[Clinical Observation of Neutropenia Patients with Hematonosis Treated with Polymyxin B Sulfate].

OBJECTIVE: To observe the clinical efficacy and safety of polymyxin B sulfate in febrile neutropenia patients with hematonosis. METHODS: Clinical data of 50 patients in the department of hematology, Fujian Medical University Union Hospital from October 2019 to September 2020 were collected. All the patients developed febrile neutropenia after chemotherapy or hematopoietic stem cell transplantation. According to the results of drug susceptible test, polymyxin B sulfate was administrated mainly when the empirical antimicrobial treatments was poor and the pathogenic microbes test was positive. RESULTS: A total of 85 times of infection occurred in 50 patients. The infection sites were lung, blood flow, intestinal tract, oral cavity, perianal, soft tissue and nasal cavity. Gram negative bacteria was the main pathogenic microbe. After administration of polymyxin B sulfate when the etiology was confirmed, the total effective rate was 68%, especially the effective rate increased significantly after more than 7 days of polymyxin B sulfate treatment. Also, 24% and 8% of the patients were discharged automatically and died respectively. The effective rate of patients receiving carbapenem antibiotics changed to polymyxin B sulfate within 14 or 7 days was 80% and 70.6%, respectively, while the effective rate of patients who changed after 2 weeks was only 33.3%. The effective rate of patients receiving tigecycline changed to polymyxin B sulfate within 14 or 7 days was 80% and 66.7%, respectively. The incidence of adverse reactions of polymyxin B sulfate was low, most of which were mild, and only one patient occurred rhabdomyolysis. CONCLUSION: Polymyxin B sulfate has good clinical efficacy and safety in febrile neutropenia patients with hematonosis.

[Expression and Clinical Significance of Heat Shock Protein 90 in Multiple Myeloma Patients].

OBJECTIVE: To investigate the expression of HSP90 in bone marrow samples of multiple myeloma (MM) patients and explore its clinical significance. METHODS: Maxvision immunohistochemistry technique was used to detect the protein expression level of HSP90 76 MM patients and 29 normal healthy donors. The clinical characteristics of the patients were collected, and the correlation between the HSP90 expression and the clinical characteristics was analyzed. RESULTS: The count of MM patients with positive HSP90 protein was significantly higher than that of normal healthy donor, and there were no significant correlation between HSP90 expression and age, sex, hemoglobin (Hb), creatinine (CREA), blood calcium, lactate dehydrogenase (LDH), bone marrow plasma cell proportion and MM subtypes (P>0.05), but HSP90 expression was correlated with ß2-microglobulin (ß2-MG) and ISS stage (P<0.05). The survival time was lower in MM patients with high expression HSP90 as compared with low expression HSP90 MM patients. CONCLUSION: HSP90 protein was over-expressed in MM patients, and was correlated with ß2-microglobulin, ISS stage and OS of MM patients.

SENP2 exerts an anti­tumor effect on chronic lymphocytic leukemia cells through the inhibition of the Notch and NF­κB signaling pathways.

Chronic lymphocytic leukemia (CLL) is one of the most often diagnosed hematological malignant tumors in the Western world and a type of inert B­cell lymphoma that commonly attacks the elderly. Small ubiquitin related modifier (SUMO)­specific protease 2 (SENP2) can act as a suppressor in various types of cancer by regulating the stability of ß­catenin to affect the Notch signaling pathway; however, it has a low expression level in CLL cells. In this study, we firstly used western blot analysis and RT­qPCR to detect the protein and mRNA expression levels of SENP2 in the peripheral blood of patients with CLL and healthy volunteers. Secondly, we overexpressed or knocked down the expression of SENP2 in CLL cells and then determined the cell invasive and chemotactic ability in a Transwell assay and chemotaxis assay. We examined the sensitivity of the cells to cytarabine and dexamethasone via a CCK­8 assay and determined the cell apoptotic condition and the expression of the Notch signaling pathway using flow cytometry and western blot analysis. The results demonstrated that the patients with CLL had relatively low expression levels of SENP2. The overexpression of SENP2 in the CLL cells decreased their invasive and proliferative ability, as well as their chemotactic response and enhanced their sensitivity to cytarabine and dexamethasone, while it promoted cell apoptosis. The silencing of SENP2 in the CLL cells generally produced the opposite results. We thus hypothesized that the overexpression of SENP2 downregulated ß­catenin expression, thus inhibiting the Notch signaling pathway in CLL cells. Moreover, the nuclear factor (NF)­κB signaling pathway was also regulated by the overexpression of SENP2. On the whole, the findings of this study indicate tha SENP2 can act as a tumor suppressor in CLL cells, and may thus prove to be a novel target for CLL treatment in clinical practice.

