LncRNA FIRRE stimulates PTBP1-induced Smurf2 decay, stabilizes B-cell receptor, and promotes the development of diffuse large B-cell lymphoma.

Sustained expression of B-cell receptor (BCR) critically contributes to the development of diffuse large B-cell lymphoma (DLBCL). However, little is known on the mechanism regulating BCR expression. In the present study, we explored the biological significance of functional intergenic repeating RNA element (FIRRE) in DLBCL and its regulation on BCR. Functional impacts of FIRRE on cell viability, transformation, and apoptosis were examined by MTT, colony formation, and flow cytometry, respectively. The interaction between FIRRE and polypyrimidine tract binding protein 1 (PTBP1) was identified by RNA pull-down and verified using RNA immunoprecipitation (RIP) assays. The effects of FIRRE and PTBP1 on Smurf2 mRNA were examined by RIP, RNA pull-down, and mRNA stability assays. Smurf2-mediated BCR ubiquitination was investigated using co-immunoprecipitation, ubiquitination, and protein stability assays. In vivo, xenograft models were used to assess the impacts of targeting FIRRE on DLBCL growth. FIRRE was specifically up-regulated in and essentially maintained multiple malignant behaviors of BCR-dependent DLBCL cells. Through the interaction with PTBP1, FIRRE promoted the mRNA decay of Smurf2, a ubiquitin ligase for the degradation BCR protein. Targeting FIRRE was sufficient to regulat Smurf2 and BCR expressions and inhibit DLBCL malignancy both in vivo and in vitro. FIRRE-PTBP1 interaction, by simulating Smurf2 mRNA decay and stabilizing BCR, promotes the development of DLBCL. Consequently, targeting this signaling mechanism may provide therapeutic benefits for DLBCL.

[Effect of rhTPO and rhIL-11 on Thrombocytopenia after Chemotherapy in Leukemia].

OBJECTIVE: To analyze and compare the efficacy of recombinant human thrombopoietin (rhTPO) and recombinant human interleukin-11 (rhIL-11) in the treatment of thrombocytopenia after chemotherapy in acute leukemia patients. METHODS: 180 patients with acute leukemia complicated with thrombocytopenia after chemotherapy in the First Affiliated Hospital of Anhui Medical University were analyzed retrospectively. Among them, 50 patients who treated with rhTPO and did not receive platelet transfusion were set as group A, 50 patients treated with rhTPO and receive platelet transfusion were set as group B, Forty patients treated with rhIL-11 without platelet transfusion were set as group C, Forty patients who treated with rhIL-11 and received platelet transfusion were set as group D. The duration of PLT below 20×109/L, the days it takes for PLT to recover to more than 100×109/L, and the incidence of different bleeding degrees were compared among several groups. RESULTS: The duration of PLT<20×109/L in group A(3.72±1.14 d) was significantly shorter than that in group C(4.93±1.33 d) (P<0.001), and there was no significant difference from group B (P>0.05). The duration of PLT<20×109/L in group B(3.06±0.91 d) was significantly shorter than that in group D(4.65±0.98 d) (P<0.001), while the difference in duration of days between group C and D was not statistically significant (P>0.05). The times for PLT to recover to 100×109/L in group A(13.46±1.67 d) were significantly shorter than that in group C(16.85±2.13 d) (P<0.05), but there was no significant difference from group B (P>0.05). The time required for PLT to recover to 100×109/L in group B(13.36±1.49 d) were significantly shorter than that in group D(16.18±1.78 d) (P<0.05), while the difference in the days required for group C and group D was not statistically significant (P>0.05). The incidence of high bleeding risk in group B was significantly lower than that in group A (22% vs 44%, P<0.05), the incidence of high bleeding risk in group D was significantly lower than that in group C (32% vs 65%, P<0.05), and the incidence of high bleeding risk in group A was significantly lower than that in group C (44% vs 65%, P<0.05). The incidence of high bleeding risk in group B(22%) was lower than that in group D(32.5%), and the difference was not statistically significant (P>0.05). CONCLUSION: In the treatment of acute leukemia patients with thrombocytopenia after chemotherapy, compared with rhIL-11, rhTPO can significantly shorten the duration for patients in a status with extremely low levels of PLT and the recovery time of PLT to normal range. In addition, PLT transfusion cannot speed up the time for patients to raise platelets to a safe range, nor can it shorten the duration of low PLT levels, but it can reduce the incidence of high bleeding risk events.

