell transplant donors or iron deficiency anemia patients) admitted to the Second Hospital of Shanxi Medical University from September 2017 to June 2021 were retrospectively collected. Reverse transcription-fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of 10 screened common hub genes in each sample, and the expressions of all genes at transcriptional level of the two groups were compared.Results:A total of 239 common DEG between PMF patients and the controls were screened out in the 3 datasets, including 153 downregulated DEG and 86 upregulated DEG. The GO enrichment analysis showed that the common downregulated DEG were significantly enriched in negative regulation of transcription, translation and fibroblast proliferation, while the upregulated DEG were mainly enriched in protein ubiquitination and ubiquitin-dependent protein catabolic process. The KEGG pathway analysis indicated that the upregulated common DEG and downregulated common DEG were both enriched in PI3K/Akt signaling pathway, cancer pathways and transcriptional misregulation in cancer. There were 8 common hub genes shared by the 2 methods among the top 10 genes ranked by MCC and Degree methods, including 6 downregulated common DEG (TP53, MYC, ATM, FYN, PTPRC and ATRX) and 2 upregulated common DEG (VEGFA and FOXO3). These above 8 common hub genes were mainly involved in PI3K/Akt signaling pathway, cell cycle and cancer transcriptional regulation signaling pathways. RT-qPCR detection of clinical peripheral blood samples showed that the relative expression levels of mRNA in 6 downregulated common DEG of PMF patients were lower compared with those in controls, while the differences were not statistically significant (all P > 0.05); the relative expression levels of mRNA in 2 upregulated common DEG of PMF patients were higher compared with those in controls, and the differences were statistically significant ( U value was 33.00, 36.00, respectively; P value was 0.021, 0.033, respectively). Conclusions:Bioinformatics and clinical sample verification show that VEGFA and FOXO3 are up-regulated in PMF patients, which are mainly involved in the PI3K/Akt signaling pathway, cell cycle and cancer transcriptional regulation signaling pathway. Both genes may be related to the development of PMF and may become potential therapeutic targets.

Objective To study the role of hematology analyzer in the preliminary diagnosis of hematological malignancies. Methods The blood routine results of 121 patients with initial hematological malignancies in the Second Hospital of Shanxi Medical University from October 2016 to February 2017 were analyzed by differential blood count (DIFF) scatter chart and alarm information of hematology analyzer SYSMEX XE-2100. Results Among 121 patients with hematological malignancies, 96 cases (79.3 %) had abnormality of DIFF scatter chart. The DIFF scatter chart of acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute promyelocytic leukemia, and chronic myeloid leukemia had their own distinct characteristics, but the features of other types were not obvious. According to blood smear, blood cells analysis alarmed that the coincidence rates of primitive cells, immature granulocytes, nucleated erythroblasts and atypical lymphocytes were 87.7 % (50/57), 76.3 % (42/55), 33.3 % (19/57), 43.1 % (31/72) respectively. Conclusion The DIFF scatter chart of hematology analyzer and alarm information play very important roles in the diagnosis of hematological malignancies.

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome(MDS).Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction(PCR)followed by sequence analysis at DNA level.The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type.ASXL1 mutation in mRNA level was detected by using reverse transcription PCR(RT-PCR)followed by sequence analysis.Results ASXL1 gene mutations were observed in 9 cases(16.9%)of 53 MDS patients.6 mutation types were detected,including 4 frameshift mutations types(2 cases with p.Glu635ArgfsX15,3 cases with p.Gly646TrpfsX12,1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10)and 2 nonsense mutation types(1 case with p.Gln1063X and 1 case with p.Gln695X).All the mutations were heterozygous,and p.Gly790TrpfsX10 and p.Gln695X were new mutation types.In addition,a single nucletide polymorphism(SNP)p.Gly652Ser was also detected in 4 cases with MDS.5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people.Frameshift mutation(p.Gly646TrpfsX12)could be detected at mRNA level by using RT-PCR.Differences were not observed in red blood cell counts,white blood cell counts,platelet counts,hemoglobin levels,reticulocyte,neutrophil granulocyte,the peripheral blood lymphocytes percentage,T-cell subsets in the peripheral blood,the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients(P >0.05).Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients,which can be detected at mRNA level.

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G₀/G₁ phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G₂/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Objective To explore the value of the plasma miR-193a-5p level on diagnosis and monitoring the response to treatment in acute myeloid leukemia (AML). Methods Peripheral blood samples were obtained from AML patients enrolled in hematology department of the Second Hospital of Shanxi Medical University from July 2015 to December 2015, including 30 de novo AML patients, 9 patients in complete remission (CR) and 6 patients in relapse. Peripheral blood samples from 15 healthy people were randomly choosed as the health control group. Plasma miR-193a-5p expression levels were detected by using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results The plasma miR-193a-5p relative expression level of AML patients group [0.465 6 (0.103 1-5.000 2)] was obviously lower than that of health control group [0.766 1 (0.052 1-3.134 4)] (U= 122, P= 0.018 7). The plasma miR-193a-5p relative expression levels of de novo group and relapse AML group were significantly lower than those of CR group and health control group (P<0.05), and there was no significant difference between the CR group and health control group (U= 56, P= 0.511 9). No significant correlation was found between the plasma miR-193a-5p level and age, gender, blast percentage of the bone marrow, peripheral blood leukocyte count, platelet count, CD34+cells'percentage and so on. Conclusion The decreased plasma miR-193a-5p expression level can be served as a new and noninvasive biomarker for the evaluation of diagnosis and treatment in AML.

