Diagnostic significance of combined anti-extractable nuclear antigens antibody, anti-cardiolipin antibody and anti-ß2-glycoprotein 1 in systemic lupus erythematosus patients.

Objective: This study aimed to investigate the diagnostic value of a combination of anti-extractable nuclear antigens (anti-ENA) antibodies, anti-cardiolipin antibodies (ACA), and anti-ß2-glycoprotein 1 (anti-ß2 GPI) antibodies in patients with systemic lupus erythematosus (SLE). Methods: A total of 646 SLE patients diagnosed in our hospital between January 2020 and April 2023 were randomly selected as study subjects, while 2075 non-SLE subjects during the same period were selected as the control group. The levels of anti-extractable nuclear antigen (ENA) antibody, ACA, and anti-ß2 GPI antibodies were measured, and their diagnostic value for SLE was analyzed using binary logistic regression and receiver operating characteristic (ROC) analysis. Results: The rates of positive anti-RNP, anti-Sm, anti-Sjögren's syndrome (SS-A), and anti-SS-B antibodies in the SLE patient group were significantly higher than those in the control group, with all differences being statistically significant (P < 0.01). The rates of positive ACA, as well as the levels of ACA IgA, ACA IgG, and ACA IgM, were significantly higher in the SLE patients group compared to the control group, with statistically differences (P < 0.05). Similarly, the rates of positive anti-ß2 GPI antibodies, anti-ß2 GPI antibody IgA, IgG, and IgM, were significantly higher in the SLE patient group compared to the control group, with all differences being statistically significant (P < 0.01). Gender, age, positive anti-JO1 antibodies, positive anti-RNP antibodies, positive anti-SS-A antibodies, positive ACA, high level ACA IgG and ACA IgM, positive anti-ß2 GPI antibodies, and high level of anti-ß2 GPI antibody IgA were identified as independent risk factors for the development of SLE (P < 0.05). The combined use of age, sex, anti-ENA antibodies, ACA, and anti-ß2 GPI antibodies yielded a sensitivity of 82.12% (80.41%-83.71%) and a specificity of 80.03% (76.77%-82.93%) for the diagnosis of SLE. Conclusion: The combination of age, sex, anti-ENA antibodies, ACA, and anti-ß2 GPI antibodies shows high diagnostic value for SLE and holds potential for clinical application as an auxiliary diagnostic tool for SLE.

A combinatorial therapeutic approach to enhance FLT3-ITD AML treatment.

Internal tandem duplication mutations of the FMS-like tyrosine kinase-3 (FLT3-ITDs) occur in 25%-30% of patients with acute myeloid leukemia (AML) and are associated with dismal prognosis. Although FLT3 inhibitors have demonstrated initial clinical efficacy, the overall outcome of patients with FLT3-ITD AML remains poor, highlighting the urgency to develop more effective treatment strategies. In this study, we reveal that FLT3 inhibitors reduced protein stability of the anti-cancer protein p53, resulting in drug resistance. Blocking p53 degradation with proteasome inhibitors restores intracellular p53 protein levels and, in combination with FLT3-ITD inhibitors, shows superior therapeutic effects against FLT3-ITD AML in cells, mouse models, and patients. These data suggest that this combinatorial therapeutic approach may represent a promising strategy to target FLT3-ITD AML.

Case report: Circulating tumor DNA technology displays temporal and spatial heterogeneity in Waldenström macroglobulinemia during treatment with BTK inhibitors.

Background: Waldenström macroglobulinemia (WM) is a rare subtype of B-cell lymphoma. Rituximab-based combination therapy and Bruton's tyrosine kinase (BTK) inhibitors have greatly improved the prognosis of WM. Despite the high response rate and good tolerance of BTK inhibitors in treatment of WM, a proportion of patients still experience disease progression. Case presentation: We report a 55-year-old man with relapsed WM. The patient achieved partial remission after six courses of CHOP chemotherapy and multiple plasma exchanges in initial treatment. He was admitted to the hospital with abdominal distension, and was diagnosed with relapsed WM and subsequently started on zanubrutinib. Disease progression and histological transformation occurred during treatment. We performed liquid biopsies on transformed plasma, tumor tissue and ascites at the same time and found high consistency between ascites and tissues. Moreover, we detected resistance mutations of BTK inhibitors (BTK, PLCG2) in ascites that were not detected in plasma or tissue. Eventually, the patient died during the 15-month follow-up after relapse. Conclusion: We describe a rare case of WM transformation to DLCBCL treated with chemoimmunotherapy and BTK inhibition. We analyzed tumor DNA obtained at different anatomic sites and circulating tumor DNA (ctDNA) derived from plasma and ascites specimens, with apparent significant temporal and spatial heterogeneity. The case specifically highlights the clinical value of ctDNA of ascites supernatant from WM patients, which is a more convenient and relatively noninvasive method compared with traditional invasive tissue biopsy.