CDK12 activates MYC to repress miR-28-5p/EZH2 and amplifies tonic BCR signaling to promote the development of diffuse large B-cell lymphoma.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes. However, its regulatory role in the progression of diffuse large B-cell lymphoma (DLBCL), which is the most prevalent subtype of non-Hodgkin lymphoma (NHL), is still elusive and controversial.The expression of CDK12 was detected by immunohistochemistry (IHC), RT-qPCR was performed to detect miR-28-5p expression of OCI-LY3 and SU-DHL-4 cells. MTT and soft agarose colony formation assays were used to detect cell proliferation. The cell apoptosis was determined by flow cytometry. The protein expressions changes of MYC, EZH2 and the biomarkers of BCR signaling were also detected. A subcutaneous transplantation tumor model of OCI-LY3 cells in nude mice was established to evaluate anticarcinogenic activities of CDK12 knockdown. Elevated expression of CDK12 was observed while miR-28-5p was downregulated in DLBCL tissues. CDK12 knockdown or miR-28-5p overexpression could inhibit proliferation and promote apoptosis of DLBCL cells. miR-28-5p inhibition could reverse the effect of CDK12 knockdown on proliferation and apoptosis of DLBCL cells. In addition, CDK12 knockdown could inhibit DLBCL tumor growth in the mice model. CDK12 activated MYC to repress miR-28-5p/EZH2 and amplified tonic BCR signaling to promote the development of DLBCL, which might provide potential therapeutic targets for future therapeutic intervention in DLBCL.

[Mechanism of apoptosis in human leukemia NB4 cells induced by total astragalosides].

OBJECTIVE: To investigate the effect of total astragalosides (TA) on proliferation and apoptosis in human leukemia NB4 cells in vitro. METHODS: The NB4 cells were treated with TA at different concentrations for 48 h in culture. Growth inhibition rates were measured by CCK-8 method. Flow cytometry was used to explore the cell apoptosis and the activity of NF-κB and Akt during apoptosis. RESULTS: TA at different concentrations (200, 400, 600, 800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner (P < 0.05), and the inhibitory rates of TA on NB4 cells were (14.54 ± 3.20)%, (24.79 ± 3.98)%, (57.28 ± 4.71)% and (88.28 ± 4.65)%, respectively. In terms of the induction of apoptosis, there was a significant difference between the TA group and blank control [(1.80 ± 1.24)%, P < 0.05]. At TA doses of 200, 400 and 600 mg/L, the apoptotic rates of NB4 cells were (10.03 ± 3.31)%, (14.87 ± 3.65)%, (23.45 ± 1.90)%, respectively. Besides, TA induced apoptosis of NB4 cells in a dose-dependent manner in the groups of 200 mg/L, 400 mg/L, 600 mg/L (P < 0.05). But there was no significant difference in apoptotic rates between the groups of 800 mg/L and 600 mg/L [(23.45 ± 1.90)%, P > 0.05]. In the group of 800 mg/L, the necrotic cells increased highly and the necrotic rate reached (45.65 ± 3.16)%. After TA treatment of NB4 cells at different concentrations (200, 400, 600 mg/L), the expression of NF-κB protein was significantly decreased compared with that of the blank control (9.79 ± 0.95, P < 0.05), while Akt protein was not significantly decreased (P > 0.05). CONCLUSION: TA can inhibit the growth of NB4 cells and induce apoptosis in NB4 cells through an Akt-independent NF-κB signaling pathway.

Clinical significance of measuring reticulated platelets in infectious diseases.

