Helicobacter pylori promotes gastric cancer metastasis via up-regulating the expression of Bmi-1 / 细胞与分子免疫学杂志

Objective To clarify whether Helicobacter pylori (H. pylori) can promote metastasis of gastric cancer cells via the high-expression of induced B cell specific Moloney murine leukemia virus integration site 1 (Bmi-1). Methods The gastric cancer tissue specimens from 82 patients were collected for this study. The protein and gene expression level of Bmi-1 in gastric adenocarcinoma tissue were detected by immunohistochemistry and real time quantitative PCR, respectively. And meanwhile the correlation between Bmi-1 levels and pathological features, and prognosis of gastric cancer were analyzed retrospectively. Then, the GES-1 cells were transfected with pLPCX-Bmi-1 plasmid and infected with H. pylori respectively. After the Bmi-1 overexpression in GES-1 cells, the invasion ability of the GES-1 cells was detected by Transwell assay, and the cell cycle and apoptosis were detected by flow cytometry. Results The mRNA and protein of Bmi-1 expression in gastric cancer tissues were higher than tumor-adjacent tissue, and the high expression of Bmi-1 was positively correlated with tumor invasion, TNM stage, tumor differentiation, lymph node metastasis and H. pylori infection. When expression of Bmi-1 was up-regulated as a result of H.pylori infection or pLPCX-Bmi-1 transfection, the GES-1 cells had higher invasiveness and lower apoptosis rate with the above treatment respectively. Conclusion H. pylori infection can inhibit the apoptosis of gastric cancer cells and promote their invasion via up-regulating expression of Bmi-1.

Utility of BMI-1 and NANOG expression levels in survival prediction of pediatric acute lymphoblastic leukemia

ABSTRACT Background Acute lymphoblastic leukemia (ALL) is the most common malignancy in children characterized by the overproduction and accumulation of immature lymphoid cells in the bone marrow and peripheral blood. The BMI-1 is an important component of the Polycomb Repressive Complex-1 (PRC1). It is an important molecule for the self-renewal of hematopoietic stem cells (HSCs). The BMI-1 expression is generally high in HSCs and decreases after cell differentiation. The BMI-1 is required for the maintenance of normal and cancer stem cells and has been reported as an oncogene in various tumors. The NANOG is a homeodomain transcription factor responsible for maintaining the stem cell compartment at the blastocyst stage of developing embryos. The NANOG gene has been proven to be transcribed in CD34+ cells and different leukemic cells. Methods The ribonucleic acid (RNA) was extracted from the peripheral blood mononuclear cells (PBMNCs) of 30 pediatric ALL patients (16 B-ALL and 14 T-ALL) and 14 healthy controls. The Bmi-1 and NANOG expression levels were determined using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results Compared to normal controls, patients with ALL exhibited upregulated levels of Bmi-1 (p = 0.03). Patients who overexpressed Bmi-1 and NANOG displayed a significantly worse survival than low-expressing patients (hazard ratio (HR) 5.74, 95% confidence interval (CI):1.48-22, p = 0.012 and HR 3.8, 95% CI:1.009-14.3, p = 0.048, respectively). Conclusions Taken together, these data suggest that the Bmi-1 and NANOG might serve as a novel survival predictor in ALL patients. Our observation also suggests that the Bmi-1 and NANOG could serve as new therapeutic targets for treatment of pediatric ALL.

Prognostic implications of immune-related eight-gene signature in pediatric brain tumors

Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors (PBT) that may lead to novel therapeutic strategies. Participants of the cohort Pediatric Brain Tumor Atlas: CBTTC (CBTTC cohort), were randomly divided into training and validation cohorts. In the training cohort, Kaplan-Meier analysis and univariate Cox regression model were applied to preliminary screening of prognostic genes. The LASSO Cox regression model was implemented to build a multi-gene signature, which was then validated in the validation and CBTTC cohorts through Kaplan-Meier, Cox, and receiver operating characteristic curve (ROC) analyses. Also, gene set enrichment analysis (GSEA) and immune infiltrating analyses were conducted to understand function annotation and the role of the signature in the tumor microenvironment. An eight-gene signature was built, which was examined by Kaplan-Meier analysis, revealing that a significant overall survival difference was seen, either in the training or validation cohorts. The eight-gene signature was further proven to be independent of other clinic-pathologic parameters via the Cox regression analyses. Moreover, ROC analysis demonstrated that this signature owned a better predictive power of PBT prognosis. Furthermore, GSEA and immune infiltrating analyses showed that the signature had close interactions with immune-related pathways and was closely related to CD8 T cells and monocytes in the tumor environment. Identifying the eight-gene signature (CBX7, JADE2, IGF2BP3, OR2W6P, PRAME, TICRR, KIF4A, and PIMREG) could accurately identify patients' prognosis and the signature had close interactions with the immunodominant tumor environment, which may provide insight into personalized prognosis prediction and new therapies for PBT patients.

BMI-1 expression increases in oral leukoplakias and correlates with cell proliferation

Abstract Oral leukoplakia (OL) is a white lesion of an indeterminate risk not related to any excluded (other) known diseases or disorders that carry no increased risk for cancer. Many biological markers have been used in an attempt to predict malignant transformation; however, no reliable markers have been established so far. Objective To evaluate cell proliferation and immortalization in OL, comparing non-dysplastic (Non-dys OL) and dysplastic OL (Dys OL). Methodology This is a cross-sectional observational study. Paraffin-embedded tissue blocks of 28 specimens of Non-dys OL, 33 of Dys OL, 9 of normal oral mucosa (NOM), 17 of inflammatory hyperplasia (IH), and 19 of oral squamous cell carcinomas (OSCC) were stained for Ki-67 and BMI-1 using immunohistochemistry. Results A gradual increase in BMI-1 and K-i67 expression was found in oral carcinogenesis. The immunolabeling for those markers was higher in OSCC when compared with the other groups (Kruskal-Wallis, p<0.05). Ki-67 expression percentage was higher in OL and in IH when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). Increased expression of BMI-1 was also observed in OL when compared with NOM (Kruskal-Wallis/Dunn, p<0.05). No differences were observed in expression of both markers when non-dysplastic and dysplastic leukoplakias were compared. A significant positive correlation between Ki-67 and BMI-1 was found (Spearman correlation coefficient, R=0.26, p=0.01). High-grade epithelial dysplasia was associated with malignant transformation (Chi-squared, p=0.03). Conclusions These findings indicate that BMI-1 expression increases in early oral carcinogenesis and is possibly associated with the occurrence of dysplastic changes. Furthermore, our findings indicate that both Ki-67 and BMI-1 are directly correlated and play a role in initiation and progression of OSCC.

MiR-218 Targeting Bmi-1 Inhibits Proliferation of Acute Promyelocytic Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the molecular mechanism by which miR-218 targeting Bmi-1 inhibits the proliferation of acute promyelocytic leukemia (APL) cells.@*METHODS@#APL cell line HL-60 was transfected by miR-218 and RNA-negative control sequences, respectively. The expression of miR-218 in cells was detected by real-time fluorescence quantitative PCR. The effect of transfected miR-218 on the proliferation of APL cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The regulation effect of miR-218 on Bmi-1 expression was determined by Western blot. The correlation of miR-218 expressions with Bmi-1 was analyzed by Spearman test. The targeted relationship between miR-218 and Bmi-1 was verified by luciferase assay.@*RESULTS@#MTT assay showed that the proliferation of HL-60 cells in vitro was inhibited by high expression miR-218 significantly. Flow cytometry showed that the G1 and G2 phase cells increased while the S phase cells decreased after transfected by miR-218. Western blot showed that the level of Bmi-1 protein in HL-60 cells decreased significantly after transfection of miR-218 (P＜0.05). Spearman correlation analysis showed that the mRNA level of miR-218 negatively correlated with the protein content of Bmi-1 (r=-0.326, P＜0.01). Luciferase assay indicated that Bmi-1 could targeted on miR-218 directly.@*CONCLUSION@#miR-218 can inhibit the proliferation, metastasis and invasion of APL cells, which can be related with the down-regulated of Bmi-1.

Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway.@*METHODS@#The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot.@*RESULTS@#The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored.@*CONCLUSION@#The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.