Effect of arsenic trioxide plus etoposide, solumedrol, high­dose cytarabine and cisplatin chemotherapy on the treatment of relapsed or ref7ractory ALK+ anaplastic large cell lymphoma.

It has been reported that arsenic trioxide (ATO) regulates lymphoma cell cycle, apoptosis, autophagy and mitochondrial activity, while it synergizes with other cytotoxic agents. In addition, ATO targets anaplastic lymphoma kinase (ALK)-fusion oncoprotein to repress anaplastic large cell lymphoma (ALCL). The current study aimed to investigate the efficacy and safety of ATO plus etoposide, solumedrol, high-dose cytarabine and cisplatin (ESHAP) chemotherapy compared with ESHAP chemotherapy alone in patients with relapsed or refractory (R/R) ALK+ ALCL. A total of 24 patients with R/R ALK+ ALCL were enrolled in the present study. Among them, 11 patients were treated with ATO plus ESHAP, while the remaining 13 patients received ESHAP chemotherapy alone. Subsequently, treatment response, event-free survival (EFS), overall survival (OS) and adverse event (AEs) rates were recorded. Both complete response (72.7% vs. 53.8%; P=0.423) and objective response (81.8% vs. 69.2%; P=0.649) rates were higher in the ATO plus ESHAP group compared with the ESHAP group. However, statistical significance was not reached. In addition, EFS was significantly prolonged (P=0.047), while OS was not significantly increased (P=0.261) in the ATO plus ESHAP group compared with the ESHAP group. More specifically, the 3-year accumulating EFS and OS rates were 59.7 and 77.1% in the ATO plus ESHAP group, respectively, and 13.8 and 59.8% in the ESHAP group, respectively. The majority of AEs, such as thrombocytopenia (81.8% vs. 46.2%; P=0.105), fever (81.8% vs. 46.2%; P=0.105) and dyspnea (36.4% vs. 15.4%; P=0.182), were more prevalent in the ATO plus ESHAP group compared with the ESHAP group. However, no statistical significance was observed. In conclusion, the current study indicated that ATO plus ESHAP chemotherapy could exert a superior efficacy compared with ESHAP chemotherapy alone in patients with R/R ALK+ ALCL.

Shegan Mahuang Decoction May Reduce Airway Inflammation in Neutrophilic Asthmatic Mice by Improving the Mitochondrial Function of Bronchoalveolar Lavage Fluid Exosomes.

Asthma is a common pulmonary disease mainly caused by the infiltration of neutrophils. There is a limit to the therapeutic effects of the available asthma drugs on neutrophilic asthma. Shegan Mahuang Decoction (SMD) is one of the representative traditional Chinese medicine (TCM) prescriptions for asthma, and it can effectively relieve the clinical symptoms of patients. However, the effect of SMD on the treatment of neutrophilic asthma remains unknown. In this study, a mouse model of neutrophilic asthma induced by lipopolysaccharide (LPS)/ovalbumin (OVA) was established, and the effect of a modified SMD prescription on the model was evaluated. After treatment, SMD was demonstrated to be therapeutically effective on asthmatic mice via airway resistance detection and lung pathology and was able to affect cytokine levels in vivo. Further experiments verified that SMD regulated the expression of mitochondrial function proteins in bronchoalveolar lavage fluid (BALF) exosomes. The results demonstrate that SMD confers a therapeutic effect on a neutrophilic asthma mouse model, and it may reduce neutrophil airway inflammation by regulating myeloid-derived regulatory cell (MDRC) function and airway exosome mitochondrial function.

MicroRNA-181b-5p insufficiency predicts treatment response failure risk and unfavorable event-free survival as well as overall survival in acute myeloid leukemia patients.

