[Strategy Optimization and Clinical Analyze of Multiple Nucleotide Polymorphism Analysis in the Chimerism Detection after Allogeneic Hematopoietic Stem Cell Transplantation].

AbstractObjective: To investigate the sample selection, result correction and clinical application value of multi nucleotide polymorphism chimerism detection method based on Next-generation sequencing. METHODS: The chimerism samples from November 2018 to June 2020 were collected, and Pearson correlation coefficient (r) was used to analyze the consistency of bone marrow and peripheral blood results detected by MNPseq; according to the different information integrity before transplantation, the calibration model was constructed to analyze the correction value of the micro chimerism results in each model; the clinical results were retrospectively analyzed to verify the reliability and practicability of chimerism results correction and the clinical value of MNPseq method. RESULTS: The results of bone marrow and peripheral blood chimerism detected by MNPseq method were consistent with each other and showed significant correlation (r=0.985, P<0.01). The three groups of calibration models were constructed according to different pre-transplant information. For the no donor and pre-transplant patients information group, the correction value was 1%; while for the group with pre-transplant patients and without donor information, 0.61% of the chimerism rate and 13 heterotopic points were used as the correction value; 0.26% of the chimerism rate and 21.57% of the heterotopic points were used as the correction value for the group with pre-transplantation patients and donor information. After correction, the number of the patients with incomplete chimerism decreased from 276 (74.19%) to 141 (37.91%) (P<0.01). Among 18 (18/141, 12.77%) patients with incomplete chimerism, the results of MNPseq in the patients were 25-39 days earlier than those in STR and flow MRD, and the result showed statistical significance. CONCLUSION: MNPseq method can be used to monitor chimerism with peripheral blood instead of bone marrow samples, and the results can be corrected to detect the changes of graft status in vivo in a more timely manner.

Small nucleolar host gene 6 promotes esophageal squamous cell carcinoma cell proliferation and inhibits cell apoptosis.

Esophageal cancer (ESCC) is one of the most common causes of cancer-associated mortality in China. The present investigation reveals that non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs), exert a significant effect on the initiation, development and metastasis of malignant tumors, including ESCC. However, to the best of our knowledge, the function of non-protein-coding genes that host small nucleolar RNAs has not been investigated in cancer, particularly in ESCC. The expression of small nucleolar host gene 6 (SNHG6) in 70 ESCC tissues and paired adjacent tissues was measured by reverse transcription quantitative polymerase chain reaction. Analysis demonstrated that SNHG6 expression was significantly increased in ESCC tissues, and associated with tumor size (P=0.040) and Tumor-Node-Metastasis stage (P<0.01). Knockdown of SNHG6 may inhibit proliferative and colony-forming abilities, and induce apoptosis, in ESCC cells. To the best of our knowledge, the data from the present study indicated for the first time that SNHG6 was upregulated in ESCC tissues and cell lines. This novel lncRNA may exert a marked effect on the generation and progression of ESCC, potentially providing a novel perspective on ESCC diagnosis and management.

[Expression of Ki-67 in Adult and Children Acute Lymphoblastic Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the relationship of Ki-67 level with clinical features, immunophenotype, gene mutation, curative efficacy and prognosis in patients with acute lymphoblastic leukemia(ALL). METHODS: Flow cytometry gated at CD45/SSC was used to detect the expression of Ki-67, and the correlation of Ki-67 expression with clinical manifestation, laboratorial indexes, curative efficacy and prognosis was analysed. RESULTS: Ki-67 expression level increased in ALL patients, the median expression rate was 29.22%, there was significant difference as compared with the healthy control (P<0.01). In adult ALL, the median expression rate of Ki-67 in the high-risk group was 31.49%, and the difference was statistically significant as compared with the low-risk group (P<0.05). In children ALL, the median expression rate of Ki-67 in high-risk group was 42.28%, and the difference was statistically significant (P<0.05). The results of unvariate analysis showed that the age, WBC count at newly diagnosed and extramedullary invasion were adverse factors affecting OS and DFS; the results of multivariate analysis showed that age and extramedullary invasion were independent risk factors for OS and DFS in patients. CONCLUSION: Age≥14 years old, intramedullary invasion are the poor factors for prognosis; the Ki-67 level is not an independent factor for the prognosis of patients.

Prognostic significance of TET2 mutations in myelodysplastic syndromes: A meta-analysis.

Tet methylcytosine dioxygenase 2 (TET2) mutations occur frequently in myelodysplastic syndromes (MDS), but its prognostic impact has not been fully assessed and was controversial. Therefore, we performed a meta-analysis to evaluate the prognostic significance of TET2 mutations in MDS. PubMed, EMBASE databases and Cochrane Library were searched for studies reporting TET2 mutations and overall survival in MDS. Hazard ratios (HR) with 95% confidence interval (CI) were determined using random-effect modeling. A total of 1494 patients from nine studies were subjected to meta-analysis. The frequency of TET2 mutations was 18.34% (274/1494) in the study. MDS with TET2 mutations had similar overall survival compared to patients without the mutations (hazard ratio 1.13, 95% CI: 0.81-1.5).Our findings suggest that TET2 mutations have no prognosis impact on OS of patients with MDS. Therefore, the status of TET2 mutations cannot be served as a prognostic marker in MDS.

[Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism].

OBJECTIVE: To explore the effect of transforming growth factor-ß activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. METHODS: The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. RESULTS: As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein expression levels were all significantly greater than that in the single-treated groups (P< 0.05). CONCLUSION: TAK1 silencing and As2O3 can separately and synergistically inhibit Kasumi-1 cell proliferation which probably relates with the inducing apoptosis via the JNK and mitochondrial pathway. Meanwhile, TAK1 silencing enhances the inhibitory effect of As2O3 on Kasumi-1 cell proliferation.

AFAP1-AS1 is upregulated and promotes esophageal squamous cell carcinoma cell proliferation and inhibits cell apoptosis.

Recent findings indicate that long noncoding RNAs (lncRNAs) were dysregulated in many kinds of tumors including esophageal squamous cell carcinoma (ESCC). LncRNA AFAP1-AS1 was found to be upregulated in hepatocellular carcinoma (HCC), lung cancer, colorectal cancer, esophageal adenocarcinoma (EAC), pancreatic ductal adenocarcinoma, and nasopharyngeal carcinoma, while its clinical value and potential function in ESCC are still unknown. Expression of AFAP1-AS1 was measured in 65 ESCC tissues and corresponding noncancerous tissues by quantitative real-time polymerase chain reaction, which revealed that AFAP1-AS1 expression was markedly elevated in ESCC tissues and significantly associated with advanced TNM stage (P = 0.004) and larger tumor size (P = 0.040). Moreover, by knocking down AFAP1-AS1 expression in ESCC cells, the proliferation and colony-forming ability were inhibited and cell apoptosis was induced. Our data indicated the first time that AFAP1-AS1, a novel oncogene, was remarkably upregulated and played a critical role in the progression of ESCC.

[Effects of As2O3 in combination with TPA on K562 cells].

This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.

[Detection of NPM1, FLT3 and C-KIT mutations in acute myeloid leukemia and their prognostic analysis].

This study was aimed to evaluate the frequencies and prognostic significance of the nucleophosmin 1 (NPM1) mutation, the fms-like tyrosine kinase 3 (FLT3) mutation and c-KIT mutation in acute myeloid leukemia (AML) and to explore their relevance to clinical characteristics, cytogenetics and survival. Genomic DNA from 78 newly diagnosed AML from August 2010 to October 2012 was screened by PCR and sequencing or capillary electrophoresis (CE) for NPM1, FLT3 and c-KIT mutations. The results showed that the incidence of NPM1 mutation was 14.1% in AML patients and 26.7% in normal karyotype AML patients. NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count and platelet counts (P < 0.05) and low expression of CD34 (P < 0.05), but no statistic difference was found in sex, percentage of bone marrow blasts, Hb, expression of CD117 and HLA-DR, complete remission rate, overall survival and relapse rate (P > 0.05). The prevalences of FLT3-ITD and FLT3-TKD mutations were 11.5% (9/78) and 3.8% (3/78) respectively, and no one patient has both of the two mutations. Patients with FLT3-ITD mutation had higher white blood cell counts and percentage of in bone marrow blasts (P < 0.05), and lower overall survival (P < 0.05), more relative to normal karyotype (P < 0.05), while no statistic difference was found in sex, age, platelet count, Hb level, complete remission rate and relapse rate (P > 0.05). No statistic analysis was performed due to the cases of less FLT3-TKD mutation. C-KIT mutation accounts for 7.7% (6/78). Patients with C-KIT mutation had a higher percentage in abnormal karyotype (P < 0.05), and higher relapse rate (P < 0.05), and lower overall survival, whereas no statistic difference was found in sex, age, percentage of bone marrow blasts, peripheral blood cell count, complete remission rate (P > 0.05). It is concluded that the detection of NPM1, FLT3 and C-KIT mutations may contribute to guiding treatment and evaluating prognosis of patients with AML.

[Effect of TAK1 gene silencing on the apoptosis of Kasumi-1 cells induced by arsenic trioxide].