Bmi-1 as A Molecular Marker for Prognosis of Pediatric ALL / 中国实验血液学杂志

OBJECTIVE@#To study whether the Bmi-1 gene can be a biomarker for analysis of clinical risk stratification and prognosis of ALL patients.@*METHODS@#The expression level of Bmi-1 gene in bone marrow samples from 127 cases of newly diagnosed ALL was detected by qRT-PCR, at the same time the expression level of Bmi-1 protein in bone marrow samples from above-mentioned cases was detected by Western blot. The collected samples were divided into 3 groups: high, intermediate and low risk according to clinical risk stratfication, the relationship between Bmi-1 expression and risk grade of ALL patients was analyzed; at the same time the collected samples were divided into 2 groups: prednisone good response (PGR) and prednisone poor respouse (PPR) according to the sensitivity of prednison test, and the sensitivily to prednisone in 2 groups was compared; moreover, the collected samples were divided into 2 groups: high level and low level according to median of Bmi-1 level, and the relation of Bmi-1 level with prognosis of patients was analyzed by using the Kaplan-Meier method.@*RESULTS@#The expression level of Bmi-1 in low risk group was lowest, while that in high risk group was highest, however that in intermediat risk group was between the low and high risk groups, statistical analysis showed significant difference (P＜0.05). The expression level of Bmi-1 in PPR group was significantly higher than that in PGR group (P＜0.001). The Kaplan-Meier analysis showed that the RFS rate in Bmi-1 high expression group was significantly lower than that in Bmi-1 low expression group (73.0% vs 90.6%) (P＜0.001).@*CONCLUSION@#The Bmi-1 can be used as a molecular marker for the analysis of chinical risk and prognosis of pediatric ALL.

Angiotensin II and TGF-β1 Induce Alterations in Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading to Cardiomyogenic Differentiation Initiation

BACKGROUND AND OBJECTIVES: Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may be a valuable source for cardiovascular tissue engineering and cell therapy. The aim of this study is to verify angiotensin II and transforming growth factor-beta 1 (TGF-β1) as potential cardiomyogenic differentiation inducers of AF-MSCs. METHODS AND RESULTS: AF-MSCs were obtained from amniocentesis samples from second-trimester pregnant women, isolated and characterized by the expression of cell surface markers (CD44, CD90, CD105 positive; CD34 negative) and pluripotency genes (OCT4, SOX2, NANOG, REX1). Cardiomyogenic differentiation was induced using different concentrations of angiotensin II and TGF-β1. Successful initiation of differentiation was confirmed by alterations in cell morphology, upregulation of cardiac genes-markers NKX2-5, TBX5, GATA4, MYH6, TNNT2, DES and main cardiac ion channels genes (sodium, calcium, potassium) as determined by RT-qPCR. Western blot and immunofluorescence analysis revealed the increased expression of Connexin43, the main component of gap junctions, and Nkx2.5, the early cardiac transcription factor. Induced AF-MSCs switched their phenotype towards more energetic and started utilizing oxidative phosphorylation more than glycolysis for energy production as assessed using Agilent Seahorse XF analyzer. The immune analysis of chromatin-modifying enzymes DNMT1, HDAC1/2 and Polycomb repressive complex 1 and 2 (PRC1/2) proteins BMI1, EZH2 and SUZ12 as well as of modified histones H3 and H4 indicated global chromatin remodeling during the induced differentiation. CONCLUSIONS: Angiotensin II and TGF-β1 are efficient cardiomyogenic inducers of human AF-MSCs; they initiate alterations at the gene and protein expression, metabolic and epigenetic levels in stem cells leading towards cardiomyocyte-like phenotype formation.