The present study aimed to explore the correlation of microRNA (miR)-181b-5p expression with treatment response and long-term prognosis in acute myeloid leukemia (AML) patients. miR-181b-5p was detected in the bone marrow of 84 AML patients before therapy. After induction therapy, the patients exhibiting complete remission (CR) were recorded. Next, event-free survival (EFS) and overall survival (OS) were calculated. miR-181b-5p had excellent potential to discriminate AML patients from healthy donors [area under the curve (AUC): 0.922, 95% confidence interval (CI): 0.873-0.971)]. In addition, miR-181b-5p expression was decreased in AML patients with the FLT3-ITD mutation (P=0.032) or WT1 mutation (P=0.017) when compared to AML patients without these genetic mutations. Meanwhile, miR-181b-5p expression was negatively correlated with the National Comprehensive Cancer Network (NCCN) risk classification of AML (P=0.036). Furthermore, miR-181b-5p expression was elevated in CR AML patients compared to non-CR AML patients (P=0.030). Moreover, higher miR-181b-5p expression was associated with favorable accumulating EFS (P=0.001) and OS (P=0.024). In addition, higher miR-181b-5p expression was independently associated with better EFS (hazard ratio: 0.698, P=0.012). In conclusion, miR-181b-5p insufficiency is associated with induction therapy response failure, unfavorable accumulating EFS and OS in AML patients.

Whole Transcriptome Data Analysis Reveals Prognostic Signature Genes for Overall Survival Prediction in Diffuse Large B Cell Lymphoma.

BACKGROUND: With the improvement of clinical treatment outcomes in diffuse large B cell lymphoma (DLBCL), the high rate of relapse in DLBCL patients is still an established barrier, as the therapeutic strategy selection based on potential targets remains unsatisfactory. Therefore, there is an urgent need in further exploration of prognostic biomarkers so as to improve the prognosis of DLBCL. METHODS: The univariable and multivariable Cox regression models were employed to screen out gene signatures for DLBCL overall survival (OS) prediction. The differential expression analysis was used to identify representative genes in high-risk and low-risk groups, respectively, where student t test and fold change were implemented. The functional difference between the high-risk and low-risk groups was identified by the gene set enrichment analysis. RESULTS: We conducted a systematic data analysis to screen the candidate genes significantly associated with OS of DLBCL in three NCBI Gene Expression Omnibus (GEO) datasets. To construct a prognostic model, five genes (CEBPA, CYP27A1, LST1, MREG, and TARP) were then screened and tested using the multivariable Cox model and the stepwise regression method. Kaplan-Meier curve confirmed the good predictive performance of this five-gene Cox model. Thereafter, the prognostic model and the expression levels of the five genes were validated by means of an independent dataset. High expression levels of these five genes were significantly associated with favorable prognosis in DLBCL, both in training and validation datasets. Additionally, further analysis revealed the independent value and superiority of this prognostic model in risk prediction. Functional enrichment analysis revealed some vital pathways responsible for unfavorable outcome and potential therapeutic targets in DLBCL. CONCLUSION: We developed a five-gene Cox model for the clinical outcome prediction of DLBCL patients. Meanwhile, potential drug selection using this model can help clinicians to improve the clinical practice for the benefit of patients.

Aggravation of airway inflammation in RSV-infected asthmatic mice following infection-induced alteration of gut microbiota.