[Clinical Significance of Corrected Serum Calcium in 320 Patients with Multiple Myeloma].

OBJECTIVE: To analyze the clinical significance of corrected serum calcium(CSCa) in patients with multiple myeloma. METHODS: The serum calcium levels of 320 patients with initial multiple myeloma were measured and corrected by serum albumin and its levels measured simultaneously. The differences of serum calcium levels were analyzed before and after the correction by serum albumin. RESULTS: There was a significant difference between serum calcium and CSCa in MM patients (2.34±0.15 vs 2.6±0.17 mmol/L). The constituent ratio of patients with hypercalcemia was from 11.3% to 23.1% after correction, the MM patients with hypocalcemia was decreased from 42.8% to 7.8% after correction, and the patients with normal calcium level were increased. There was a significant difference between serum calcium level and CSCa in I, II, III stages of MM patients respectively(P<0.05). In the 320 patients, the incidence of anemia was 80%, renal failure was 20.9%, and myeloma bone disease was 68.8%. Calcium concentration in both anemia and renal insufficiency was higher than the normal group, and the difference was more significant after correction. In 220 cases of MM receiving chemotherapy, the median progression-free survival (PFS) was 15 months, and overall survival(OS) time was 20 months. The PFS and OS time of the patients with hypercalcemia were shortened, and the difference was very significant after correction(P<0.01). CONCLUSION: Corrected serum calcium can more sensitively to reflect the diseases serious extent, thus indicating prognosis has better effect.

miR-203 inhibits cell growth and regulates G1/S transition by targeting Bmi-1 in myeloma cells.

The oncogene B-cell-specific Moloney murine leukemia virus insertion site­1 (Bmi­1) is overexpressed in multiple myeloma (MM). Our previous study demonstrated that Bmi­1 silencing sensitized MM cells to bortezomib. Translational regulation has emerged as a prominent underlying mechanism of Bmi­1 regulation, particularly via microRNA targeting. The present study determined that Bmi­1 may be directly targeted by miR­203 using a luciferase assay. In addition, enforced expression of miR-203 led to significant downregulation of Bmi­1 protein and mRNA expression levels. Furthermore, restoration of miR-203 significantly inhibited cell growth and G1/S transition in MM cells. miR­203 was downregulated in MM patients, and a negative correlation between the expression of miR­203 and Bmi­1 was observed. The results of the present study indicated that miR­203 exerts growth­inhibiting effects in MM through the suppression of Bmi­1 expression. In conclusion, the present study demonstrated that Bmi­1 is a direct functional target of miR-203 in MM.

miR-299-5p promotes cell growth and regulates G1/S transition by targeting p21Cip1/Waf1 in acute promyelocytic leukemia.

MicroRNAs (miRs) are often located in genomic breakpoint regions and are hypothesized to be important regulators involved in the regulation of critical cell processes, including cell apoptosis, proliferation and differentiation. miR-299 has been reported to be upregulated in acute promyelocytic leukemia (APL); however, the function and mechanistic role of miR-299 in APL remains unknown. The present study demonstrated mir-299 significantly induced cell growth and cell cycle progression at the G1/S transition in APL cells. Notably, the present study revealed that miR-299-5p induces these effects, whereas miR-299-3p does not. Additional studies demonstrated that in APL cells the tumor suppressor p21Cip1/Waf1 is a downstream target of miR-299; miR-299 binds directly to the 3' untranslated region of p21Cip1/Waf1, and reduces protein, but not mRNA, levels of p21Cip1/Waf1. The present findings demonstrate that miR-299 exerts growth-promoting effects in APL cells through the suppression of p21Cip1/Waf1. Overall, the present study demonstrates that p21Cip1/Waf1 is a direct functional target of miR-299 in APL.