Analysis of the Epithelium-Mesenchymal Transition Process on Oral Squamous Cell Carcinomas

Abstract The aim of this study is to evaluate the immunohistochemical expression of E-cadherin, N-cadherin and Bmi-1, and their association with clinical parameters and with the degree of histopathological differentiation in oral squamous cell carcinomas. 65 squamous cell carcinoma samples were used for constructing a tissue microarray block, and then immunohistochemistry was performed for different markers. A semi-quantitative analysis of the amount of positive tumor cells was performed by two blind and calibrated observers (Kappa>0.75). The statistical Mann-Whitney and Kruskal-Wallis tests were used to evaluate the data. The correlation between variables was investigated by the Spearman test, and the significance level set at p<0.05. We observed higher expression of Bmi-1 in tumors located in the palate (p<0.0001). In addition, poorly differentiated tumors had a greater amount of Bmi-1 positive cells (p=0.0011). Regarding the other correlations between variables, no significant associations were detected. In conclusion, poorly differentiated squamous cell carcinomas located in the palate have higher immunostaining of Bmi-1, which can characterize activation of the Epithelial-Mesenchymal Transition process in these tumors.

Resumo O objetivo deste estudo foi avaliar a associação entre a expressão imunoistoquímica de E-caderina, N-caderina e Bmi-1, com os parâmetros clínicos e o grau de diferenciação em carcinomas espinocelulares bucais. Sessenta e cinco amostras foram selecionadas para a construção de um bloco de microarranjo tecidual, e a técnica de imunoistoquímica foi realizada para os diferentes marcadores. Uma análise semi-quantitativa das células tumorais positivas foi realizada por dois observadores calibrados e cegos (Kappa>0.75). Os testes estatísticos Mann-Whitney e Kruskal-Wallis foram utilizados para a análise dos dados e a correlação entre as variáveis foi investigada com o teste de Spearman. O nível de significância foi determinado em p <0.05. Observamos maior expressão de Bmi-1 em tumores localizados em palato (p <0.0001). Além disso, tumores pobremente diferenciados apresentaram maior quantidade de células positivas para Bmi-1 (p=0.0011). Não encontramos outras correlações ou associações significativas. Em conclusão, carcinomas espinocelulares pobremente diferenciados e localizados no palato apresentam maior marcação imunoistoquímica de Bmi-1, o que pode caracterizar a ativação do processo de transição epitélio-mesênquima nesses tumores.

Are 30 minutes of rest between two incremental shuttle walking tests enough for cardiovascular variables and perceived exertion to return to baseline values?

Objective: To verify whether 30 minutes of rest between two incremental shuttle walking tests (ISWT) are enough for cardiovascular variables and perceived exertion to return to baseline values in healthy subjects in a broad age range. Method: The maximal exercise capacity of 334 apparently healthy subjects (age ≥18) was evaluated using the ISWT. The test was performed twice with 30 minutes of rest in between. Heart rate (HR), arterial blood pressure (ABP), dyspnea, and leg fatigue were evaluated before and after each test. Subjects were allocated to 6 groups according to their age: G1: 18-29 years; G2: 30-39 years; G3: 40-49 years; G4: 50-59 years; G5: 60-69 years and G6: ≥70 years. Results: All groups had a good performance in the ISWT (median >90% of the predicted distance). The initial HR (HRi) of the second ISWT was higher than the first ISWT in the total sample (p<0.0001), as well as in all groups (p<0.0001). No difference was observed in the behavior of ABP (systolic and diastolic) and dyspnea between the two tests, but this difference occurred for leg fatigue (greater before the second ISWT) in G1 (p<0.05). Most subjects (58%) performed better in the second test. Conclusion: 30 minutes of rest between two ISWTs are not enough for all cardiovascular variables and perceived exertion to return to baseline values. However, this period appears to be sufficient for blood pressure and performance to recover in most subjects. .

Knockdown of Bmi1 inhibits bladder cancer cell growth both in vitro and in vivo by blocking cell cycle at G1 phase and inducing apoptosis / 华中科技大学学报（医学）（英德文版）

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.