This study aimed to explore the association between the percentage of reticulated platelets (RP%) and infection, and analyze the value of combined measurement of RP% with other inflammatory indicators in diagnosing infection. A total of 190 patients with signs and symptoms suspicious of infection were included in the infection group, and 70 healthy subjects with comparable percentages of gender and age were included in the control group. Peripheral white blood cell (WBC) count, percentage of neutrophils (N%), platelet count, C-reactive protein (CRP), procalcitonin (PCT), RP%, and axillary temperature were recorded. Dynamic changes in RP% with infection were measured to analyze the association between RP% and infection. The receiver operating characteristic curve was used to evaluate the value of each inflammatory indicator in diagnosing infection and analyze the diagnostic value of the combined adoption of multiple inflammatory indicators. RP% was significantly higher in the infection group than in the noninfection and control groups. The sensitivity and specificity for diagnosing infection were, respectively, 91.78% and 93.18% when RP% and CRP were used in combination, 90.41% and 90.90% when RP% and PCT were used in combination, and 100% and 100% when RP%, CRP, and PCT were used in combination. RP% changed dynamically with the progression of infection and recovered to lower than 5.5% at 2 to 7 days before the body temperature recovered to a normal level. The diagnostic value of RP% was the highest. A combined use with CRP/PCT could improve the sensitivity and specificity in the early diagnosis of infectious diseases.

[Expression of CXCR7 in Acute Monocytic Leukemia and Its Effects on THP-1 Cell Functions].

OBJECTIVE: To study the expression of stromal cell derived factor-1α (SDF-1α) receptor CXCR7 in acute monocytic leukemia (AML-M5), and its effects on proliferation, apoptosis, invasion of acute monocytic leukemia cell line THP-1. METHODS: CXCR7 protein and mRNA expression levels in THP-1 cells and peripheral blood mononuclear cells (PBMNC) from the newly diagnosed AML-M5 patients and normal individuals were detected by flow cytometry, Western blot and RT-PCR respectively. CCK8, Annexin V/PI double staining and Transwell assay were used to observe the effects of CXCR7 on the proliferation, apoptosis, and invasion of THP-1 cells in vitro. RESULTS: The expression of CXCR7 on immature cell surface of the newly diagnosed AML-M5 patients was significantly higher than that in the control group (P<0.05). CXCR7 was also highly expressed on THP-1 cells surface. The CXCR7 protein and mRNA levels in THP-1 cells and PBMNC of AML-M5 patients were significantly higher than those in the control group (P<0.05). The THP-1 cell proliferation activity was higher in SDF-1α-treated group, but this activity could be inhibited by CXCR7 antibody (P<0.01). CXCR7 antibody did not affect THP-1 cell apoptosis (P>0.05). CXCR7 antibody could inhibit SDF-1α -induced THP-1 cell invasiveness (P<0.01). CONCLUSION: CXCR7 highly expresses in AML-M5 patients and THP-1 cells, and involves in cell proliferation and invasion. The blocking CXCR7 expression can reduce the risk of AML-M5 cell infiltration.

[Clinical Efficacy and Safety of Rituximab Combined with Fludarabine and Cyclophosphamide for Treatment of Chronic Lymphocytic Leukemia].

OBJECTIVE: To explore the clinical efficacy and safety of rituximab combined with fludarabine and cyclophosphamide for the treatment of the chronic lymphocytic leukemia (CLL). METHODS: Forty cases of CLL patients treated in our hospital from March 2010 to March 2014 years were selected and divided into the observation group (20 cases) and control group (20 cases) by random number table method. The patients in control group were treated with CHOP chemotherapy, the patients in observation group were treated with rituximab combined with fludarabine, cyclophosphamide treatment. The therapeutic efficacy of patients in 2 groups was analyzed according to the peripheral hemogram indexes, symptom and sign disappeared time as well as adverse reaction incidence. RESULTS: the remission rate in observation group was 90.00%, which was significantly higher than that in control group (70.00%) (P < 0.05); the peripheral hemogram indexes in 2 groups before treatment showed no significant difference (P > 0.05), and were significantly improved after treatment, but the white blood cell count and lymphocyte absolute number were significantly lower in observation group as compared to the control group (P < 0.05); symptom and sign disappeared time in observation group were significantly shorter as compared with the control group (P < 0.05); adverse reaction incidence in obseovation group was significantly lower as compared with control group (P < 0.05). CONCLUSION: application of rituximab combined with fludarabine and cyclophosphamide in the treatment of CLL shows the higher curative effect, can effectively improve the symptoms and reduce the incidence of adverse reactions. It is worthy to be popularized.