[Expression and Clinical Significance of Bmi-1 and P14 in Extranodal NK/T-cell Lymphoma].

OBJECTIVE: This study was aimed to investigate the expression and clinical significance of Bmi-1 and P14 in extranodal NK/T-cell lymphoma (ENKTCL) tissue. METHODS: Maxvision immunohistochemistry technique was used to detect the expression level of Bmi-1 and P14 in the tissues of 21 patients with ENKTCL and 11 normal lymph nodes. The correlation of Bmi-1 or P14 expression with the clinical features and the correlation between Bmi-1 and P14 expression were analyzed. RESULTS: The expression of Bmi-1 protein was higher in tissues of ENKTCL than that in tissues of lymph nodes, and the Bmi-1 expression levels did not correlate with patients' sex, age, lactate dehydrogenase (LDH), International Prognostic Index (IPI) scores and B symptoms (P > 0.05), except for clinical stage (P < 0.05). The P14 protein expression level was lower in ENKTCL tissues than in normal lymph node tissues, which did not correlate with age, sex, LDH, IPI scores, clinical stage and B symptoms. Correlation test showed a negative correlation between Bmi-1 and P14 (r = -0.472, P = 0.031). CONCLUSION: Bmi-1 protein over-expresses in ENKTCL tissues that may display a negative-regulation effect on P14 in the genesis and progress of ENKTCL.

[Growth Inhibition of Multiple Myeloma Cells Caused by MicroRNA-15a and Its Mechanisms].

OBJECTIVE: To explore the effect and possible mechanisms of miR-15a on growth of multiple myeloma(MM) cells. METHODS: MM cell lines (U266 and RPMI8226) were transfected by lentiviral particles. MM stable cell lines were selected and collected by flow cytometry (FCM). Proliferation of MM cells before and after miR-15a high expression was detected by CCK-8 method. Apoptosis of MM cells before and after miR-15a high expression was detected by AO/EB dying, Hoechst 33258 dying and FCM, respectively. Cell cycle of MM cells before and after miR-15a high expression was detected by FCM. The expressions of miR-15a, BMI-1 and BCL-2 mRNA of MM cells before and after miR-15a high expression were detected by real-time PCR. The expressions of BMI-1 protein of MM cells before and after miR-15a high expression were detected by Western blot. RESULTS: MM stable cell lines with miR-15a high expression was acquired. CCK-8 result showed that high expression of miR-15a could inhibit growth of MM cells (U266 and RPMI8226). AO/EB dying, Hoechst 33258 dying and FCM testing results showed that high expression of miR-15a could significantly induce apoptosis of MM cells (U266 and RPMI8226). The apoptosis rates of U266 and RPMI8226 cells in high expression group and control group were 90.52% vs 37.08% and 59.40% vs 44.17%, respectively. Meanwhile, FCM testing results showed that high expression of miR-15a could induce G1 arrest of MM cells (U266 and RPMI8226), which proportion of G1 phase were 41.50%±0.64%, 45.31%±0.77%, respectively. Real-time PCR results showed that during the growth inhibition process of MM cells caused by miR-15a high expression, the expression of BCL-2 mRNA decreased, but there was no significant changes in the expression of BMI-1 mRNA, while the expression of BMI-1 protein decreased significantly. CONCLUSION: High expression of miR-15a can induce cell cycle arrest and apoptosis of MM cells, then inhibit their growth. The mechanisms may be related with the negative regulation of BMI-1 and BCL-2 genes in post-transcription level caused by miR-15a.

Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia.