The treatment effects and mechanisms of pyrroloquinoline quinone on defective teeth and mandible in Bmi-1 knockout mice / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the treatment effects and mechanisms of pyrroloquinoline quinine(PQQ) on defective teeth and mandible in Bmi-1 knockout mice.</p><p><b>METHODS</b>Male and female Bmi1(+/-) mice were paired with each other from the same nest. At the age of 7 weeks, the mice were divided into three groups, the wild type mice received normal diet(10 mice, WT group), Bmi1(-/-) mice received normal diet (10 mice, BKO group), and the Bmi1(-/-) mice received normal diet and PQQ diet(10 mice, BKO+PQQ group). X-ray and micro- CT were used to detect mandible and dental size and bone mineral density. HE staining, histochemical and immunohistochemical methods were respectively used to detect alveolar bone thickness of cortical bone, predentin thickness of mandibular first molar, mandibular osteoblast number and osteoclast number. Flow cytometry was used to detect reactive oxygen species(ROS) levels of various organs(femur, thymus and liver). The data were statistically analyzed with one-way ANOVA and t test.</p><p><b>RESULTS</b>Compared with BKO mice, BKO+PQQ mice partially rescued total body phenotype, increased body weight and prolonged survival time. X- ray and micro- CT showed the size of the mandible and teeth and bone mineral density of PQQ+BKO mice increased compared with BKO mice. In PQQ+BKO mice, mandibular alveolar bone cortical thickness [(68.65 ± 0.25) µm] was significantly different from that in BKO mice [(42.45 ± 0.35) µm] (P<0.01). There was significant difference in predentin thickness of mandibular first molar between PQQ+BKO mice [(4.25 ± 0.15) µm] and BKO mice [(31.55 ± 0.35) µm] (P<0.001). The number of osteoblasts in the mandible of BKO+PQQ mice [(38.45 ± 0.25) cell/mm³] was significantly higher than that in the BKO mice [(18.15 ± 0.55) cell/mm³] (P<0.01). However, the number of osteoclasts in the BKO+PQQ mice [(9.45 ± 0.25) cell/mm³] was significantly lower than that in the BKO group [(14.25 ± 0.35) cell/mm³] (P<0.01). Compared with the BKO group, ROS levels of the femur, thymus and liver in the BKO+PQQ mice were significantly decreased (P<0.01).</p><p><b>CONCLUSIONS</b>The results indicate that PQQ may have treatment effects on defective teeth and mandible through promoting osteoblast bone formation and reducing osteoclast bone resorption, scavenging ROS and reducing DNA damage.</p>

Effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells (CSC-LCs).@*METHOD@#The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 gene (LV-Bmi-1shRNA) was constructed and was used to infect CD44+ nasopharyngeal carcinoma cells which were sorted by flow cytometry. A lentiviral which included a random sequence was also designed to serve as a negative control. We employed fluorescence microscope and flow cytometry to detect infection efficiency; real-time PCR was used to detect Bmi-1 and its downstream gene while each protein expression level was confirmed by western blotting protocol; CCK-8 proliferation assay was applied to measure proliferation capacity; tumor spheroid assay was used to evaluate the self-renewal capacity. Colony formation assay was used to measure cell colony formation capability; flow cytometry analyzed cell cycle distribution.@*RESULT@#The constructed LV-Bmi-1shRNA successfully infected into the CD44+ nasopharyngeal carcinoma cells. The infection efficiency could reach above 95%; LV-Bmi-lshRNA effectively inhibited Bmi-1 mRNA and protein expression, while the downstream gene p16INK4a and p14ARF mRNA as well as protein expression level were upregulated (P < 0.05). Notablely, the proliferation, colony formation, self-renewal capabilities of the experimental group decreased significantly (P < 0.05). In addition, the cell cycle arrested at the G0-G1 phase.@*CONCLUSION@#Gene silencing of Bmi-1 inhibited the proliferation, colony formation and self-renewal capabilities of the CD44+ nasopharyngeal carcinoma CSC-LCs, inhibited the cell cycle processes, which may mediate through Bmi1-p16INK4a/p14ARF-p53 pathway. Our experimental results indicated that Bmi-1 gene may play an important role in the maintenance of the stem cell-like characteristics of CD44+ nasopharyngeal carcinoma cells. Bmi-1 gene may be a potential new target for the treatment of nasopharyng al carcinoma in the future.