[Value of Neutrophil/Lymphocyte Ratio and Platelet/Lymphocyte Ratio for Prognostic Evaluation of Diffuse Large B-cell Lymphoma].

OBJECTIVE: To investigate the predictive value of neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) for the patients with diffuse large B-cell lymphoma (DLBCL). METHODS: The clinical data of 57 DLBCL patients admitted in the First Affiliated hospital of Anhui Medical University were analyzed retrospectively. According to ROC curve, the cut-off value for NLR and PLR was deterimined, and the patients were divided into high and low NLR/PLR groups before first chamotherapy. Then the relation of NLR and PLR with overall survival (OS) and progression-free survival (PFS) was analyzed by univariate and multivariate COX regression. RESULTS: The optimal cut-off value for NLR and PLR was 2.915 and 270.27, respectively. NLR at the diagnosis was found to be an independent predictor for OS and PFS by univariate and multivariate analysis, while the PLR was an independent predictor for PFS, but did not affect the OS. CONCLUSION: NLR and PLR may provide additional prognostic information for DLBCL patients.

[Influence of Anticoagulants on Detection of ITP Platelet-Specific Autoantibodies and Relationship of Autoantibody Types with Glucocorticoid Efficacy].

OBJECTIVE: To investigate the influence of divalent cation chelator EDTA and heparin sodium on the detection of ITP platelet-specific autoantibodies by the modified monoclonal antibody immobilization of platelet antigen assay (MAIPA) and to explore the relationship between types of platelet specific autoantibodies and glucocorticoid efficacy. METHODS: The samples were obtained from EDTA- and heparin- anticoagulant ITP patients, respectively, so as to detect the platelet-specific autoantibodies (GPIIb/IIIa and GPIbα) in 140 ITP samples by modified MAIPA, then the differences between these two methods were compared. RESULTS: Out of 140 cases in EDTA group, 55 cases were positive for GPIIb/IIIa, while 76 cases in heparin group were positive for GPIIb/IIIa, 42 cases in both group were repeatable; among them 13 cases were positive in EDTA group and negative in heparin group, while 34 cases were positive in heparin group and negative in EDTA group, there was significant difference between them (x (2) = 9.38, P < 0.05), 62 cases in 140 cases of EDTA group were positive for GPIba, while 51 cases in heparin group were positive for GPIba, 42 cases in both group were repeatabe; among them 20 cases were positive in EDTA group and negative in heparin group, while 9 cases were positive in heparin group and negative in EDTA group, there was no significant difference between them (x (2) = 3.44, P > 0.05). A total of 320 cases received a standard glucocorticoid treatment, out of them 143 cases were positive for GPIbα with effective rate 39.9%, 177 cases were negative for GPIbα with effective rate 79.7%, there was statisticalty significant difference between them (x (2) = 53.115, P < 0.05). CONCLUSION: EDTA anticoagulant (a divalent cation chelator) has a significant influence on detection of ITP platelet-specific autoantibodies (GPIIb/IIIa) by a modified MAIPA method and the GPIbα antibody positive may be one of the important factors that results in un-sensitivity of ITP patients to glucocorticoid treatment.

[Role of BCL-2, caspase-3 and NF-κB in astragaloside inducing apoptosis of human NB4 cells].

