Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: Lipofectamine TM 2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are 1.5×10 4 (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01).MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.
Background:Triple-negative breast cancers (TNBC) are the most aggressive subtype of breast cancer, accounting for 15% - 20% of all cases, and have no response to available hormonal therapies and anti-HER2-targeted therapies due to the absence of corresponding targets. Over half of TNBC patients have overexpressed EGFR, but they are insensitive to EGFR inhibitors from monotherapy. Mammalian target of rapamycin (mTOR) connected with EGFR in the downstream signaling and involved in the progress of TNBC. The purpose of this study is to determine the combined effect of everolimus and geftinib in a TNBC cell model and investigate the possible mechanism. Results: This work showed the expression EGFR and p-mTOR protein in TNBC tissues were significantly higher than that in non-TNBC(p<0.05), while the expression of mTOR, S6K1, pEGFR and p-S6K1 were significantly higher in the EGF stimulation. EGFR and p-mTOR protein are related to poor prognosis. EGFR inhibitor gefitinib and mTOR inhibitor everolimus significantly inhibited the proliferation of human triple-negative breast cancer MDA-MB-468 cells and arrested cells in G0/G1 phase when applied separately and in combination in a dose-dependent manner (P<0.05). Meanwhile, the rate of apoptosis of MDA-MB-468 cells was significantly incresased separately by two drugs (P<0.01). Furthermore, the combination of everolimus and geftinib reduced the phosphorylation of mTOR downstream proteins. Instead, the phosphorylation of 4E-BP1 was enhanced after the everolimus and geftinib treatment, indicated an alternative activation pattern. Conclusions: These results suggested that dual inhibition of mTOR and EGFR could be a promising approach to treat TNBC.
Objective To detect the activation of the EGFR and mTOR signaling pathways in the triple negative breast cancer cell line MDA-MB-468 and investigate the inhibitory effect of gefitinib, an epidermal growth factor receptor inhibitor, and everolimus, a target protein inhibitor of rapamycin, on triple negative breast cancer cells. Methods Triple negative human breast cancer MDA-MB-468 cells were cultured and blank control group, single EGFR inhibitor gefitinib group, single mTOR inhibitor everolimus group, and two drug combination group were set up respectively to detect the effects of single and combined drugs on cell proliferation activity, cell cycle and apoptosis, and the expression of EGFR and mTOR signal pathway proteins in cell lines after single and combined drug intervention was detected again by Western blot. Results The level of EGFR and p-mTOR protein in triple negative breast cancer was higher than in non triple negative breast cancer ( P <0.05). The level of mTOR, S6K1, p-EGFR, p-S6K1 was significantly increased when treated with EGF (0ng/mL, 10ng/mL, 100ng/mL) for 1h, compared to without EGF stimulation ( P <0.05). The level of p-EGFR, p-mTOR, p-S6K1 protein increased significantly when the cells were exposed to EGF for 2h, respectively ( P <0.05). EGFR inhibitor gefitinib alone and the mTOR inhibitor everolimus alone could significantly inhibit the proliferation of human triple negative breast cancer MDA-MB-468 cells in a dose-dependent manner ( P <0.05). The level of p-4EBP1 protein in EGFR and mTOR signal pathway was significantly increased after the intervention of gefitinib alone, everolimus alone, and the combination of two drugs ( P <0.05). Conclusion EGFR and mTOR signaling pathways can be activated in triple negative breast cancer; Both the EGFR inhibitor gefitinib alone and the mTOR inhibitor everolimus alone can significantly inhibit the proliferation of human triple negative breast cancer MDA-MB-468 cells. The combination of the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus may achieve anti-tumor effect similar to that of single drug by reducing the drug dose.
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