Expression and Clinical Significance of Bmi-1 and P14 in Extranodal NK/T-cell Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the expression and clinical significance of Bmi-1 and P14 in extranodal NK/T-cell lymphoma (ENKTCL) tissue.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Bmi-1 protein over-expresses in ENKTCL tissues that may display a negative-regulation effect on P14 in the genesis and progress of ENKTCL.</p>

Construction of shRNA-Bmi-1 plasmid and its effect on proliferation of ECA109 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the role of Bmi-1 gene in the proliferation of squamous carcinoma cells and whether the silencing Bmi-1 can inhibit the growth of squamous cell carcinomas cells.</p><p><b>METHODS</b>The expressions of Bmi-1 in primary cultured Fibroblasts, karatinocyte cell line Hacat,squamous carcinoma cell line A431, and ECA109 were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Recombinant plasmid inserted with Bmi-1 gene short hairpin RNA (shRNA) expression vector PGPU6/GFP/Neo-shBmi-1 was constructed and transfected into ECA109 cells with control set. After transfection for 48 and 72 hours,the mRNA and protein levels of Bmi-1 were examined with RT-PCR and Western blot analysis, respectively. The proliferation of the ECA109 cells was evaluated by MTT method and flow cytometry.</p><p><b>RESULTS</b>Bmi-1 was highly expressed in A431 and ECA109 cells than in Fibroblast cells and Hacat cells. The mRNA and protein expressions of Bmi-1 were significantly silenced in ECA109 cells after recombinant expression vector PGPU6/GFP/Neo-shBmi-1 transfection (P<0.05). Compared with the control groups,the proliferation of ECA109 transfected with PGPU6/GFP/Neo-shBmi-1 was significantly inhibited (P<0.05), and cells in G1 phase increased while in S phase decreased (P<0.05).</p><p><b>CONCLUSIONS</b>Bmi-1 is involved in the proliferation of squamous carcinoma cells. After the silencing of Bmi-1 expression,the proliferation ECA109 cells is suppressed due to the altered cell cycle.</p>

Effect of silencing Bmi-1 expression in reversing cisplatin resistance in lung cancer cells and its mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of silencing Bmi-1 expression in reversing cisplatin resistance in human lung cancer cells and explore the possible mechanisms.</p><p><b>METHODS</b>Cisplatin-resistant A549/DDP cells with small interference RNA (siRNA)-mediated Bmi-1 expression silencing were examined for cisplatin sensitivity using MTT assay and alterations in cell cycle distribution and apoptosis with flow cytometry, and the changes in cell senescence was assessed using β-galactosidase staining. The protein expressions of Bmi-1, P14(ARF), P16(INK4a), P53, P21, Rb and ubi-H2AK119 in the cells were determined with Western blotting.</p><p><b>RESULTS</b>A549/DDP cells showed significantly higher Bmi-1 expression than A549 cells. After siRNA-mediated Bmi-1 silencing, A549/DDP cells showed significantly enhanced cisplatin sensitivity with an increased IC50 from 40.3±4.1 µmol/L to 18.3±2.8 µmol/L (P<0.01) and increased cell percentage in G0/G1 phase from (48.9±2.3)% to (78.7±7.6)% (P<0.01). Silencing Bmi-1 did not cause significant changes in the cell apoptosis rate but induced obvious senescence phenotype in A549/DDP cells with down-regulated expression of ubi-H2AK119 and up-regulated expressions of P14(ARF), P16(INK4a), P53, P21 and Rb.</p><p><b>CONCLUSION</b>Silencing Bmi-1 by RNA interference can induce cell senescence and resensitize A549/DDP cells to cisplatin possibly by regulating INK4a/ARF/Rb senescence pathway.</p>

Sphere-forming-like cells (squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma / 国际口腔科学杂志·英文版

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell (CSC) traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas (SCCs) and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation (CD) 44, CD133, acetaldehyde dehydrogenase 1 (ALDH1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), Nestin, octamer-binding transcription factor 4 (Oct4) and reduced expression protein-1 (Rex-1) expression with reverse transcription-polymerase chain reaction (RT-PCR); chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers (CD44, Bmi-1, Nestin, Oct4 and Rex-1), capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts (with histological features resembling those of the original VX2 rabbit buccal SCC) from the transplantation of 10(3) undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.