NOTCH1 mutations occur in approximately 10% of patients with chronic lymphocytic leukemia (CLL). However, the relationship between the genetic aberrations and tumor cell drug resistance or disease progression remains unclear. Frameshift deletions were detected by gene sequencing in the NOTCH1 PEST domain in three naive CLL patients. These mutations were associated with chromosomal abnormalities including trisomy 12 or 13q deletion. Of note, one of the patients developed Richter's transformation during FCR treatment. Immunofluorescent and western blot analyses revealed a markedly higher intracellular domain of NOTCH (ICN) expression in the mutated cells compared with their unmutated counterparts and normal CD19+ B lymphocytes (P<0.01 and P<0.001, respectively). In addition, strong DNA-κB binding activities were observed in the mutant cells by gel shift assays. RT-PCR analysis revealed elevated RelA mRNA expression in the mutant cells, while RelB levels were variable. Reduced levels of RelA and RelB mRNA were observed in unmutated CLL and normal B cells. Compared to unmutated CLL and normal B cells, increased apoptosis occurred in the mutant cells in the presence of GSI (ICN inhibitor) and PDTC (NF-κB inhibitor), particularly under the synergistic effects of the two drugs (P=0.03). Moreover, IKKα and IKKß, the active components in the NF-κB pathway, were markedly inhibited following prolonged treatment with GSI and PDTC. These results suggested that NOTCH1 mutations constitutively activate the NF-κB signaling pathway in CLL, which is likely related to ICN overexpression, indicating NOTCH1 and NF-κB as potential therapeutic targets in the treatment of CLL.

[Anti-leukemia mechanism of miR-17 and miR-20a silencing mediated by miRNA sponge].

This study was aimed to quantitatively detect the expression levels of pre-miR-17 and pre-miR-20a in acute leukemia patients and eight kinds of leukemia cell lines, and to investigate the anti-leukemia mechanism of miR-17 and miR-20a silence mediated by miRNA Sponge. Quantitative real-time PCR was used to detect the mRNA expression levels of pre-miR-17 and pre-miR-20a in patients with various types of leukemia and leukemia cell lines. The Jurkat cells over-expressing miR-17 and miR-20a were transfected with recombinant lentivirus-transfecting units targeted at miR-17 and miR-20a plus 6 µg/ml of polybrene. Then the proliferation ability and cell cycle of Jurkat cells was evaluated by CCK-8 and flow cytometry respectively. The results showed that the expression level of pre-miR-17 and pre-miR-20a in all leukemia patients was significantly higher than that in normal group(P < 0.05), the expression of pre-miR-17 and pre-miR-20a in acute lymphoid leukemia was significantly higher than that in acute myeloid leukemia(P < 0.05), and the pre-miR-17 and pre-miR-20a expression level did not correlate significantly with high white blood cell count>20.0×10(9)/L(P > 0.05). The miR-17 and miR-20a silencing mediated by miRNA Sponge led to a significant decrease of cell growth, restored G1 accumulation and increase of cell apoptosis. It is concluded that the expression of miR-17 and miR-20a is upregulated in leukemia patients, which may contribute to leukemogenesis. Over-expressed miR-17 and miR-20a promote cell growth and cell cycle progression, and inhibit apoptosis through negatively-regulating P21 and E2F1 after-transcriptionally.

ShRNA-mediated Bmi-1 silencing sensitizes multiple myeloma cells to bortezomib.

The introduction of bortezomib has resulted in a paradigm shift in the treatment of multiple myeloma (MM) and has contributed to the improved survival of patients with MM. Inevitably, resistance to therapy develops, and thus the clinical efficacy of bortezomib is hampered by drug resistance. The oncogene B-cell­specific Moloney murine leukemia virus insertion site­1 (Bmi-1) has been implicated in the pathogenesis of various human malignancies. Furthermore, RNA interference (RNAi)­mediated Bmi-1 silencing has been shown to sensitize tumor cells to chemotherapy and radiation. The role of Bmi-1 in influencing the response to bortezomib therapy has not been investigated to date. In the present study, Bmi-1 was silenced in two MM cell lines (U266 and RPMI8226) using short hairpin RNA (shRNA) targeting Bmi-1 (shBmi-1). A cell counting kit-8 (CCK-8) assay was performed to analyze cell proliferation and evaluate the 50% inhibitory concentration (IC50) values of bortezomib. Cell cycle progression and apoptosis were analyzed by flow cytometry (FCM), and the mRNA and protein expression of associated genes (Bmi-1, p14, p21, Bcl-2 and Bax) was quantified by RT-qPCR and western blot analysis, respectively. The IC50 values significantly decreased in the cells transfected with shBmi-1 (p<0.05). The depletion of Bmi-1 sensitized the MM cells to bortezomib, which increased the G1 phase duration and enhanced bortezomib­induced apoptosis (p<0.05). The expression of p21 and Bax (apoptosis inducer) was upregulated, whereas that of the anti-apoptotic protein, Bcl-2, was downregulated in the Bmi-1­silenced cells (p<0.05). The depletion of Bmi-1 enhanced the sensitivity of MM cells to bortezomib by inhibiting cell proliferation and inducing cell cycle arrest and apoptosis. Our data suggest that Bmi-1 may serve as an important novel therapeutic target in MM.

