Résumé
ObjectiveTo study the effects of BMI1 on the proliferation and drug resistance of cervical cancer (CC) and endometrial cancer (EC) cells. In addition, the mechanism of paclitaxel (PTX) resistance induced by BMI1 was explored. MethodsIn this study, we utilized the GTEx, Cbioportal, TCGA, and CPTAC databases to comprehensively analyze the mutation rate as well as mRNA and protein expression profiles of BMI1 in CC and EC. Subsequently, immunohistochemistry (IHC) analysis was employed to evaluate the protein expression levels of BMI1 in 40 pairs of CC and 40 pairs of EC tissue samples. Western blot was conducted to investigate alterations in downstream factor protein levels upon BMI1 knockdown in CC and EC cells. Furthermore, functional experiments were performed to elucidate the role of BMI1 in CC and EC cells. Finally, we assessed the synergistic anti-growth effect by combining BMI1 knockdown with paclitaxel treatment in vitro. ResultsThe Cbioportal database revealed that BMI1 amplification, misinterpretation, and splicing occurred in 1.5% of CC patients and 1.9% of EC patients. Mining the data from TCGA and CPTAC databases, high mRNA levels of BMI1 were associated with the pathological type of CC and lower overall survival, and high protein levels of BMI1 were related to EC’s pathological type and tumor grade. Furthermore, the BMI1 protein level is overexpressed in cancer tissues of CC and EC compared with normal tissues, as detected by IHC analysis. Besides, drug sensitivity experiments showed that overexpression of BMI1 resulted in decreased sensitivity of HeLa and HEC-1-A cells to a variety of anticancer drugs, including paclitaxel. In order to further analyze the relationship between BMI1 and paclitaxel resistance, Western blot was used to detect the changes in the protein levels of downstream factors of BMI1 in HeLa and HEC-1-A cells after BMI1 knockdown. The results showed that the level of anti-apoptotic factor Bcl-2 protein decreased, while that of pro-apoptotic factor BAX increased with BMI1 knockdown. Additionally, we showed that high expression of BMI1 promoted the proliferation and migration of CC and EC cells in vitro. Moreover, CC and EC cells with low BMI1 expression were more sensitive to the paclitaxel. ConclusionThe expression of BMI1 is significantly upregulated in tumor tissues from patients with cervical and endometrial cancer, and silencing BMI1 makes CC and EC cells more sensitive to paclitaxel via enhancing pro-apoptotic regulation.
Résumé
Objective:To investigate the impact of BMI1 expression in OSCC on the recruitment and differentiation of tumor-associat-ed macrophages(TAMs).Methods:BMI1 expression in 519 cases of OSCC tissues and 44 normal controls was analyzed using online datasets of GEPIA 2.0,and validated in 3 cases of OSCC samples and controls by qRT-PCR and western blotting.The function of BMI1/NF-κB axis during OSCC carcinogenesis was investigated by CCK8 assays,wound healing test and transwell assays.Macrophage phenotypes and recruitment were determined using qRT-PCR and western blotting following coculture of the cells with human monocyte cells(THP-1)by OSCC conditioned medium.Moreover,a cell line-derived xenograft(CDX)model was used to detect the effect of BMI1 on tumor growth in vivo.Results:Compared with the normal tissues and cells,the expression level of BMI1 in OSCC tissues and cells was significantly upregulated.BMI1 knockdown impaired the proliferation,migration,and invasion abilities of OSCC cell lines in NF-κB-dependent manner.Furthermore,OSCC cells with high BMI1 expression inhibited the migration of THP-1 cells,promoted M2-like macrophage polarization through NF-κB pathway in vitro.Xenograft experiments further confirmed the inhibitory effect of BMI1 knockdown on the tumorigenesis ability of OSCC cells in vivo.Conclusion:BMI1 promotes M2-like polarization by regulating NF-κB and may be used as a potential therapeutic target for antitumor immunity.