BACKGROUND: This study aimed to confirm the relationship between asthma, respiratory syncytial virus (RSV) infection, and the gut environment by analyzing the alterations in the gut microbiota of RSV-infected asthmatic mice. METHODS: Twenty-four male BALB/c mice were randomly separated into a control group (CON), ovalbumin (OVA) group, and an OVA + RSV group, (n=8 mice/group). At the end of experiments, we evaluated the RSV-infected asthma model using Wright-Giemsa staining, histopathology and immunoglobulin E (IgE) level using enzyme-linked immunosorbent assays (ELISA). Next, airway hyper-responsiveness (AHR) was measured using Buxco's modular and invasive system. Furthermore, IL cytokine expression were measured using ELISA. Moreover, feces were collected for 16S ribosome RNA (16S rRNA) sequencing and data analysis. RESULTS: We observed that the total BAL fluid lung cells in the OVA + RSV group was significantly higher than other group. We revealed that the inflammatory infiltration, edema, and collagen hyperplasia were more severe in the OVA + RSV group. The AHR of RSV-infected mice was aggravated compared with the other groups, (P<0.05 and P<0.01). We observed a higher expression of IgE, interleukin (IL)-5, IL-13, IL-25, and IL-33 levels in mice from the OVA and OVA + RSV groups (P<0.05 and P<0.01). The associations between Prevotellaceae_UCG_001, which is positive, and IgE, IL-13, IL-33 (P<0.001), IL-5 (P<0.01), and IL-25 (P<0.05) were highly significant. Lachnospiraceae_NK4A136_group is also positive and was significantly associated with IgE and IL-33. Helicobacter and Uncultured_Bacteroidales_bacteriumare_group, which are negative, were associated with IL-25 (P<0.05). CONCLUSIONS: Our results indicated that RSV-infected mice with asthma may have changes in the gut microbiota's major components and may influence the mutual relationship between the core operational taxonomic units (OTUs) and IgE as well as inflammatory cytokines.

Circular RNA profile of acute myeloid leukaemia indicates circular RNA annexin A2 as a potential biomarker and therapeutic target for acute myeloid leukaemia.

This study was to investigate the circular RNA (circRNA) expression profile and the potential circRNAs as biomarkers and therapeutic targets for acute myeloid leukaemia (AML). CircRNA expression profile in bone marrow mononuclear cells from 5 AML patients and 5 healthy donor controls (HCs) was evaluated by microarray. Then, 10 candidate circRNAs (top 5 upregulated and top 5 downregulated) from microarray were validated by RT-qPCR in 130 AML patients and 50 HCs. Finally, the effects of circRNA annexin A2 (circ-ANXA2) knockdown on cell proliferation, apoptosis, chemosensitivity to cytarabine, daunorbicin and potential target microRNAs were assessed in THP-1 and KG-1 cells. By microarray, 173 upregulated and 181 downregulated circRNAs were found in AML patients than HCs, and these circRNAs were found in AML patients compared with HCs, and these circRNAs were implicated in AML-related pathways such as ErbB and EGFR pathways. By RT-qPCR, 9 of 10 candidate circRNAs (including circ-RPS6KB1, circ-CSMD2, circ-PTK2, circ-ANXA2, circ-PWP2, circ-RBM5, circ-ZZEF1, circ-GSK3B and circ-FOXP1) were dysregulated in AML patients compared with HCs. Circ-ANXA2 correlated with higher disease risk, poor risk stratification, lower complete remission level, shorter event-free survival and overall survival in AML. In cellular experiments, circ-ANXA2 was upregulated in AML cell lines, and its knockdown suppressed proliferation, enhanced apoptosis of THP-1 and KG-1 cells and increased their chemosensitivity to cytarabine and daunorbicin. Additionally, circ-ANXA2 knockdown promoted microRNA (miR)-23a-5p and miR-503-3p expression in THP-1 and KG-1 cells. In conclusion, our findings provide a macroscopic view of the circRNA expression profile in AML, and demonstrate that circ-ANXA2 may be a potential biomarker and therapeutic target for AML.

Efficacy and safety of combined immunosuppressive therapy plus umbilical cord blood infusion in severe aplastic anemia patients: A cohort study.

The present study aimed to evaluate the efficacy and safety of combined immunosuppressive therapy (IST) plus umbilical cord blood infusion (UCBI) in severe aplastic anemia (SAA) patients. A total of 68 patients with SAA were enrolled in the current prospective cohort study and divided into the IST (n=35; positive control) and IST+UCBI (n=33; experimental) groups according to the treatment conditions. Patients in the IST group were treated with rabbit antithymocyte globulin (r-ATG) at a dose of 2.5 mg/kg through intravenous infusion once a day for five days. This was combined with oral cyclosporine A (CsA) at a dose of 3-5 mg/kg twice a day for 2 years. Patients in the IST+UBCI group were treated with r-ATG and CsA at the same doses and frequencies as the IST group plus one UCBI 1 day after the final treatment with r-ATG. At 6 months post treatment, the complete response and overall response rate (ORR) of the IST+UCBI group were markedly higher compared with those in the IST group. Furthermore, patients in the IST+UCBI group achieved absolute neutrophil count (ANC) and platelet count responses more rapidly as compared with the IST group. However, no difference in the hemoglobin (Hb) response was identified between the two groups. In addition, SAA patients achieved responses in the ANC and platelet count more rapidly in comparison with very severe aplastic anemia (VSAA) patients, while the number of days to Hb responses were similar in the SAA and VSAA patients. Multivariate logistic regression analysis also revealed that IST+UCBI treatment was an independent predicting factor for patients achieving complete response or partial response, whereas VSAA was an independent predictor of a worse ORR. Platelet and reticulocyte were also independent predicting factors. Finally, the survival of patients was similar between the groups, and no difference in the safety of the treatment was observed. In conclusion, combined IST plus UCBI treatment may be applied as an effective and safe therapy for SAA patients.