Trends in in-hospital mortality among patients with stroke in China.

BACKGROUND: The incidence and burden of stroke in China is increasing rapidly. However, little is known about trends in mortality during stroke hospitalization. The objectives of this study were to assess trends of in-hospital mortality among patients with stroke and explore influence factors of in-hospital death after stroke in China. METHODS: 109 grade III class A hospitals were sampled by multistage stratified cluster sampling. All patients admitted to hospitals between 2007 and 2010 with a discharge diagnosis of stroke were included. Trends in in-hospital mortality among patients with stroke were assessed. Influence factors of in-hospital death after stroke were explored using multivariable logistic regression. RESULTS: Overall stroke hospitalizations increased from 79,894 in 2007 to 85,475 in 2010, and in-hospital mortality of stroke decreased from 3.16% to 2.30% (P<0.0001). The percentage of severe patients increased while odds of mortality (2010 versus 2007) decreased regardless of stroke type: subarachnoid hemorrhage (OR 0.792, 95% CI = 0.636 to 0.987), intracerebral hemorrhage (OR 0.647, 95% CI = 0.591 to 0.708), and ischemic stroke (OR 0.588, 95% CI = 0.532 to 0.649). In multivariable analyses, older age, male, basic health insurance, multiple comorbidities and severity of disease were linked to higher odds of in-hospital mortality. CONCLUSIONS: The mortality of stroke hospitalizations decreased likely reflecting advancements in stroke care and prevention. Decreasing of mortality with increasing of severe stroke patients indicated that we should pay more attention to rehabilitation and life quality of stroke patients. Specific individual and hospital-level characteristics may be targets for facilitating further declines.

[Expression of MCL-1 and microRNA-29a in extranodal NK/T-cell lymphoma tissue].

This study was aimed to explore the expression level and correlation of MCL-1 and miR-29a in extranodal NK/T-cell lymphoma (ENKTCL) tissue. Maxvision immunohistochemistry technique and real time fluorescent quantitative PCR were used to detect the expression level of MCL-1 and miR-29a in tissue of 20 patients with ENKTCL and 10 patients with proliferative lymphadenitis, respectively. The results showed that the expression of MCL-1 protein were higher in patients with ENKTCL than that in patients with proliferative lymphadenitis, but there were no significant correlation between MCL-1 overexpression and age, sex, Ann Arbor stage and International Prognostic Index (IPI), respectively. Correlation analysis indicated that there was significant negative correlation between miR-29a expression and MCL-1 expression (r = -0.59, P = 0.016). It is concluded that miR-29a may target MCL-1 gene, regulate its expression, then participate in tumorigenesis and development of ENKTCL.

[Construction of miRNA sponge targeting miR-20a and stable expression in Jurkat leukemia cell line].