Résumé
Objective To investigate the mechanisms of miRNA-15a and miRNA-16 in the process of reversing cisplatin resistance in ovarian cancer.Methods Human ovarian cancer cisplatin-resistant cell lines CoC1/DDP were transfected with miRNA-15a and miRNA-16 mimics and treated with cisplatin.qRT-PCR was used to detect the expression levels of miRNA-15a and miRNA-16 in normal CoC1/DDP cell group,cisplatin treated group,negative control group,miRNA-15a transfected group,miRNA-16 transfected group and overexpressed Bmi-1 plasmid.Western blot was used to detect the expression level of Bmi-1 in each group,CCK-8 and Annexin V/PI were used to detect cell survival and apoptosis,and γ-H2AX immunofluorescence was used to detect cell apoptosis.Results The CoC1/DDP ovarian cancer cell line shows low expression of miRNA-15a and miRNA-16,and high expression of Bmi-1 protein,which makes it resistant to cisplatin.When the levels of miRNA-15a and miRNA-16 are overexpressed,the Bmi-1 protein decreases(P<0.05),leading to a decrease in cell survival rate(P<0.05),a significant increase in DNA apoptosis(P<0.05),and more severe DNA damage(P<0.05).Overexpression of Bmi-1 plasmid can increase cell viability(P<0.05)and reduce the rate of cell apoptosis(P<0.05).Conclusion The Bmi-1 protein may be a target for the regulation of miRNA-15a and miRNA-16,and overexpression of miRNA-15a and miRNA-16 can increase the sensitivity of ovarian cancer cells to cisplatin by reducing the Bmi-1 protein.This provides a new idea for predicting molecular markers of cisplatin resistance in ovarian cancer and overcoming drug resistance targets in ovarian cancer.
Résumé
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.
Sujets)
Humains , Mâle , Femelle , Leucémie-lymphome lymphoblastique à précurseurs B et T , Réaction de polymérisation en chaine en temps réel , Protéines du groupe Polycomb , Complexe répresseur Polycomb-1 , Protéine homéotique NanogRésumé
Liver is the most common metastatic site for colorectal cancer (CRC), there is no satisfied approach to treat CRC liver metastasis (CRCLM). Here, we investigated the role of a polycomb protein BMI-1 in CRCLM. Immunohistochemical analysis showed that BMI-1 expression in liver metastases was upregulated and associated with T4 stage, invasion depth and right-sided primary tumor. Knockdown
Résumé
: Aim To detect the effect of B-cell-specific moloney murine leukemia virus in section site 1 (Bmi-1) on the migration and invasion of renal cancer cell line 786-0 and ACHN and its possible mechanism. Methods siRNA interfered with the expression of Bmi-1 in renal cancer cells, and the effects of Bmi-1 on the migration and invasion of renal cancer cells were detected by scratch healing and Transwell chamber assay. The expression of invasion and metastasis related proteins in 786-0 and ACHN after Bmi-1 knockdown were detected by Western blot. Results Protein expression of Bmi-1 increased in renal cancer cells. In vitro synthesis of specific Bmi-1 gene siRNA significantly inhibited the expression of Bmi-1 in 786-0 and ACHN cells. After silencing Bmi-1, the scratch healing ability and migration and invasion activity of renal cancer cells were significantly lower than those of control cells. The results of Western blot showed that epithelial markers E-cadherin increased after Bmi-1 silencing, while the expression of Vimentin decreased, and the phosphorylation level of Akt was also reduced. Conclusions Silencing Bmi-1 inhibits the migration and invasion of renal cancer cells, and its effect is related to the activation of Akt signaling pathway.
Résumé
Objective The purpose of this study was to investigate the mechanism of Bmi-1 regulating the sensitivity of non-small cell lung cancer (NSCLC) to chemotherapy by observing its effects on multidrug-resistance protein 1 (MDR1) and apoptosis-related proteins. Methods Small interfering RNAs (siRNA) targeting Bmi-1 were transfected into A549 and A549/DDP cells of NSCLC and the logarithmic-phase cells were randomly divided into a siRNA-Bmi-1, a siRNA-negative control and a blank control group. The A549 cells were treated with siRNA-Bmi-1, DDP or Bmi-1+DDP, or left untreated (the control). CCK8 assay was employed to measure the 50% inhibitory concentration (IC50) of cisplatin in the A549 and A549/DDP cells before and after treatment. The apoptosis of the cells was detected by flow cytometry, the mRNA expression of Bmi-1 determined by RT-PCR, and the relationship of Bmi-1 with MDR1 and cleaved caspase-3 proteins analyzed by Western blot. Results After transfection, the relative mRNA and protein expressions of Bmi-1 in the A549 and A549/DDP cells were significantly lower in the siRNA-Bmi-1 than in the blank control group (P < 0.05), but higher in the A549/DDP than in the A549 cells. The survival rates of the A549 and A549/DDP cells were decreased with the increased concentration of cisplatin (P < 0.05), even lower in the Bmi-1+DDP than in the DDP subgroup (P < 0.05). The apoptosis rate of the A549 cells was markedly higher in the Bmi-1+DDP than in the DDP, Bmi-1 and control groups ([39.65±3.41]% vs [23.11±1. 62] %, [2.05±1.56]% and [1.98±1.05]%, P < 0.05). After 24 hours of treatment with DDP, both the expressions of Bmi-1 and MDR1 were remarkably elevated, while the down-regulation of Bmi-1 significantly decreased the expression of MDR1 and increased that of cleaved caspase-3. Conclusion The expression of siRNA-Bmi-1 makes non-small lung cancer cells more sensitive to cisplatin, which might be associated with its inhibition of MDR1 expression and activation of apoptosis-related proteins.
