Objective： To study the inhibition of proliferation of breast cancer by small interfering RNA（siRNA） targeting human prolactin （hPRLR） and the underlying mechanisms. Methods：The siRNA targeting hPRLR was chemica...Objective： To study the inhibition of proliferation of breast cancer by small interfering RNA（siRNA） targeting human prolactin （hPRLR） and the underlying mechanisms. Methods：The siRNA targeting hPRLR was chemically synthesized and transfected into MCF-7 cells, the expression of hPRLR was analyzed by real-time quantitive PCR, cell growth inhibition was measured with MTT assay, cell cycle of the transfected cells was examined by flow cytometry, meanwhile, expression of cyclin D1 was tested by semi-quantitative RT-PCR, Results：24 h after transfection with 100 nmol/L siRNA-PRLR, the expression of hPRLR mRNA was suppressed by 65%, cells in G1 phase increased, but cells in S phase decreased. Down regulated hPRLR expression exhibited significant inhibition in cell proliferation. And the expression of cyclin D 1 was down regulated. Conclusion：The results indicate that siRNA-hPRLR is a useful tool for silencing hPRLR expression and inhibiting cell proliferation in breast cancer MCF-7 cell line, and it may be a possible new approach for breast cancer gene therapy.展开更多
Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferatio...Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.展开更多
Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined appl...Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.展开更多
基金This project was supported by Scientific Foundation of Nanjing Medical University(CX2002004)
文摘Objective： To study the inhibition of proliferation of breast cancer by small interfering RNA（siRNA） targeting human prolactin （hPRLR） and the underlying mechanisms. Methods：The siRNA targeting hPRLR was chemically synthesized and transfected into MCF-7 cells, the expression of hPRLR was analyzed by real-time quantitive PCR, cell growth inhibition was measured with MTT assay, cell cycle of the transfected cells was examined by flow cytometry, meanwhile, expression of cyclin D1 was tested by semi-quantitative RT-PCR, Results：24 h after transfection with 100 nmol/L siRNA-PRLR, the expression of hPRLR mRNA was suppressed by 65%, cells in G1 phase increased, but cells in S phase decreased. Down regulated hPRLR expression exhibited significant inhibition in cell proliferation. And the expression of cyclin D 1 was down regulated. Conclusion：The results indicate that siRNA-hPRLR is a useful tool for silencing hPRLR expression and inhibiting cell proliferation in breast cancer MCF-7 cell line, and it may be a possible new approach for breast cancer gene therapy.
基金This work was supported by NationalNatural Science Foundation of China (No. 30200095).
文摘Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.
文摘Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.