This study was aimed to construct miRNA sponge targeting miR-20a and to establish a stable cell line Jurkat-S, paving the way for further research on function of miR-20a and application of RNAi in gene therapy. One pair of two-repeated oligonucleotide sequences containing bulged sites that are mispaired opposite miR-20a positions 9-12 was designed and synthesized with enzyme cutting sites. The annealed oligonucleotide fragments were subcloned into pCDNA3.0-L expressing vector. After double-enzyme cutting, the vector was ligated to the annealed oligonucleotide fragments again. Enzyme cutting and luciferase activity assay were performed for identification after four repeats. Then the ligated fragment was subcloned to lentivirus expressing vector. Virus particles were collected after the control or sponge vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells using Lipofectamine 2000. The Jurkat cells were transfused with recombinant lentivirus-transfusing units plus 6 µg/ml of Polybrene. Real-time PCR and Western blot were used to detect the mRNA and protein expression of P21 and E2F1 after lentivirus transfusion respectively. As a result, luciferase activity assay demonstrated that the sponge targeting miR-20a was constructed successfully and the virus was packaged in 293T. The titer of virus was 5×10(7) TU/ml. Stable transfected Jurkat-S cell line was established. As was expected, the mRNA and protein level of P21 and E2F1 was upregulated significantly in Jurkat-S cells. It is concluded that the miR-20a sponge is constructed successfully, and Jurkat-S stable cell line is established, in which the expression of miR-20a is inhibited stably.

Effect of anti-obesity drug on cardiovascular risk factors: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Anti-obesity drugs are widely used to prevent the complications of obesity, however, the effects of anti-obesity drugs on cardiovascular risk factors are unclear at the present time. We carried out a comprehensively systematic review and meta-analysis to assess the effects of anti-obesity drugs on cardiovascular risk factors. METHODOLOGY AND PRINCIPAL FINDINGS: We systematically searched Medline, EmBase, the Cochrane Central Register of Controlled Trials, reference lists of articles and proceedings of major meetings for relevant literatures. We included randomized placebo-controlled trials that reported the effects of anti-obesity drugs on cardiovascular risk factors compared to placebo. Overall, orlistat produced a reduction of 2.39 kg (95%CI-3.34 to -1.45) for weight, a reduction of 0.27 mmol/L (95%CI: -0.36 to -0.17) for total cholesterol, a reduction of 0.21 mmol/L (95%CI: -0.30 to -0.12) for LDL, a reduction of 0.12 mmol/L (95%CI: -0.20 to -0.04) for fasting glucose, 1.85 mmHg reduction (95%CI: -3.30 to -0.40) for SBP, and a reduction of 1.49 mmHg (95%CI: -2.39 to -0.58) for DBP. Sibutramine only showed effects on weight loss and triglycerides reduction with statistical significances. Rimonabant was associated with statistically significant effects on weight loss, SBP reduction and DBP reduction. No other significantly different effects were identified between anti-obesity therapy and placebo. CONCLUSION/SIGNIFICANCE: We identified that anti-obesity therapy was associated with a decrease of weight regardless of the type of the drug. Orlistat and rimonabant could lead to an improvement on cardiovascular risk factors. However, Sibutramine may have a direct effect on cardiovascular risk factors.

[Clinical analysis on 40 patients with chronic lymphocytic leukemia].

This study was aimed to analyze the clinical and laboratorial characteristics of patients with chronic lymphocytic leukemia (CLL), as well as their relationship with outcomes of patients. The clinical and laboratorial data of 40 CLL patients admitted from 2004 to 2010 in our hospital were analyzed retrospectively. The results indicated that the most of CLL attacked the elderly male patients with median age 66 (from 42 to 80). Flow cytometric analysis showed that 25 cases were positive for typical immunophenotype of CLL. On the other hand, all the patients clearly expressed CD19 and CD5, 7 cases (17.5%) and 14 cases (35%) were positive for the expression of CD38 and Zap70 respectively. 8 cases harbored a mutated immunoglobulin heavy-chain (VH) gene, among them 4 cases belong to VH3 family. Interphase FISH analysis showed that P53 deletion, RB1 deletion, trisomy 12 and normal chromosome were detected in 6, 3, 1, and 5 cases, respectively. The median PFS in 31 patients received treatment of fludarabine based chemotherapy was 48 months (95%CI: 39 - 57 months), among them 27 cases (87.1%) achieved CR + PR. While PFS was 14 months (95%CI: 10 - 18 months, P < 0.001) in 9 patients received other treatment regimen, out of them only 3 cases (33.3%) achieved CR + PR. Patients with normal level of serum ß2-microglobulin at diagnosis showed significantly higher overall survival (78%, 95%CI: 69% - 87%) in 36 months than those with abnormal level of serum ß2-microglobulin (47%, 95%CI: 35% - 59%, P = 0.004). Significant difference in the rate of CR + PR was noted in the Zap70 positive group (50%) and in negative group (88.5%, P = 0.006). All of 8 patients with IgVH mutation displayed CR after treatment, while 4 cases (66.7%) archived CR among 6 patients without IgVH mutation. It is concluded that CLL is characterized by high heterogeneity in both clinical features and molecular markers, which are associated with prediction of outcomes for patients. The treatment with fludarabine-based chemotherapy results in a major benefit and long survival for patients with CLL.