Résumé
Objective@#To explore the effects of Bmi1 on proliferation of mouse cranial suture mesenchymal cells.@*Methods@#Primary posterior frontal and sagittal suture derived cells were isolated from the 2-5 d old C57BL/6 suckling mice (n=6) of the same brood and cultured. Flow cytometry and multilineage differentiation assay were performed to identify the mesenchymal stem cells (MSCs) characteristics of the 2 kinds of cranial suture-derived cells. The mRNA expression of stem cell related genes, Bmi1, Twist1, Gli1 and Axin2 were detected by real-time quantitative polymerase chain reaction (RT-PCR). Then, the proliferation and downstream protein expression were analyzed after down-regulation of Bmi1 in the sagittal suture derived MSCs by transfecting Bmi1 siRNA. The t test was used to compare the mean between two groups. Statistical significance was set at P<0.05.@*Results@#The mouse cranial suture derived cells were successfully cultured in vitro. These cells expressed typical MSCs markers, CD44, CD90, CD73, except for CD34. These cells had osteogenic, adipogenic and chondrogenic differentiation potency. RT-PCR results showed that the mRNA expressions of Bmi1 (0.006 30±0.000 58 vs 0.002 60±0.000 34, t=5.430, P=0.005 6), Twist1(0.000 31±0.000 04 vs 0.000 15±0.000 02, t=3.343, P=0.028 8), Axin2(0.000 33±0.000 03 vs 0.000 17±0.000 05, t=3.067, P=0.037 4) and Gli1 (0.001 10±0.000 13 vs 0.000 60±0.000 33, t=3.956, P=0.016 7) were significantly decreased in the posterior frontal suture MSCs compared with those in sagittal suture derived cells. Among them, Bmi1 has the largest decline. After down-regulation of Bmi1 in sagittal suture MSCs, the protein expression level of Ink4a was significantly up-regulated compared with the control group, and the cell proliferation ability was significantly decreased.@*Conclusions@#Inhibition of Bmi1 expression can up-regulate the expression of Ink4a protein and decrease the proliferation ability of suture MSCs, which may lead to craniosynostosis.
Résumé
BACKGROUND: ATP-binding cassette transporters are important in the mechanism of multidrug resistance. ABCB1 displays a high affinity for imatinib. BMI1 is a polycomb group protein thought to be overexpressed in leukemic cells. METHODS: This study was conducted to investigate the prognostic value of ABCB1 and BMI1 expressions in chronic myeloid leukemia (CML). Expression levels were measured in 81 patients newly diagnosed with CML and 20 healthy controls by real time reverse transcription- PCR. RESULTS: The ABCB1 expression levels did not differ between patients with CML and controls. Low ABCB1 mRNA levels were observed in patients who achieved an optimal response compared to suboptimal and resistant cases (P=0.005). Non-responders showed the highest ABCB1 levels. ABCB1 expression did not affect the progression-free survival (PFS) of patients. BMI1 expression was higher in patients than that in controls (P=0.001). Patients in advanced phases expressed higher levels of BMI1 than those in the chronic phase (P=0.004). High BMI1 expression was associated with a shorter PFS. CONCLUSION: ABCB1 mRNA expression may serve as a predictor of the optimal response to imatinib treatment in patients with CML. BMI1 expression was higher in the accelerated and blastic crisis phases of CML and associated with a shorter PFS.
