The Long Noncoding RNA MEG3 and its Target miR-147 Regulate JAK/STAT Pathway in Advanced Chronic Myeloid Leukemia.

BACKGROUND: Long non-coding (lnc) RNAs plays an important role in chronic myeloid leukemia (CML). In this study, we aimed to uncover the mechanism of the lncRNA maternally expressed 3 (MEG3) and its target microRNA-147 (miR-147) in CML. METHODS: Sixty CML patients and 10 healthy donors were included in the study. The methylation of MEG3 and miR-147 promoter was determined by methylation-specific PCR. The relationship of MEG3 and miR-147 was explored by luciferase assay. The interactions of proteins were studied by RNA pull-down assay, RNA immunoprecipitation and co-immunoprecipitation. FINDINGS: Patients in accelerated phase CML (CML-AP) and blast phase CML (CML-BP) showed lower expressions of MEG3 and miR-147 and higher expressions of DNMT1, DNMT3B, MBD2, MECP2 and HDAC1 compared to the controls. These patients also showed a higher degree of methylation of MEG3 and miR-147 while there was a reduction after chidamide treatment. Furthermore, the overexpression of MEG3 and miR-147 inhibited cell proliferation both in vivo and in vitro, promoted apoptosis and decreased the expressions of DNMT1, DNMT3A, DNMT3B, MBD2, HDAC1 and MECP2. We also found MEG3 interacted with DNMT1, JAK2, STAT3, HDAC1, and TYK2, and JAK2 was bound to STAT3, STAT5 and MYC. More interestingly, JAK2 was bound to TYK2 by the bridge of MEG3. INTERPRETATION: LncRNA MEG3 and its target miR-147 may serve as a novel therapeutic target for CML blast crisis, and chidamide might have a potential clinical application in treating CML blast crisis.

Association between methylation of tumor suppressor gene SOCS1 and acute myeloid leukemia.

Suppressor of cytokine signaling­1 (SOCS1) is a widely recognized tumor suppressor gene. Silencing of SOCS1 expression as a result of promoter methylation is associated with occurrence and development of solid tumors such as liver, cervical and pancreatic cancer. However, the association between SOCS1 gene methylation and acute myeloid leukemia (AML) has not been well explored. In the present study, we examined whether gene expression and methylation status of SOCS1 was altered in AML, and whether this was related to disease occurrence and development. To assess this hypothesis, we analyzed SOCS1 in four groups of AML patients: i) Initial treatment group (IT); ii) relapsed/refractory group (RR); iii) remission group (RE); and iv) normal control group (NC). We also used leukemia cell lines U937 and THP­1 to study the underlying molecular mechanism of SOCS1 in AML, mainly the JAK2/STAT pathway. We used several techniques such as quantitative PCR (qPCR), methylation­specific PCR (MS­PCR), western blotting, flow cytometry and cell transfection techniques to analyze the expression and methylation status of SOCS1. We found that the SOCS1 gene methylation rate in the IT and RR groups was significantly higher than that in the RR and NC groups (48, 80 vs. 0 and 0%, respectively). Furthermore, mRNA and protein expression was significantly lower in the IT and RR groups when compared to the RE and NC groups. We also found that the JAK2/STAT signaling pathway was negatively affected by SOCS1. SOCS1 gene methylation caused gene silencing of SOCS1 which overcame the suppression of the downstream JAK2/STAT signaling pathway by SOCS1, and promoted the growth and proliferation of AML cells.

[Cyclin E overexpression and centrosome amplification in squamous cell carcinoma of oral cavity].

OBJECTIVE: To study the correlation between cyclin E protein overexpression and centrosome amplification in oral squamous cell carcinoma (OSCC). METHODS: Formalin-fixed, paraffin-embedded tissues from 12 normal oral epithelium cases and 46 cases of OSCC were studied. Their centrosome status was analyzed by indirect immunofluorescence double staining with antibodies to centrosome protein gamma-tubulin and cytokeratin. The expression of cyclin E protein was studied by immunohistochemical methods. The correlation between cyclin E protein expression and centrosome amplification in OSCC was statistically analyzed by SPSS 12.0. RESULTS: Thirty-seven of the 46 OSCC cases (80.4%) studied showed evidence of centrosome amplification, as signified by enlargement and/or increase in number of centrosomes, while normal oral epithelium possessed centromeres of normal size and number. Positive staining for cyclin E protein was observed in 30 of the 46 OSCC cases (65.2%), while all the normal oral epithelium cases were cyclin E protein-negative. The percentage of centrosome amplification in OSCC with positive cyclin E protein staining (90.0%, 27/30) was higher than that in OSCC with negative cyclin E protein staining (62.5%, 10/16) (chi(2) = 5.014, P < 0.05). Centrosome amplification showed positive correlation with cyclin E protein overexpression (r = 0.330, P < 0.05). CONCLUSION: Up-regulation of cyclin E protein may represent one of the possible mechanisms for centrosome amplification in OSCC.

[Clinical Analysis of 12 cases of Systemic Lupus Erythematosus Associated with Thrombotic Thrombocytopenic Purpura].