OBJECTIVE: To study the effect of transforming growth factor-ß activated kinase-1 (TAK1) gene silencing on the proliferation and apoptosis of Kasumi-1 cells induced by arsenic trioxide (As2O3). METHODS: Acute myeloid leukemia with t(8;21) cell line Kasumi-1 cells were treated with As2O3 or in combination with TAK1 siRNA interference technology. The experiment was divided into four groups: Kasumi-1 cells without any treatment, TAK1 specific siRNA transfection alone, Kasumi-1 cells treated with different concentration of As2O3, TAK1siRNA transfection combined with As2O3. CCK-8 was used to detect the cell viability. The expression of phosphorylated c-Jun N-terminal kinase (P-JNK) was determined by Western Blot. Cell apoptosis and growth were examined by morphological and colony formation assay. RESULTS: After Kasumi-1 cells were treated with As2O3, the rate of cell inhibition was concentration-dependent, and the 50% inhibitory concentration was 3.5 µmol/L. The highest expression level of P-JNK appeared in 30 minutes after cells were treated with As2O3. The apoptosis rates of Kasumi-1 cells without any treatment, TAK1 siRNA interference alone group, As2O3 alone group and the combined group were (5.02 ± 1.13)%, (6.18 ± 0.28)%, (48.33 ± 2.70)% and (86.07 ± 2.21)%; colony formation rates were (73.83 ± 2.78)%, (76.03 ± 1.46)%, (55.07 ± 1.50)% and (22.20 ± 1.15)%; apoptosis rate of TAK1 siRNA group and the untreated group has no significant difference (P = 0.052); colony formation rate between TAk1 siRNA group and the untreated group has no significant difference (P = 0.179), but the difference in other groups was significant (P = 0.000). CONCLUSION: Silencing the expression of TAK1 can enhance the anti-proliferative and pro-apoptotic effect of As2O3 on Kasumi-1 cells, and its mechanism may be through the TAK1 downstream JNK signal pathway.

Prognostic value of ADAM17 in human gastric cancer.

A disintegrin and metalloproteinase-17 (ADAM17, also named as tumor necrosis factor-alpha-converting enzyme) is a member of the ADAM family. Of all ADAMs, the strongest evidence for a role in malignancy exists for ADAM17. Especially, it has been demonstrated that ADAM17 expression was significantly increased in human gastric cancer. The aim of this study was to investigate the association between ADAM17 expression and the clinicopathological features of patients with gastric cancer. The expression of ADAM17 was detected by real-time quantitative RT-PCR in gastric cancer and adjacent non-cancerous tissues. In addition, ADAM17 expression was analyzed by immunohistochemistry in 220 clinicopathologically characterized gastric cancer cases. The expression levels of ADAM17 mRNA and protein in gastric cancer tissues were both significantly higher than those in non-cancerous gastric mucosa. In addition, positive expression of ADAM17 correlated with the degree of tumor differentiation, depth of invasion, lymph node metastases, distant metastases, and TNM stage (all P<0.05). Furthermore, multivariate analysis suggested that lymph node metastases, distant metastases, TNM stage, and ADAM17 expression were independent prognostic indicators for gastric cancer. Our data suggest for the first time that the increased expression of ADAM17 in gastric cancer is associated significantly with aggressive progression and poor prognosis. ADAM17 may be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of gastric cancer.

[Clinical significance of IDH1 and IDH2 mutations in patients with acute myeloid leukemia].

OBJECTIVE: To assess the frequencies and prognostic significance of the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations in acute myeloid leukemia (AML) and to explore their relevance to clinical, cytogenetic and molecular feature. METHODS: Genomic DNA from 96 newly diagnosed AML patients from Sep. 2009 to Jan. 2011 was screened by RT-PCR and sequencing for IDH1 and 1DH2 mutation. RESULTS: The prevalence of IDH1 (p. P127 and p. I130) and IDH2 mutations (p. R140) was 14.6% (14/ 96) and 2.17% (2/96) respectively. The IDH1 mutations of p. P127 and p. I130 were not reported so far in literature. Of 14 IDH1 mutation patients, 10 were with normal karyotype and the differences had statistical significance (P=0.021). Two patients with IDH2 mutation were also with normal karyotype. IDH2 mutations were in older patients at diagnosis. Patients with IDH mutation had higher white blood cell counts, lower platelet counts, expression of HLA-DR, CD34, CD33 and CD13, lower remission rate and higher relapse rate. CONCLUSION: IDH mutation is recurring genetic change in AML and indicates poor prognosis.

[Expression of NF-kappaB mRNA in acute myeloid leukemia].

This study was aimed to investigate the expression level of NF-kappaB mRNA in acute myeloid leukemia (AML) using real time fluorescence quantitative polymerase chain reduction (qPCR) detection and to explore the effect of NF-kappaB mRNA in pathogenesis of AML. The fresh bone marrow was collected from 60 newly diagnosed patients with AML, the total RNA was extracted by means of RTIzoL, the cDNA was synthesized, the expression of NF-kappaB mRNA was detected by qPCR using GAPDH as internal reference. 12 normal healthy persons were selected as controls. The results showed that the expression of NF-kappaB mRNA in patients with AML was higher than that in normal healthy persons with significant difference (p<0.05), the expression of NF-kappaB mRNA in patients with AML-M4 and -M5 were higher than that in patients with AML-M1, -M2 and -M3. It is concluded that the expression of NF-kappaB mRNA is higher in patients with AML. The up-regulation of NF-kappaB expression in patients with AML may play an important role in pathogenesis of AML.

[MEKK2 regulates the production of interleukin 2].

OBJECTIVE: To study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody. METHODS: The MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells. CONCLUSION: MEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.