Sujets)
Humains , Transporteurs ABC , Survie sans rechute , Multirésistance aux médicaments , Mésilate d'imatinib , Leucémie myéloïde chronique BCR-ABL positive , Réaction de polymérisation en chaîne , Réaction de polymérisation en chaine en temps réel , ARN messagerRésumé
Objective The role of BMI1 gene in the development of gastrointestinal stromal tumor (GIST) has not yet been clarified. This study aimed to explore the expression of BMI1 gene in gastrointestinal stromal tumor,and analyze its relationship with clinical pathological features of GIST. Methods The clinical data of 68 GIST patients treated in The First People's Hospital of Nan-ning from August 2012 to October 2015 were analyzed retrospectively. The expression of BMI1 in normal gastrointestinal tissues and GIST tissues were detected with immunohistochemistry method,and analyzed the relationship between various clinicopathological pa-rameters of GIST and BMI1. The expression of BMI1 protein was detected by Western blot. Results The positive rate of BMI1 was much higher in GIST group than in non-GIST (76.47% vs 36.84%,P<0.05). The difference in the expression of BMI1 protein between the different risk groups was statistically significant (P<0.05). The positive expression rate was the highest in the high-risk group (93.75%),but had no statistically significant difference among different genders,age,locations,histological types and whether me-tastasis (P>0.05). Expression of proliferation genes such as PCNA,CyclinD1 mRNA in BMI1 positive group were higher than those in BMI1 negative group,the expression of Pro-apoptotic genes such as Caspase-7,Smac mRNA were lower than those in BMI1 negative group,the expression of anti-apoptosis genes such as Livin,p53,Bcl-2 mRNA were higher than those in BMI1 negative group (P<0.05). Conclusion The expression of BMI1 protein was increased in GIST tissue. It is correlated with the risk classification,and is an important factor affecting the prognosis of patients.
Résumé
Long non-coding RNA antisense non-coding RNA in the INK4 locus (ANRIL) has been reported to promote tumorigenesis via regulating microRNA (miR)-99a in gastric cancer cells. However, the role of each component involved in it is still not well understood. This study aimed to verify the role of ANRIL in gastric cancer as well as the underlying mechanisms. ANRIL levels in clinical gastric cancer tissues and cell lines were tested by qPCR. Effects of ANRIL silence on cell viability, migration and invasion, apoptosis, and miR-99a expression in MKN-45 and SGC-7901 cells were measured using CCK-8, Transwell assay, flow cytometry, and qPCR assays, respectively. Then, effects of miR-99a inhibition on ANRIL-silenced cells were evaluated. B-lymphoma Mo-MLV insertion region 1 (BMI1) expression, after abnormal expression of ANRIL and miR-99a, was determined. Finally, expression of key proteins in the apoptotic, Notch, and mTOR pathways was assessed. ANRIL level was elevated in gastric cancer tissues and cell lines. Knockdown of ANRIL suppressed cell viability, migration, and invasion, and increased apoptosis through up-regulating miR-99a. Furthermore, ANRIL silence down-regulated BMI1 via up-regulating miR-99a. BMI1 silence down-regulated Bcl-2 and key kinases in the Notch and mTOR pathways and up-regulated p16 and cleaved caspases. We verified the tumor suppressive effects of ANRIL knockdown in gastric cancer cells via crosstalk with miR-99a. Together, we provided a novel regulatory mechanism for ANRIL in gastric cancer, in which ANRIL silence down-regulated BMI1 via miR-99a, along with activation of the apoptotic pathway and inhibition of the Notch and mTOR pathways.
Sujets)
Humains , Tumeurs de l'estomac/métabolisme , Régulation négative , microARN/métabolisme , Sérine-thréonine kinases TOR/métabolisme , ARN long non codant/génétique , Carcinogenèse/génétique , Tumeurs de l'estomac/anatomopathologie , Transfection , Régulation de l'expression des gènes tumoraux , Régulation positive , Apoptose/génétique , Lignée cellulaire tumorale , Invasion tumoraleRésumé
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.