OBJECTIVE: To investigate the clinical manifestations, treatment strategies and outcomes of 12 patients with systemic lupus erythematosus (SLE) associated with thrombotic thrombocytopenic purpura(TTP). METHODS: The clinical data from 12 cases of SLE associated with TTP admitted in the Second Hospital of Hebei Medical University from January 2002 to August 2015 were retrospectively analyzed. RESULTS: 12 cases of SLE associated with TTP included 11 females and 1 male, their median age was 34.5 years old, among them 5 cases of TTP were diagnosed during the treatment of SLE, 7 cases of TTP were comfirmed together with SLE on admission. The hemolytic anemia, thrombocytopenia and neurological deficits appeared in all the patients, the renal impairment was observed in 10 cases, the schistocytes of peripheral blood smears (>1%) were present in 9 cases, a severely reduction of ADAMTS 13 activity (<5%) with inhibitor-positive had been demonstrated in 5 cases, all of the 12 patients were treated with glucocorticoid, and 11 cases were treated in combination with other drug(10 cases combined with cytotoxics, 1 case with intravenous gamma globulin, 1 case with rituximab), plasma exchange were used in 10 cases, and 2 cases died, 2 cases without receiving plasma exchange all died, renal damage was observed in all the dead patients. CONCLUSION: Clinical manifestation and repeated examinations of peripheral blood smears are helpful for early diagnosis of SLE associated with TTP, the plasma exchange combined with glucocortcoids is an effective treatment method, the renal impairment may be a risk factor related with poor prognosis.

[Effect of Rapamycin on Expression of Survivin and Caspase-3 and Its Influence on K562 Cell Ultrastructure].

OBJECTIVE: To investigate the effect of rapamycin on the expression of survivin and caspase-3 at mRNA level in K562 cells and the influence of rapamycin on K562 cell ultrastructure. METHODS: The effects of rapamycin at various concentration on K562 cell proliferation were analyzed by CCK8; the morphological characteristics of K562 cells was observed by transmission electron microscopy; the expression of survivin and caspase-3 at mRNA level in K562 cells treated with rapamycin was detected by RT-PCR. RESULTS: The proliferation of K562 cells was significantly inhibited by rapamycin. The apoptosis level of K562 cells increased with increase of rapamycin concentration, the expression of survivin at mRNA level decreased with increase of rapamycin concentration (P < 0.05). The expression of caspase-3 at mRNA level increased with increase of rapamycin concentration. CONCLUSION: Rapamycin can prornote K562 cell apoptosis through up-regulating caspase-3 level and reduceing survivin level.

[Expression and clinical significance of MMP-2 and MMP-9 in B acute lymphoblastic leukemia].

This study was purposed to investigate the expression and clinical significance of MMP-2 and MMP-9 in patients with B-acute lymphoblastic leukemia (B-ALL). The expression of MMP-2 and MMP-9 in bone marrow mononuclear cells of B-ALL patients and normal controls was detected by RT-PCR. The gelatinolytic activity was detected by zymography. The results showed that the expression of MMP-2 in de novo and relapsed B-ALL patients was markedly higher than that in normal controls (P < 0.05). The expression of MMP-9 in de novo and relapsed B-ALL patients was markedly lower than that in normal controls (P < 0.05). The expression of MMP-2 and MMP-9 in patients with extramedullary infiltration was significantly higher than that in patients without extramedullary infiltration. The incidence of extramedullary infiltration in patients with MMP-2/MMP-9 (+) was markedly higher than that in patients with MMP-2/MMP-9 (-). The expression of MMP-9 was markedly higher in high-risk patients than that in standard-risk patients (P < 0.05), but the expression of MMP-2 had no significant difference between the high-risk and standard-risk patients (P > 0.05). It is concluded that MMP-2 and MMP-9 may be secreted by B lymphoblasts and may involve in the extramedullary infiltration. MMP-9 may correlate with poor prognosis.

[Expression of interleukin-12p40 and interferon-gamma in local lesions of human oral lichen planus].

OBJECTIVE: To investigate the significance of cytokine interleukin-12p40 (IL-12p40) and interferon-gamma (IFN-gamma) in tissues formation and development of human oral lichen planus (OLP). METHODS: The tissues of 11 cases of normal oral epithelium and 43 cases of OLP were investigated for the expression of IL-12p40 and IFN-gamma proteins by using Envision two-step immunohistochemistry. The correlations between the expressions of these two cytokines, and their clinical and pathological significance in OLP were analyzed. RESULTS: 1) IL-12p40 and IFN-gamma proteins were up-regulated in OLP comparing with that in normal oral mucosa and there was statistical significance between their difference (P < 0.05). 2) The percentage of positive IL-12p40 staining in OLP of IFN-gamma positive group was higher than IFN-gamma negative group and there was statistical significance between their difference (Chi2 = 5.828, P = 0.016). A positive correlation was found between IL-12p40 and IFN-gamma proteins in OLP (Spearman r = 0.357, P = 0.019). 3) The percentage of positive IL-12p40 staining in OLP with short course (< 6 months) was higher than that in OLP with long course (> 6 months; Chi2 = 7.935, P = 0.005), and a significant association was found between IFN-gamma over expression and the degeneration of base cells in OLP lesions (Chi2 = 9.070, P = 0.011). CONCLUSION: These results indicate that at the primary phase of OLP, IL-12 may drive the pathological destruction in OLP lesions by elevating IFN-gamma protein locally. IFN-gamma may play an important role for the pathological destruction in OLP lesions.