RNAi-mediated silencing of the Bmi-1 gene causes growth inhibition and enhances doxorubicin-induced apoptosis in MCF-7 cells

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G1 /S phase transition. The number of target cells was found to increase in phase G 0 /G 1 and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer

Cutaneous Manifestations of Diabetes Mellitus in a Tertiary Care Hospital in Eastern India: A Cross-sectional Study

Background: Diabetes mellitus is a chronic metabolic disorder and is a major public health problem worldwide. Skin diseases can appear as the first sign of diabetes or can develop at any time in the course of the illness. Purpose: This study aimed to determine the pattern and prevalence of skin manifestations in patients with diabetes mellitus. Methods: An observational cross-sectional study was done on 80 Type 1 and Type 2 diagnosed diabetes mellitus patients and with informed consent, they were thoroughly examined for their respective skin disorders. Results: Among 80 patients, 35% of patients had infectious skin lesions, whereas 20% had noninfectious dermatosis only. More than one type of dermatosis was seen in 45% of patients. Candidal balanoposthitis was the most common fungal infection. Limitation: Limited sample in a single center. Conclusion: Infections were more prevalent compared to noninfectious disease in a population with uncontrolled diabetes mellitus. Pruritus was the most common disorder among the other diabetic complications

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

105-112The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer

Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular<i style=""> </i>characteristics

862-870The whole length of MCHR2 gene cDNA fragment was amplified by PCR using human fetal brain cDNA library as template. The pcDNA3.1 (+)/MCHR2 eukaryotic expression vector was constructed successfully. The recombinant pcDNA3.1 (+)/MCHR2 plasmid was transfected into Chinese hamster ovary (CHO) cell by lipofectamineTM2000, after G418 selection and then the CHO cell line expressing MCHR2 gene was established. The MCHR2 gene expression was tested by RT-PCR, western blotting and immunofluorescence. The maximum binding (Bmax) of CHO cell line was 309.97±1.14 fM·mg-1protein and the dissociation constant (Kd value) was 0.170±0.0006 nM. MCH could stimulate Ca2+ release, its 50% effective concentration (EC50) was 2.32±0.01 nM. The construction of the CHO cell line and the research of MCHR2 molecular characteristics have established a good experimental basis for the further research about the function of MCHR2 gene.</b