[Treatment of Ph-adult acute lymphoblastic leukemia patients with Tongji-96 regimen].

OBJECTIVE: To investigate the efficacy and side effects of the consecutive chemotherapeutic protocol, Tongji-96, for adult patients with Philadelphia chromosome negative acute lymphoblastic leukemia (Ph-aALL). METHODS: A retrospective analysis was conducted on 95 cases of Ph-aALL patients treated between January 2004 and December 2012 with Tongji-96 regimen in Tongji hospital, Shanghai. RESULTS: Among these 95 patients, the overall complete remission (CR) rate was 92.6%, 7-year overall survival (OS) and event-free survival (EFS) rates were (39.3±5.9)% and (31.5±5.3)%, respectively, with the median survival of 28 months. Based on multivariable COX proportional hazards regression model analysis, patients with the poor karyotype and failed to achieve CR after first course induction therapy had a higher risk of mortality compared to those who had good or normal cytogenetics and achieved CR after 1 course of induction treatment [the risk ratios (RR) were 3.380 (95% CI 1.530-7.463, P=0.003) and 3.005 (95% CI 1.522-5.933, P=0.002)，respectively]. By means of Kaplan-Meier analysis and Log-rank test，patients aged less than 60 years and successively achieved CR after first induction therapy had more favorable 7-year OS and EFS rates. Patients with normal karyotype and hyperdiploidy had significantly higher 2-year OS and EFS rates compared with those with complex karyotype, t(4;11) translocation and other karyotypes. CONCLUSION: Age (60 years as the cut-off)，treatment courses for achieving CR and cytogenetics were predictive factors for the prognosis of Ph-aALL from this retrospective study. As a comprehensive and sequential therapy protocol, Tongji-96 regimen was proved to obtain long-term survival, reduce risks for relapse and improve outcomes for part Ph-aALL patients.

Retrospective comparison of fludarabine in combination with intermediate-dose cytarabine versus high-dose cytarabine as consolidation therapies for acute myeloid leukemia.

This retrospective study compared efficacy and safety of fludarabine combined with intermediate-dose cytarabine (FA regimen) versus high-dose cytarabine (HiDAC regimen) as consolidation therapy in acute myeloid leukemia (AML) patients who achieved complete remission. Disease-free survival (DFS) and overall survival (OS) based on age (≥ 60, <60 years) and cytogenetics were evaluated from data between January 2005 and March 2013. Total 82 patients (FA, n = 45; HiDAC, n = 37; 14-65 years) were evaluated. Five-year DFS was 32.0% and 36.2% for FA and HiDAC groups, respectively (P = 0.729), and 5-year OS was 39.5% and 47.8% (P = 0.568), respectively. Among older patients (≥ 60 years), 3-year DFS was 26.0% for FA group and 12.5% for HiDAC group (P = 0.032), and 3-year OS was 34.6% and 12.5%, respectively (P = 0.026). In FA group, hematological toxicities were significantly lower. FA regimen was as effective as HiDAC regimen in patients with good/intermediate cytogenetics and significantly improved DFS and OS in older patients.

Genetic inhibition of vascular endothelial growth factor receptor-1 significantly inhibits the migration and proliferation of leukemia cells and increases their sensitivity to chemotherapy.