[A case of Richter syndrome transformed from chronic lymphocytic leukemia with karyotype aberration of trisomy 12].

This study was aimed to investigate the relationship between Richter's syndrome (RS) transformation and clinical characteristics as well as karyotype of patient with chronic lymphocytic leukemia (CLL). By the follow-up of a patient with CLL, the clinical characteristics, karyotype, treatment pattern and its effect, as well as disease progression were monitored regularly with serological test, flow cytometry and FISH technique. The results indicated that the patient typically presented with history of CLL at initial diagnosis, with expression of CD5(+), CD19(+) and CD23(+), Binet stage C, as well as karyotype aberration of trisomy 12, and poorly responded to 4 cycles of standard chemotherapy of FCR regimen. The disease progression was confirmed at 5 months with the symptoms of fever in the absence of infection, elevated lactate dehydrogenase level and rapidly enlarging lymphnodes which showed typically diffuse large B cell lymphoma by the biopsy. It is concluded that karyotype aberration of trisomy 12 is one of the risk factors for RS transformation, and treatment pattern of the patient with CLL may be associated with the transformation of RS.

[Establishment of a U266 cell line with stable Bmi-1 silencing by lentivirus-mediated RNA interference].

This study was aimed to construct lentivirus-mediated shRNA expression vector targeting Bmi-1 and establish a stable cell line U266-li, so as to pave the way for further research on function of Bmi-1 and application of shRNA to gene therapy. One pair of oligonucleotide sequences targeted at human Bmi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected after the control or shRNA vectors were co-transfected with the psPAX2 packaging plasmid and the plasmid pMD2.G was enveloped into HEK-293T cells by using Lipofectamine2000. The U266 cells were transduced with 5 × 10(6) recombinant lentivirus-transducing units plus 6 µg/ml of polybrene. Real-time PCR and Western blot were used respectively to detect the expression of Bmi-1 and P14 after lentivirus transduction. DNA sequencing demonstrated that the lentivirus RNAi vector of Bmi-1 was constructed successfully and the virus was packaged in 293T cells. The titer of virus was 5 × 10(7) TU/ml. Stable transfected U266 cell line was established. As was expected, the mRNA and protein levels of Bmi-1 was reduced significantly in U266 cells after lentivirus transduction, whereas the mRNA and protein levels of P14 was upregulated. It is concluded that the lentiviral RNAi vector of Bmi-1 is constructed, and U266 stable cell line is established.

[Establishment and application of lentivirus luciferase reporter-mediated miRNA target gene screening system].

This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.

[Mechanism associated to enhancing the sensitivity of myeloma cells U266 to bortezomib by 2-methoxyestradiol].

This study was aimed to explore the synergistic effect of 2-methoxyestradiol (2-ME2) and bortezomib (Bor) on the proliferative inhibition and apoptosis of U266 cell line and its possible mechanism. The cells were treated with 2-ME2, Bor alone and 2-ME2 combined with Bor, respectively. The cell viability and proliferative curve were detected by CCK8, the cell apoptosis was detected by caspase 3/7 activity test, cell cycle status was analyzed by flow cytometry, and real-time PCR was used to detect the mRNA expression of P21, BAX and BCL-2. The results showed that compared with cells treated with 2-ME2 or Bor alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.05), and apoptosis rate markedly increased (p < 0.05), cell cycle was arrested at G(1)-S phase, the mRNA expressive level of P21 and BAX increased, while the expression of BCL-2 decreased. It is concluded that 2-ME2 combined with Bor synergistically inhibits cell proliferation and induces apoptosis in U266 cell line. The possible mechanism may be associated with its effect of up-regulating P21 and BAX expressions.