miRNA-199 a-5 p inhibit the invasion of MDA-MB-231 cells via regulating ERK5 through SP1 / 临床与实验病理学杂志

Purpose To study the effect and mechanism of miR-199a-5p on the invasion of breast cancer MDA-MB-231 cells. Meth-ods miR-199a-5p mimic was transfected into MDA-MB-231 cells. Influence of miR-199a-5p on the invasion of MDA-MB-231 cell was displayed by Transwell, the expression of epithelial-mesenchymal transition ( EMT) molecular markers ( E-cadherin, vimentin) regulated by miR-199a-5p was determined using immunofluorescence and Western blot. Western blot was employed to assess the levels of ERK5, pERK5, EGF and SP1 in MDA-MB-231 cells dealt with miR-199a-5p mimic and LNA-siRNA. Chromatin immunoprecipita-tion (CHIP) was applied for displaying the reaction of SP1 with ERK5 promoter. Results miR-199a-5p could inhibit the invasion of MDA-MB-231 cells, decrease the expression of vimentin and enhance E-cadherin. Meanwhile, miR-199a-5p decreased the expression of ERK5 and pERK5, the levels of EGF and SP1 were also downregulated. On the contrary, the levels of EGF, SP1, ERK5 and pERK5 were enhanced by employing LNA-siRNA targeting miR-199a-5p. SP1 could bind with ERK5 promoter. Conclusions miR-199a-5p could reduce the expression of ERK5 and pERK5 through regulating EGF and SP1, which functioning the inhibitory effect on invasion of MDA-MB-231 breast cancer cells.

Expression and correlation of PKCζ, MMP-2 and MMP-9 in breast cancer / 临床与实验病理学杂志

Purpose To investigate the expressions of PKCζ, MMP-2, and MMP-9 in breast cancer and the relationship with the inva-sion and metastasis of breast cancer. Methods The expression of PKCζ, MMP-2 and MMP-9 in 100 cases with breast cancer was as-sessed with immunohistochemistry PV 9000 method. PKCζ-siRNA was transfected into MDA-MB-231 cell lines, called siPKCζ/MDA-MB-231. While siRNA construct containing a scrambled sequence was transfected into MDA-MB-231 cells to generate control cells, which were designated as Scr/MDA231 cells. Western blotting was used to measure the expression of PKCζ in transfected cells, and the Transwell invasion assay was used to detect the invasion ability in vitro. The content of MMP-2, MMP-9 were measured by ELISA. Results The expression rates of PKCζ, MMP-2 and MMP-9 in breast cancer tissues were 62.5%, 37.5% and 32.5%, and there were significant differences among them (P0.05). The expres-sion of PKCζ, MMP-2 and MMP-9 were lower in siPKCζ/ MDA-MB-231 group than those in scr/ MDA-MB-231 group, and the in vitro invasion ability was significantly decreased (P<0.05). Conclusions PKCζ can promote the invasion and metastasis of breast canc-er, and correlated with the expression of MMP-2, MMP-9(P<0.05).

Highexpression of Snail leads to the P-gp modulateds MDR in breast cancer cell MCF-7 / 中国药理学通报

Aim To explore the relationship between Snail and P-gp in breast cancer cell,and to reveal the effect of epithelial-mesenchymal transformation(EMT)on the multidrug-resistance(MDR)of breast cancer cell.Methods The eukaryotic expression vector pCDNA3.1-Snail was constructed, and then transfected into human breast cancer cell line MCF-7 to constuct MCF-7/Snail.Both cell lines MCF-7 and MCF-7/Snail were induced by adriamycin(ADM).Cell cytotoxicity assay and ADM efflux assay were used to measure the ability of drug resistance.The positive rate of P-gp of the two cell lines was detected by flow cytometry;the mRNA of MDR1 and Snail was evaluated by real-time PCR.Results MCF-7,the expression of MDR1 mRNA and Snail mRNA in MCF-7/Snail cell lines significantly increased;the expression of P-gp was increased too;the RR increased to 109.2;fluorescence intensity intracellular was reduced to 7.1.Conclusion After transfected the eukaryotic expression vector,the capacity of MCF-7/Snail strongly increases compared with that of MCF-7.

THE STUDY OF THE MECHANISM OF 17?-ESTRADIOL ON REVERSAL OF THE BREAST CANCER RESISTANT PROTEIN-MEDIATED MULTIDRUG RESISTANCE / 解剖学报

Objective To explore the mechanism of the reversal of breast cancer resistant protein-mediated multidrug resistance by 17?-estradiol. Methods Two BCRP expressing cell lines of MCF-7/MX20 and MCF-7/BCRP were established in which breast canrcer resistant protein(BCRP) was promoted by BCRP promoter and cytomegalovirus(CMV) promoter respectively.These drug resistant cell lines were cultured in medium containing 17?-estradiol.Fourty-eight hours later,cytotoxicity assay,mitoxantrone efflux assays,quantitative RT-PCR were performed to observe the reversal function of BCRP by 17?-estradiol on MCF-7/MX20 and MCF-7/BCRP respectively. Results After being treated with 17?-estradiol,the intensity of mitoxantrone in MCF-7/BCRP was weaker than that in MCF-7/MX20 and the BCRP mRNA level in MCF-7/BCRP was high than that in MCF-7/MX20.The results of these experiments revealed that 17?-estradiol could reverse the BCRP mediated multidrug resistance(MDR) in MCF-7/MX20 cells but not in MCF-7/BCRP ones.Conclusion 17?-estradiol may reverse the phenotype of BCRP through regulation of the promoter of BCRP gene but not acted as the substrate of BCRP.