Sujets)
Humains , Mâle , Femelle , Adulte , Adulte d'âge moyen , Sujet âgé , Sujet âgé de 80 ans ou plus , Carcinome épidermoïde/anatomopathologie , Transition épithélio-mésenchymateuse , Tumeurs de la bouche/anatomopathologie , Marqueurs biologiques tumoraux/métabolisme , Cadhérines/métabolisme , Immunohistochimie , Complexe répresseur Polycomb-1/métabolisme , Analyse sur puce à tissusRésumé
Colorectal stem cells have many bio-markers, including Lgr5 which expression is associated with THE stage of disease , also regulating the cell cycle , anothers is +4 stem cell , which is associated with tumor heteroge-neity, also expressed Bmi1, arresting cell cycle.Besides there is Msi1.Many studies show that those markers are highly expressed in colorectal cancer , which activate Notch and Wnt signaling pathway , and can promote the pro-gress of tumor .
Résumé
Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma Mo-MLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism. Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5, 1.5, 3.0 Pa) and under different time phase (1, 2, 6, 24 h) were loaded by parallel-plate flow chamber system. The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt) and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting. The signaling inhibitors, wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor), were used to investigate possible mechanical signal transduction pathway. Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h. All FSS with different magnitude could increase Bmi-1 expression, especial at high FSS (3.0 Pa). Meanwhile, FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs. After treated with wortmannin, the expression of Bmi-1 was inhibited prominently, however, PD98059, the expression of Bmi-1 did not change. Conclusions FSS can activate the expression of Bmi-1, the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS, and Akt signal molecule plays an important role during the process. These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.
Résumé
Objective To detect the expression of BMI-1, and to discuss its role in the development of gastric cancer. Methods The expression of BMI-1 protein and mRNA in gastric cancer and adjacent tissues was detected by immunohistochemical P-V method (78 cases) and semi-quantitative RT-PCR (30 cases). Results The positive expression rate of BMI-1 protein in gastric cancer patients was 66.7 % (52/78), and it was 19.2 % (15/78) in adjacent tissues, the difference was statistically significant (χ2= 35.815, P= 0.000). The expression of BMI-1 mRNA in 30 gastric cancer tissues was 0.23 ± 0.12, it was 0.03 ± 0.12 in adjacent tissues, the difference was statistically significant (t=8.372, P=0.000). The expression of BMI-1 protein and mRNA was correlated with the degree of tumor differentiation, depth of invasion, lymph node metastasis and TNM staging (all P< 0.05). Conclusion The expression of BMI-1 is closely related to the occurrence and development of gastric cancer, which may provide guidance for molecular pathogenesis, targeted therapy and prognosis of gastric cancer.
Résumé
Objective To investigate the effects of BMI-1 expression inhibition by RNA interference on the radiosensitivity of esophageal cancer TE-13 cells and its mechanism.Methods The siRNA based on the sequence of BMI-1 mRNA was synthesized to transfect cultured TE-13 cells as BMI-1 siRNA group,a negative one was synthesized to transfect cultured TE-13 cells as negative control group (NC group),and untransfected TE-13 cells were named as control group.The expression of the BMI-1 mRNA and protein in TE-13 cells was measured by quantitative real-time PCR and Western blot,respectively.The cell proliferation and the radiosensitivity of TE-13 cells were measured by MTS and colony-forming assay,respectively.Flow cytometry was used to analyze cell cycle and apoptosis.The expression of BCL-2 and BAX in TE-13 cells was measured by Western blot.Comparison between groups was made by analysis of variance.Results The BMI-1 siRNA group had significantly lower expression of BMI-1 mRNA and protein than the control group and the NC group (P=0.000,0.000).The proliferation of TE-13 cells in the BMI-1 siRNA group decreased significantly after irradiation (P=0.031).The colony-forming assay showed that the BMI-1 siRNA group had a significantly higher radiosensitivity than the control group and the NC group (P=0.000).After irradiation,the BMI-1 siRNA group had a significantly lower percentage of cells in G2/M phase than the control group and the NC group (P=0.000,0.000).The BMI-1 siRNA group had a significantly increased apoptosis rate (P=0.000,0.000),significantly reduced expression of BCL-2(P=0.000,0.000),and significantly increased expression of BAX after irradiation (P=0.000,0.000).Conclusions BMI-1 siRNA can inhibit the expression of BMI-1 gene in esophageal cancer TE-13 cells,eliminate the cell cycle arrest in G2/M phase,induce cell apoptosis after ionizing irradiation in vitro,and increase the radiosensitivity,which may be related to the regulation of the expression of BCL-2 and BAX.