The role of Bmi-1 gene in submandibular gland of mice / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine the role of Bmi-1 in the submandibular gland (SMG) of mice.</p><p><b>METHODS</b>SMG of 4-week wild-type (WT) and Bmi-1 null (Bmi-1(-/-)) mice was analyzed on the weight, salivary flow rate, hematoxylin-eosin staining morphological differences and the changes in proliferation and aging by histology, immunohistochemistry and Western blotting.</p><p><b>RESULTS</b>Compared with WT mice, the average static salivary flow rate [WT:(0.21 ± 0.02) µg/min,Bmi-1(-/-): (0.10 ± 0.02) µg/min] (P = 0.001) and the submandibular gland weight [WT: (1.89 ± 0.15) µg], Bmi-1(-/-): [(1.34 ± 0.07)µg] (P = 0.003) of the male Bmi-1(-/-) mice were significantly decreased, the number of gland duct increased, and the granular convoluted duct showed reduced diameter and branches. More senescence-associated β-galactosidase positive cells existed in SMG of Bmi-1(-/-)mice (WT:0.00, Bmi-1(-/-): 0.18 ± 0.02), and Ki-67 immunopositive cells decreased in SMG of Bmi-1(-/-) mice (WT:0.40 ∼ 0.47, Bmi-1(-/-): 0.18 ∼ 0.20) (P = 0.000). The expression of p16 (WT:1.00 ± 0.12, Bmi-1(-/-): 0.00 ± 0.00) (P = 0.003) and p19 (WT:0.97 ± 0.09, Bmi-1(-/-): 5.09 ± 0.21) (P = 0.004) were up-regulated dramatically in SMG of the Bmi-1(-/-) mice.</p><p><b>CONCLUSIONS</b>Bmi-1 gene deficiency causes abnormal function of SMG by inducing senescence phenotype of SMG.</p>

MicroRNA-218 expression and its role in hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of microRNA-218 (miR-218) and its role in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Forty-six pairs of fresh surgical specimens of HCC and adjacent tissues were examined for miR-218 expression using qRT-PCR. A miR-218 mimic was transfected into HepG2 cells, and the cell viability and apoptosis were analyzed by MTT assay and flow cytometry, and the potential targets of miR-218 were detected by qRT-PCR and Western blotting.</p><p><b>RESULTS</b>The expressions of miR-218 in HCC tissues were significantly down-regulated compared to those in the adjacent tissues (P<0.05). Down-regulation of miR-218 was found to correlate significantly with the tumor size (>5 cm) and an advanced TNM stage (III+IV) (P<0.05). Ectopic expression of miR-218 in HepG2 cells resulted in suppressed cell proliferation and enhanced cell apoptosis as well as the down-regulation of Bmi-1 and CDK6 mRNA and protein expressions (P<0.05).</p><p><b>CONCLUSION</b>The low-expression of miR-218 is correlated with malignant clinicopathological characteristics of HCC, and miR-218 may inhibit cell proliferation and promote cell apoptosis by down-regulating Bmi-1 and CDK6 in HCC.</p>

Effects of Bmi-1 RNAi gene on laryngeal carcinoma Hep-2 cells / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To construct the Bmi-1 RNAi expression vector and investigate its influence on the proliferation and invasiveness of laryngeal carcinoma Hep-2 cells.@*METHOD@#The recombinant vshRNA-Bmi-1 plasmid of Bmi-1 RNAi was constructed by the lentiviral expression system, pHelper1.0/pHelper2.0/pGCL2GFP. Bmi-1mRNA and protein expressions of stably transfected laryngeal carcinoma cells were identified tespectively by real-time PCR and Western blot analyses. The changes of the proliferation and invasiveness of laryngeal carcinoma were detected by clone formation test and an invasion assay.@*RESULT@#The Bmi-1 mRNA expressions of stably transfected laryngeal carcinoma Hep-2 cells were significantly decreased. The expression of Bmi-1 protein in laryngeal carcinoma Hep-2 cells was significantly decreased. And the inhibitory rates were 79% and 88% respectively. Whereas the proliferation and invasiveness of Hep-2 cells were significantly reduced.@*CONCLUSION@#The Bmi-1 RNAi expression vector were constructed successful. Reduced Bmi-1 expression of Hep-2 cells demonstrated the role of Bmi-1 RNAi in restraining proliferation and invasiveness of laryngeal carcinoma cells.