ABSTRACT
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.
ABSTRACT
A síndrome mielodisplásica (SMD) primária compreende um grupo heterogêneo de doenças clonais de célula tronco hematopoética. É caracterizada por uma hematopoese ineficiente, presença de displasias na medula óssea (MO), citopenias no sangue periférico (SP). Cerca de 10-40% dos casos evoluem para leucemia mielóide aguda (LMA). O objetivo deste trabalho foi analisar a expressão dos genes BMI1 e MLL em pacientes com SMD primária, no sentido de avaliarmos, em especial, o papel do gene BMI1 no desenvolvimento da SMD e sua evolução para LMA, correlacionando com características citogenéticas, celulares e clínicas. Nossos resultados mostraram a presença de cariótipos anormais em 48,6% nos pacientes com SMD primária. As alterações cromossômicas mais frequentes foram: del(5q), -7, del(11q), del(17p) e cariótipos complexos. Altas taxas de apoptose foram encontradas no estágio inicial, quando comparadas com os estágios mais avançados da doença. A medula óssea de pacientes com SMD hipocelular apresentou um percentual de apoptose mais elevado quando comparado com a medula óssea de indivíduos saudáveis. No entanto, o percentual de apoptose da medula óssea hipocelular não foi maior que o encontrado em pacientes com medula óssea hiper/normocelular. Células comprometidas com o programa de diferenciação estiveram associadas com altas taxas de apoptose, sugerindo que a apoptose seria uma consequência da hematopoese ineficiente, onde o sistema hematopoético seria capaz de eliminar no início da doença células displásicas...
The primary myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic stem cell diseases. It is characterized by inefficient hematopoiesis, the presence of dysplasias in bone marrow (BM), peripheral blood cytopenias (PB). Approximately 10-40% of cases progress to acute myeloid leukemia (AML). The aim of this study was to analyze the expression of BMI1 and MLL genes in patients with primary MDS, to assess, in particular, the role of BMI-1 gene in the development and evolution from MDS to AML and correlate with cytogenetic, cellular and clinics characteristics. Our results showed the presence of abnormal karyotypes 48,6% in patients with primary MDS. The most frequent chromosomal abnormalities were del (5q), -7, del (11q), del (17p) and complex karyotypes. High rates of apoptosis were found in the initial stage, when compared with the more advanced stages of the disease. The bone marrow hypocellularity of patients with MDS showed a higher percentage of apoptosis when compared with the bone marrow of healthy individuals. However, the percentage of apoptosis of bone marrow hypocellularity was not higher than that found in patients with bone marrow hyper/normocellular. Committed to differentiation program cells were associated with high rates of apoptosis, suggesting that apoptosis would be a consequence of inefficient hematopoiesis, thereby the hematopoietic system could eliminate the dysplastic cells at the initial of the disease...
Subject(s)
Humans , Male , Female , Child , Adult , Cytogenetics , Genes , Myelodysplastic SyndromesABSTRACT
Objective To study the apoptotic activity of hydroxycamptothecin (HCFT) against human gastric carcinoma and its mechanism of action.Methods The apoptotic activity of HCPT against gastric carcinoma SGC-7901 cells was detected by MTT assay.The apoptosis occurrence was determined by Annexin V/PI staining and flow cytometry analysis.The mRNA expression of Bmi-1 was detected by real-time PCR analysis.Western blot analysis was used to detect the protein expression of Bmi-1.Results The IC50 values of HCPT against gastric carcinoma SGC-7901 cells was (4.87 ± 0.35) μ mol/L.After treatment with 0,5,10,20 μ mol/L HCPT for 48 h,the percent of apoptosis was elevated from (1.62 ± 0.37)％ to (21.45 ±4.54)％,(36.67 ±5.38)％,(54.26 ±7.14)％ and the 2-△Ct values of Bmi-1 mRNA was decreased from 0.614 ±0.022 to 0.445 ±0.018,0.376 ±0.012,0.215 ±0.010,there had significant difference among the different treatment (P ＜ 0.01).Treated with 0,5,10,20 μ mol/L HCPT for 48 h,the protein expression of Bmi-1 was down regulated by HCPT treatment in a dose-dependent manner by Western blot analysis.Conclusion HCPT shows effective activity to induce apoptosis in gastric carcinoma cells,which is mainly related to the down regulation of Bmi-1.