This study was purposed to investigate the apoptosis-inducing effect of astragalosides on acute promyelocytic leukemia(APL) cell line NB4 and its mechanism. NB4 cells were treated with different concentrations (200, 300, 400 µg /ml) of astragalosides for 48 h. The cell proliferation was assayed by using CCK-8 method; the cell apoptosis was analyzed by flow cytometry with Annexin V-FITE/PI double staining. The mRNA expression of BCL-2 and the relative activity of BCL-2, NF-κB and caspase-3 were detected by RT-PCR and Western blot, respectively. The results showed that after treated with astragalosides for 48 h, astragalosides inhibited NB4 cell proliferation in concentration-dependent way, the apoptosis rate of NB4 cells gradually elevated from 4.69% to 40.85% with the increasing of astragalosides concentration. Simultaneously, the mRNA expression of BCL-2 was down-regulated, Western blot analysis showed that the protein expression levels of BCL-2 and NF-κB decreased after astragalosides treatment, while caspase-3 protein expression level increased. It is concluded that the molecular mechanism of the astragalosides-induced apoptosis in NB4 cells may be associated with down-regulation of the expression of BCL-2 and NF-κB, finally the relative activity of caspase-3 activated.

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China.

BACKGROUND: Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China. METHODS: All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications. RESULTS: The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of ß2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS. CONCLUSIONS: Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.

[NB4 cell apoptosis induced by bortezomib combined with As(2)O(3) and its mechanism].

This study was aimed to investigate the apoptosis induced by bortezomib combined with As(2)O(3) in APL cell line NB4 and its mechanism. The apoptotic cells were detected by flow cytometry with Annexin V/propidium iodide double staining; the morphology of apoptotic cells was observed by Hoechst staining, Western blot was used to measure activation of caspase-3 and -9 as well as expression of NOXA; the siRNA technique was used to specifically silence NOXA gene; the lipofectamine 2000 was used to transfect pEGFP-Noxa plasmid and pEGFP vacant vector. The results showed that the bortezomib combined with As(2)O(3) could induce significant apoptosis of NB4 cells and activation of caspase 3 and caspase 9, but As(2)O(3) (0.5 µmol/L) alone could not cause marked activation of caspase cascade and apoptosis of NB4 cells. The expression level of NOXA in NB4 cells induced by bortezomib combined with As(2)O(3) was up-regulated; the activation level of caspase-3 and apoptotic rate of NB4 cells treated by bortezomib combined with As(2)O(3) decreased after specifically silencing the NOXA gene. The high expression of NOXA induced by transfection of plasmid could enhance the caspase 3 activity induced by As(2)O(3) alone. It is concluded that bortezomib can enhance sensitivity of NB4 cells to apoptosis induced by As(2)O(3) which may be related with up-regulation of proapoptotic protein NOXA.

[Detection of ABL kinase domain point mutations in chronic myeloid leukemia patients receiving imatinib treatment].

This study was purposed to evaluate ABL tyrosine kinase point mutations in imatinib-treated chronic myeloid leukemia (CML) patients and their clinical significance. 51 bone marrow samples from 28 imatinib-resistant patients and 10 newly diagnosed CML patients were collected. ABL kinase domain of bcr-abl allele was amplified by nested reverse transcription-polymerase chain reaction, followed by purifying, directly sequencing and sequence homology analysis of amplified products in order to determine the existence and type of point mutation. The results showed that the point mutations were found in 12 of 38 patients, and all the 12 ones progressed to advanced disease or death. 2 patients showed Met351Thr mutation, 7 patients showed Glu252His, 2 patients showed Glu279Lys, the other types were Glu255Val and Glu355Gly, each of which was tested in one patient. The incidence of the point mutation was 17.6%, 45.5% and 44.4% in chronic, accelerated and blast phase respectively. The incidences of point mutation in hematologically and genetically resistant patients were 50% (5/10) and 44.4% (8/18), and the 95% confidence interval (CI) was 12.3% - 87.7% and 19% - 69.9% respectively. It is concluded that ABL kinase point mutation is an important mechanism of imatinib resistance, monitoring the ABL kinase domain point mutation is helpful to estimating the prognosis and adjusting the therapeutic strategy.