Little is known about the role of vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1) in acute leukemia. In this study, using real-time PCR and ELISA, we found that VEGF and VEGFR-1 are highly expressed in U937 leukemia cells and primary leukemia cells (M4/M5 subtypes), which are associated with an increased migration rate and extramedullary disease. In order to elucidate the role of VEGFR-1 in acute leukemia, we used a lentivirus-mediated shRNA expression system to specifically inhibit VEGFR-1 expression in the U937 cell line. In addition, a series of in vitro experiments were conducted, including cell proliferation and migration assays and drug treatments. Our results showed that shRNA reduced the proliferation and migration of U937 cells. RNA interference targeting VEGFR-1 in combination with bevacizumab did not exert synergistic antitumor effects. However, shRNA enhanced the sensitivity of the U937 cells to cytarabine by decreasing the IC50 of cytarabine, reducing the number of cells in the S phase and suppressing the expression of the survivin gene. Taken together, these results suggest that VEGFR-1 interference may serve as a novel antitumor therapeutic strategy for the treatment of leukemia.

Sequential arabinosylcytosin with or without fludarabine in paracmastic patients with acute myeloid leukemia.

The purpose of this study is to assess how fludarabine (Fa) influences arabinosylcytosin's (Ara-C) mode of action. Plasma, cerebrospinal and urine samples were withdrawn from two study groups at specific time points and analyzed by HPLC. Group A was treated with Ara-C only whereas Group B was treated with Fa+Ara-C. The two study groups are all undergoing complete remission (CR). The Ara-C dose for Group A was 3g/m2 x 2, and the AUC(0-4) was 5.131 +/- 0.936. The Ara-C dose for Group B was 2g/m2 x 2, and the AUC(0-4) was 12.245 +/- 3.863. The AUC(0-4) for Group B is more than twice the AUC(0-4) for Group A, and these results indicate that Fa conduces a synergistic increase in the concentration and AUC of Ara-C in plasma and in cerebrospinal fluid. The pharmacokinetics between the different dose treatments was statistically different (P = 0.016). The differences in the ratios of C(Ara-u) to C(Ara-C), and in the Tmax between Groups A and B could indicate whether or not Ara-C combined with Fa. Although Group B demonstrates a higher AUC(0-4) with lower doses of Ara-C (2 g/m2 x 2), the adverse drug reaction (ADR) and bone inhibition were not more pronounced in Group B compared to Group A. These results are based on a limited number of case studies, hence, additional studies are necessary to support and prove this hypothesis.

[Preliminary study of biological mechanism and apoptotic effect induced by YM155 in SUDHL-4 cell line].

OBJECTIVE: To explore the apoptotic effect of follicular lymphoma and related mechanism induced by YM155 in vitro and provide laboratory rationales for the clinical treatment of follicular of lymphoma with YM155 in the future. METHODS: SUDHL-4 cells were cultured to logarithmic phase and transferred to 96-well plates. There were a series of YM155 concentration gradients: 100, 10, 1, 0.1 and 0 ng/ml and cultured for 24, 48 and 72 h. After the addition of CCK-8 reagent for 2 h at each time point, optical density values were obtained from the cell growth inhibition curves depending on time and drug concentration and the half growth inhibition concentration (IC(50)) values calculated. SUDHL-4 cells were co-cultured with YM155 (1 ng/ml) for 0, 24, 48 and 72 h respectively. Then flow cytometry (FCM) was used to detect apoptosis. SUDHL-4 cell line was treated with YM155 for 24 and 48 h to extract the total RNA. The mRNA expressions of bcl-2, bcl-xl, bid, bax and survivin gene at the time point of 48 h and the survivin mRNA expression at 24 h were detected by reverse transcription-PCR (RT-PCR). The protein expressions of survivin, caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected at each time point with Western blot respectively. RESULTS: SUDHL-4 cell line showed significant growth inhibition effect depending on time and dose. And the 24, 48, 72 h IC(50) was 6.1, 2.7 and 1.2 ng/ml respectively. SUDHL-4 cells stained AnnexinV-FITC and PI examined by FCM demonstrated that the proportion of AnnexinV-FITC positive cells gradually increased with time (17.3% ± 2.1%, 35.7% ± 3.3%, 54.6% ± 4.3% vs 2.1% ± 0.3%, all P < 0.05). And the results of real-time fluorescent PCR proved that YM155 decreased the expression of survivin gene obviously (24 h: 0.72 ± 0.02, 48 h: 0.56 ± 0.01 vs 1.00, both P < 0.05) but had little effects on the gene expressions of bax, bid, bcl-2 and bcl-xl. The Western blot results further confirmed that the protein expressions of survivin and caspase-3 decreased with time while caspase-9 and cleaved caspase-9 showed no obvious changes. But cleaved caspase-3 increased significantly. CONCLUSIONS: YM155 displays significant apoptotic effects in SUDHL-4 cell lines. The mechanism may be the direct activation of caspase-3 through the down-regulation of survivin. And the apoptotic pathway is probably not regulated by bcl-2 family.