CD5 Antigens ; Chromosome Aberrations ; Humans ; Lymphoma, Large B-Cell, Diffuse ; complications ; genetics ; Splenic Infarction ; complications

OBJECTIVETo identify the MPL L391-V392ins12 spliceosome and analyze its frequencies in patients with myeloproliferative neoplasms (MPN).

METHODSMPL aberrant spliceosome was identified through reverse transcription polymerase chain reaction (RT-PCR)combined with cloning sequencing. The mutation of this spliceosome in 248 MPN patients and 200 normal people was determined by allele-specific polymerase chain reaction (AS-PCR).

RESULTSA novel aberrant spliceosome of MPL gene (MPL L391-V392ins12)was identified, i.e. 36 bp intron was retained between exon7 and exon8, and there were 12 amino acids (EGLKLLPADIPV)inserted. MPL L391-V392ins12 mutation was detected in 19 (7.66%)of the 248 patients with MPN, including 1 (1.92%) of 52 patients with PV, 14 (9.66%) of 145 with ET, and 4 (7.84%) of 51 with PMF. And the mutation was not detected in the group of 200 normal people.

CONCLUSIONMPL L391-V392ins12 spliceosome is an aberrant spliceosome present in the MPN. It can be detected in PV, ET and PMF, and more frequently in ET and PMF. This mutation may play an important role in the process of MPN.

Humans ; Mutation ; Myeloproliferative Disorders ; genetics ; Neoplasms ; genetics ; Polymerase Chain Reaction ; Receptors, Thrombopoietin ; genetics ; Spliceosomes

Objective To explore the relationship between T-cell acute lymphoblastic leukemia and the Notch signaling pathway.Methods Human T-cell acute lymphoblastic leukemia SupT1 cells were infected with the lentiviral vector made up specific Notch1-shRNA gene and nonspecific Notch1-shRNA gene.The inhibitive rate of SupT1 cells was detected by CCK-8.The rates of early apoptotic cells (Annexin V+/ 7-AAD-) and late apoptotic cells (Annexin V+/7-AAD+) were analyzed by flow cytometry and the expression levels of Notch1 receptor gene and downstream target genes were assessed by quantitative reverse transcription and polymerase chain reaction (QT-PCR).Results The cell inhibition rates of Notch1 interference group,control group and empty vector group at 96 h were 0.902±0.013,0,and 0.486±0.084,respectively,and it was increased obviously in Notch1 interference group (both P ＜ 0.05).The cell early apoptosis rates of the three groups were (15.27±0.31) ％,(5.57±0.25) ％,(5.80±0.20) ％,respectively.The cell early apoptosis rate of Notch1 interference group was increased obviously compared with the control group and empty vector group (both P ＜ 0.05).While the cell late apoptosis rates had no significant difference among the three groups (P ＞ 0.05).The mRNA expression levels of Notch1 receptor gene and its target genes (Hesl,c-myc,NF-κB) at 48 h,72 h and 96 h were higher than those in the control group and empty vector group (all P ＜ 0.05).Conclusions The specificity of Notch1-shRNA can effectively decrease the Notch1 mRNA expression,and reduce the expression level of downstream target genes.Notch1 cut can inhibit the proliferation of SupT1 cells,and promote the early apoptosis.

Objective To investigate the relevance between CD25 and common markers of immune phenotype in acute myeloid leukemia (AML), and the relationship between CD25 and FLT3-ITD gene. Methods The expression of mono-clonal antibodies, including, CD7, CD117, CD33, CD34, CD38, cyMPO, CD13, CD36, CD56, CD11b, CD19, CD16, CD14, CD64 and interleukin-2 three receptor chain CD25 (αchain), CD122 (β chain), CD132 (γ chain) were detected by immunofluorescence flow cytometry in 36 cases of AML bone marrow blast. The expression of every group of pa-tients with FLT3-ITD gene were detected by PCR. Results In 36 cases of AML patients, the rate of CD25+was 13.89%(5/36), 12.5% (1/8) in AML-M2 group, 18.18% (2/11) in AML-M4 group, 20% (2/10) in AML-M5 group, respectively, and expression rate of CD25+had been no statistically differences between those subgroups (P> 0.05). The percentage of bone marrow blasts which express CD7, CD117, CD33, CD34, CD38, cyMPO, CD13, CD36, CD56, CD11b, CD19, CD16, CD14, CD64, CD122, CD132 monoclonal antibodies has no significant difference between CD25+AML-M2 group and CD25-AML-M2 group, CD25+AML-M4 group and CD25-AML-M4 group, CD25+AML-M5 group and CD25-AML-M5 group, respectively (P> 0.05). Seven cases of AML patients with FLT3-ITD mutation, in five cases of CD25+AML pa-tients three case accompany FLT3-ITD+ mutation, the rates of FLT3-ITD+in CD25+AML was 60%, and higher than CD25-AML group (P <0.05). Conclusion (1)The expression rate of CD25 in AML patients is lower, and no significant correlation in patients in different AML FAB subtypes and common myeloid phenotype markers. (2)Since the FLT3-ITD mutation rates are higher in CD25+AML patients, there is important significance to clear the correlation between CD25 and FLT3-ITD gene.