Résumé
Background and purpose:B cell-specific MLV integration site 1 (BMI-1) gene plays an important role in DNA damage after exposure to irradiation. The present study aimed to investigate the effect ofBMI-1 on radio-sensitivity of esophageal carcinoma cell after down-regulation of BMI-1 expression by silencing siRNA.Methods:Three pairs of siRNA based on the sequences of the BMI-1 mRNA were synthesized (siRNA1, siRNA2 and siRNA3) by compa-ny, and transfected into cultured TE13 cells as the BMI-1 siRNA groups, and a negative one was synthesized to be used as the negative control (NC) group. The untransfected group was named as the control group. BMI-1 mRNA and protein expression in esophageal cancer TE13 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot in different groups. This study used flow cytometry assay to analyze cell cycle of transfected cells, and examined cellular growth and radiosensitivityin vitro by MTT and clone formation assay. mRNA and protein expression of p16 and CDK4 in esophageal cancer TE13 cells were detected by RT-PCR and Western blot.Results:The results of RT-PCR and Western blot showed that the expressions of BMI-1 at gene and protein levels were inhibited after silencing the BMI-1 gene. The mRNA and protein expression of BMI-1 in BMI-1 siRNA3 group were both significantly lower than that in BMI-1 siRNA1 and 2 groups. There was no significant difference in the cell proliferation among control, NC and BMI-1 siRNA3 groups. The values ofD0,Dq, and SF2 in BMI-1 siRNA3 group were 1.761, 2.122 and 0.6255, respectively, obvi-ously lower than those in control group (2.514, 2.694 and 0.8268) and those in NC group (2.506, 2.664 and 0.8231), while the value of N in BMI-1 siRNA3 group (3.336) was higher than that in control group (2.92) and that in NC group (2.895), which showed higher radiosensitivity in BMI-1 siRNA3 group. In addition, the cell cycle was arrested at G2/M phase after irradiation in control and NC groups. The percentage of G0/G1 phase in BMI-1 siRNA3 group was higher than that of control group and NC group, while the percentage of G2/M phase was lower than those in the latter. The up-regulation of p16 and down-regulation of CDK4 at gene and protein levels were detected after knockdown of BMI-1 expression by siRNA (P<0.01).Conclusion:siRNA could inhibitBMI-1 gene expression in esophageal cancer TE13 cells and enhance radiosensitivity, followed by eliminating the cell cycle arrest at G2/M stage after irradiationin vitro, which is related to the regulation of the protein expression ofp16 andCDK4.
Résumé
Objective To detect the expressions of c-myc,Bmi-1,serum insulin-like growth factor-Ⅰ (IGF-Ⅰ) and insulin-like growth factor binding protein-3 (IGFBP-3) in diffuse large B-cell lymphoma (DLBCL),and to analyze their relations with clinical stages,efficacy and prognosis.Methods 102 cases of incipient patients with DLBCL and 60 patients or health examination volunteers were chosen as DLBCL group and control group,respectively.Immunohistochemical method was used to detect expressions of c-myc and Bmi-1 in DLBCL wax samples.Chemiluminescence immunoassay method was used to determine the levels of serum IGF-Ⅰ and serum IGFBP-3.The expression differences of these factors between DLBCL group and control group and their relations with pathological types,clinical stage,IPI and chemotherapy were analyzed.Results The positive rates of c-myc and Bmi-1 were 71.6 % (73/102) and 61.8 %(64/102) in the tissues of DLBCL,respectively.The positive rates of c-myc and Bmi-1 in non-GCB group were higher than those in GCB group [c-myc:80.0 % (48/60) vs 59.5 % (25/42);Bmi-1:71.7 % (43/60) vs 50.0 % (21/42)].With the increase of IPI score,the expressions of c-myc and Bmi-1 were enhanced,but there were no statistical differences between Ⅲ-Ⅳ group and Ⅰ-Ⅱ group (P > 0.01).The differences of 3-year progression free survival (PFS) rate and 3-year overall survival (OS) rate between c-myc gene or Bmi-1 gene normal and abnormal had statistical significance,and 3-year PFS rate and 3-year OS rate of double-hit of c-myc gene and Bmi-1 were lower.C-myc gene and Bmi-1 gene aberrant were the independent prognosis factors.The levels of serum IGF-Ⅰ and serum IGFBP-3 in DLBCL group were significantly lower than those in the control group (P < 0.01),however,the levels were increased after chemotherapy (P < 0.01).Serum IGF-Ⅰ and serum IGFBP-3 levels had no significant differences between non-GCB group and GCB group (P > 0.01).Their levels in stage Ⅳ group or high risk group were significantly lower than those in other groups.Serum IGF-Ⅰ level and serum IGFBP-3 level had no significant differences between the c-myc gene or Bmi-1 gene abnormal group and normal group (P > 0.01),but their levels were lower in both c-myc gene and Bmi-1 gene abnormal group than those in normal group.Conclusions C-myc and Bmi-1 are related with the biological characteristics and prognosis of DLBCL.Serum IGF-Ⅰ level and serum IGFBP-3 level reflect clinical stages of DLBCL and the efficacy in a certain degree.The expressions of c-myc and Bmi-1 have some correlation with the levels of serum IGF-Ⅰ and IGFBP-3 in DLBCL.