[The effect of apoptosis induced by IL-21 in SUDHL-4 cell line and its mechanism].

OBJECTIVE: To investigate the apoptosis effect of diffuse large B-cell lymphoma cell line (DLBCL) SUDHL-4 induced by IL-21 and its related mechanism. METHODS: SUDHL-4 cells were treated with IL-21 at different concentration (1000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) for 24 h, 48 h, 72 h, respectively. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The cell growth curves were drawn and half inhibitory concentration (IC(50)) values were calculated. The cell apoptosis were detected by flow cytometry (FCM), the expression of the caspase-9, caspase-3, cleaved caspase-3, Bcl-2, Bcl-XL, Bid, Bax and c-myc protein in SUDHL-4 cells treated with IL-21 by western blot, the mRNA expression of Bcl-2, Bcl-XL, Bid, Bax, c-myc by Survivin gene with RT-PCR. RESULTS: IL-21 markedly inhibited SUDHL-4 cell growth in a time- and dose-dependent manner. The 48 hIC(50) was 140.9ng/ml; The FCM showed that the apoptosis proportion of SUDHL-4 cells treated with 100 ng/ml of IL-21 apoptosis (AnnexinV-FITC(+) positive cells) gradually increased (48 h: 19.7 ± 2.3%). The protein expression of caspase-9, caspase-3, Bcl-2 and Bcl-XL decreased in a time-dependent manner. The Bax and c-myc protein markedly increased, but the Bid protein level did not change. IL-21 up regulated c-myc and Bax gene expression, however down regulated Bcl-2 and BCL-XL gene expression, but the gene expression of Bid and Survivin hadn't been changed significantly. CONCLUSIONS: IL-21 can inhibit proliferation and induce apoptosis of SUDHL-4 cell. The mechanism may involve in endogenous mitochondrial pathway mediated by the c-myc and the Bcl-2 genes.

[Silencing of survivin gene in Jeko-1 cell line with small interfering RNA].

This study was aimed to investigate the impact of specific siRNA on survivin gene in transfected lymphoma cell line and provide experimental evidences for future treatment of mantle cell lymphoma. The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into Jeko-1 that showed high survivin expression in mRNA level. The levels of survivin mRNA and protein expression were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. The apoptosis effect was examined by calculating the ratio of Annexin V-FITC/PI positive cells using flow cytometry. The inhibition of cell proliferation was assayed with CCK-8 reagent after transfection. The results showed that expression of survivin mRNA was markedly suppressed by the siRNA. The relative expression levels were 0.49 ± 0.03, 0.38 ± 0.02 and 0.17 ± 0.02 at time points of 24, 48 and 72 h respectively, compared with the control group; the inhibitive rates of cell proliferation were (31.2 ± 2.1)%, (43.3 ± 3.4)% and (52.6 ± 2.5)%; the apoptotic rates of cells were (6.3 ± 0.5)%, (13.5 ± 1.1)% and (23.6 ± 1.6)% respectively; survivin protein expression levels were gradually reduced. It is concluded that the siRNA targeting survivin down-regulates the expressions of survivin mRNA and protein evidently. The siRNA of survivin displays the potent ability to inhibit the proliferation of lymphoma cell line Jeko-1; survivin may become a potential molecular target for the therapy of lymphoma in the future.

[Study of hypermethylation of SOCS gene in typical myeloproliferative disease].