Résumé
Objective: To study the effect of B cell-specific murine leukemia virus integration site-1 (BMI-1) gene expression inhibited by RNA interference on doxorubicin (Dox)-resistance of hepatocellular carcinoma cells, and to explore its related molecular mechanism. Methods: The Dox-resistant MHCC-97H cell line was established and named as 97H/Dox, while the parental MHCC-97H cell line (named as 97H) was used as the control. The drug-resistance, cell colony-forming capacity and invasive ability were measured by MTT method, cell colony formation assay and in vitro Transwell invasive experiment, respectively. The mRNA and protein levels of tumor-related genes were detected by real-time fluorescent quantitative-PCR (RFQ-PCR) and Western blotting. Then the small interference RNA (siRNA) targeting BMI-1 gene (BMI-1-siRNA) was transfected into 97H/Dox and 97H cells. After BMI-1 gene silencing, the changes of Dox-resistance of 97H/Dox and 97H cells were detected by MTT method, and the change of expressions of tumor-related proteins in 97H cells was detected by Western blotting. Results: The 97H/Dox cell line had a stable multiple-drug resistance, and its colony formation capacity was higher than that of the control cell line 97H (P < 0.05). The levels of BMI-1, ATP-binding cassette superfamily G member 2 (ABCG2) and matrix metalloproteinase-2 (MMP-2) were significantly increased in 97H/Dox cells compared with those in the control 97H cells (all P < 0.05). Knockdown of BMI-1 gene by siRNA significantly increased the sensitivity to Dox in 97H/Dox and 97H cells (both P < 0.05), and resulted in a significant reduction of the expression levels of phospho-Akt (p-Akt), phospho-c-Jun N-terminal kinase (p-JNK) and ABCG2 in 97H cells (all P < 0.05). Conclusion: The upregulation of BMI-1 gene expression may be involved in the acquisition of Dox-resistance in hepatocellular carcinoma 97H cells. Inhibition of BMI-1 expression can enhance the sensitivity to Dox in hepatocellular carcinoma cells. Therefore, interfering the expression of BMI-1 gene may be a potential strategy for enhancing the response to chemotherapy in treatment of hepatocellular carcinoma.
Résumé
BMI1 belongs to the polycomb-repressive complex 1 (PRC1) family of genes that are conserved chromatin silencers. These are essential for maintaining both the normal and cancerous stem cell state. In this study, we evaluated the effect of siRNA-mediated BMI1 knockdown on tumor cell properties such as invasion, migration, and apoptosis, as well as on cell signaling pathways responsible for tumor progression in the human glioma cell line, U251. Knockdown of BMI1 induced apoptosis by activating cleavage of PARP and caspase-3. It also decreased the expression of anti-apoptotic proteins, survivin, XIAP, Bcl-xL, and Mcl1. Additionally, BMI1 knockdown significantly decreased cell invasion and cell migration ability. BMI1 knockdown also decreased the phosphorylation of Akt/FOXO1/3a signaling proteins. Our results suggest that BMI1 knockdown induces apoptosis and decreases cell invasion and cell migration. Moreover, we believe these phenomenona are associated with decreased phoshorylation of Akt signaling proteins, which contributes to cancer progression.