OBJECTIVE: To investigate the clinical role of hypermethylation of suppressor of cytokine signaling (SOCS) on typical myeloproliferative disease (MPD) patients and its mechanism. METHODS: Methylation specific PCR was used to detect SOCS1, 2, 3 methylation, direct DNA sequencing was performed to detect JAK2V617F mutation, real-time fluorescence quantitative PCR were applied to evaluate transcriptional activity of SOCS1, 2, 3. RESULTS: Among 100 MPD patients, hypermethylation of SOCS1 was detected in 27 (27%), hypermethylation of SOCS2 in 9 (9%), hypermethylation of SOCS3 in 34 (34%); JAK2V617F mutation in 64 (64%). Hypermethylation of SOCS1, 3 greatly inhibited gene expression compared with unmethylated ones (P < 0.05). Presence of JAK2V617F mutation markedly down-regulated SOCS1, 3 gene mRNA expression compared with wild JAK2V617F (P < 0.05). CONCLUSION: Hypermethylation of SOCS1, 3 and JAK2V617F mutation exist in MPD, which inhibited SOCS1, 3 gene expression. SOCS hypermethylation and JAK2V617F mutation can activate JAK-STAT signaling pathways, these observations may provide a potential therapeutic direction.

[Effect of hypoxia on HIF-1α and its sumoylation in Jurkat cells].

OBJECTIVE: To investigate the effects of hypoxia on sumoylation of HIF-1α, as well as transcriptional activity and protein stability of HIF-1α in Jurkat cells, and explore its effect and significance on modulating the transcriptional activity of down stream target gene. METHODS: CoCl(2) was used as a chemical inducer to simulate the hypoxia environment. Real-time fluorescence quantitative PCR and western blot were used to evaluate transcriptional activity and protein stability of HIF-1α, levels of SUMO-1 and SENP1 protein, and gene transcripts of VEGF, mdr1, mdr3, Mcl-1 and survivin respectively. RESULTS: After 4 h, 8 h, 16 h, 24 h and 72 h after hypoxia induction, the gene transcripts of HIF-1α were 0.79 ± 0.19, 2.65 ± 2.05, 4.19 ± 4.72, 2.77 ± 3.37, 0.09 ± 0.05 and 0.69 ± 0.55-fold (P > 0.01) of that of 0h in Jurkat cells, respectively, while the protein stability increased first, then decreased (P < 0.01). SENP-1 protein up-regulated first, then down-regulated, and the SUMO-1 protein changed in an opposite trend. Excepting for survivin gene, transcriptional activities of VEGF, mdr1, mdr3, and Mcl-1 were affected by the stability of HIF-1α protein. CONCLUSION: Hypoxia induces changes in SENP-1 expression, which increases the stability of HIF-1α by decreasing the sumoylation of HIF-1α and affects biological process by regulating the transcriptional activities of VEGF, mdr1, mdr3 and Mcl-1 gene.

[Effect of VEGFR1 gene silencing by shRNA on proliferation, migration and apoptosis of U937 line].

OBJECTIVE: To investigate the effects of lentivirus-mediated RNA interference targeting vascular endothelial growth factor receptor 1 (VEGFR1) gene on the proliferation, migration and apoptosis of leukemic cell line U937. METHODS: Short hairpin RNAs (shRNA) targeting VEGFR-1 was synthesized and cloned into pRNAT-U6.2 lentiviral vector. The expression vectors were transfected into 293T cell line to produce packaged lentivirus. After infected with the packaged lentivirus, the expression of VEGFR-1 gene of U937 cells at mRNA and protein level was detected by real-time PCR and Western blot. VEGF production by the cells was determined by ELISA. Cell proliferation and survival under regular culture and in the presence of cytarabine (Ara-C) was determined by CCK-8 assay. Migration assays were performed by 5 µm pore transwell inserts. RESULTS: The lentiviral shRNA vector targeting VEGFR-1 was successfully constructed and transfected into U937 cells. The shRNA vector effectively inhibited the expression of VEGFR-1 gene in U937 cell line at mRNA and protein levels. As compared to that of the control, the proliferation rate of U937-shVEGFR-1 cells reduced; The VEGF production and migrated cell number of U937-shVEGFR-1 cells decreased dramatically. After treated with Ara-C, the inhibition rate and apoptotic rate of U937-shVEGFR-1 cells increased significantly. The number of migrated cells in the KD group under regular culture and in the presence of VEGF was markedly lower than that in the NC group and CON group. Bevacizumab could decrease the number of migrated cells in the NC group and CON group, but could not in the KD group. CONCLUSIONS: Lentivirus-mediated RNA interference targeting VEGFR1 gene reduces the proliferation, migration of U937 cell line and enhances its sensitivity to Ara-C.