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Objective:To establish osimertinib-resistant non-small cell lung cancer (NSCLC) cell line and explore its drug resistance mechanism.Methods:The human NSCLC cell line H1975 was used as the research object, and low-concentration osimertinib was used to continuously select secondary drug-resistant cell lines. Osimertinib drug sensitivity of cells was detected by MTS method. Cell proliferation was detected by live cell workstations. Flow cytometry was used to detect cell cycle and apoptosis. Protein mass spectrometry was used to construct differentially expressed protein profiles between parental and drug-resistant cells and some resistance-related proteins were validated by real time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blot.Results:Secondary drug-resistant H1975/OSI cell line were successfully established. Compared with the parental cells, the resistance index of H1975/OSI cells increased by 27.25 times ( P<0.01), the cell proliferation ability decreased but the apoptosis resistance increased ( P=0.01), and no new drug-resistance related gene mutation in H1975/OSI cells. Meanwhile, the differential protein expression profiles of H1975 and H1975/OSI cells were built, and 307 upregulated proteins and 295 down-regulated proteins were found in resistant cells. When fibroblast specific protein-1 (FSP1) gene with expression up-regulation was diturbed in H1975/OSI cells, the cell IC50 value of osimertinib decreased 3.51 times ( P=0.02) , and when FSP1 was overexpressed in the H1975 cells, the IC50 value of osimertinib increased by 3.75 times ( P<0.01). Conclusions:We successfully established human NSCLC osimitinib-resistant cell line H1975/OSI. Protein differential expression profiles between H1975 and H1975/OSI was constructed successfully. It was found that FSP1 was involved in mediating the resistance of H1975/OSI to osimertinib.
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Objective To investigate the effect and mechanism of miR-23b on the proliferation and migration of triple negative breast cancer (TNBC) cells.Methods Real time polymerase chain reaction (PCR) was used to detect the expression of miR-23b in triple negative breast cancer tissues.MDA-MB-231/miR-23b,BT549/miR-23b cell lines are constructed.Proliferation assay,scaling healing experiment and Transwell migration assay were used to detect the effect of miR-23b on the proliferation and migration of triple negative breast cancer cells.Dual-luciferase reporter gene assay was employed to examine the interactions between miR-23b and forkhead box C2 (FOXC2).Real time PCR and Western blot were performed to detect the effect of miR-23b on the expression of FOXC2.Results The expression level of miR-23b in triple negative breast cancer tissues was significantly less than that in adjacent normal tissues.miR-23b could reduced the proliferation and migration of triple negative breast cancer cells.Dual-luciferase assay confirmed that miR-23b could regulate the expression and activity of FOXC2.The expression of FOXC2 in mRNA and protein level was inhibited by miR-23b.Conclusions miR-23b can inhibit the expression of FOXC2 and affect the proliferation and migration of triple negative breast cancer cells.
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Aim To investigate the anti-proliferative effect of salinomycin on doxorubicin-resistant human breast cancer MCF-7 /DOX cells.Methods MCF-7 and MCF-7 /DOX cells were treated or untreated with salinomycin.Cell viability was detected by MTS assay. Cell apoptosis was detected by Annexin V-FITC /PI as-say.Reactive oxygen species (ROS)was measured by DCFH-DA staining.Mitochondrial membrane potential was measured by JC-1 assay.The expression of apopto-sis related proteins BAX, BCL-2, caspase-3, and caspase-9 were evaluated by Western blot analysis. Results The cell viability was significantly reduced by salinomycin treatment in a dose-dependent manner. The flow cytometry results showed that salinomycin in-duced MCF-7 /DOX cell apoptosis,increased ROS pro-duction,and decreased mitochondrial membrane poten-tial.Furthermore,salinomycin decreased the expres-sion of BCL-2,and increased the expression of BAX, cleaved caspase-3,and cleaved caspase-9.Moreover, the antioxidant N-acetylcysteine (NAC ) markedly blocked the above effects.Conclusions Our results suggest that salinomycin-induced apoptosis in MCF-7 /DOX is associated with induction of ROS production, and activation of mitochondria apoptosis pathway, which may become a potential chemotherapeutic agent for the therapy of doxorubicin resistant breast cancer.
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Objective To investigate the relationship of glycoprotein serglycin (SRGN) expression with invasion and metastasis of breast cancer cells,and the role of microRNA in the regulation of SRGN expression.Methods Real-time quantitative polymer ase chain reaction (PCR) and Western blot were used to detect the differences in SRGN expression between higher metastasis Michigan cancer foundation-7 (MCF-7)/5-Fu breast cancer cell lines and weaker metastasis MCF-7 cell line.The siRNA interference experiment and in vitro Transwell experiment were used to detect effect of SRGN on the ability of invasion and metastasis of breast cancer cells.Bioinformatics software was used to predict miRNAs targeting SRGN,and integrated microRNA differentially expressed chip data between breast cancer cell MCF-7 versus MCF-7/5-Fu.The miRNA quantitative PCR was used to determine the differences of candi date miRNA expression.After transfection of microRNA minics,Western blot was used to test candidate microRNA target SRGN.Transwell experiment was used to test the effects of candidate microRNAs on tumor cell invasion and metastasis.Results SRGN was increased significantly in MCF-7/5-Fu cells,and the invasion and metastasis of tumor cells were inhibited when SRGN was interfered.In addition,miR181 b/c expressed in MCF-7/5-Fu cells was reduced significantly,negatively correlated with SRGN expression,and targeted SRGN expression.It inhibited invasion and metastasis of tumor cells.Conclusions MicroRNA181b/c inhibits metastasis of breast cancer by targeting SRGN.
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Objective To explore the effect of dendritic cells (DC) primed by total RNA extracted from human colon cancer Lovo cell on specific cytotoxicity of cytokine-induced killer cells (CIK) in vitro.Methods Cord blood mononuclear cells extracted by Ficoll density gradient centrifuge were induced into CIK and DC cells separately, and their Immunophenotype was detected by Flow cytometer. Trizol harvested total RNA from colon cancer cell Lovo and the RNAs were loaded to DCs obtained from cord blood as tumor anti gens. Effectors were grouped accordingly as CIK cells co-cultured with DCs transfected with Lovo RNA, CIK cells co- cultured with unloaded DCs and CIK cells. Targets was Lovo cells. In vitro cytotoxicity of CHK cells was extured with DCs loaded with Lovo RNA(76.49%±4.21%), DC + CIK group was lower(53.84% ± 2.15%),and CIK cells group possessed the lowest cytotoxicity(32.20% ± 3.07%), showing statistic significance( P ＜0.05). Conclusion Extraction of total RNA from tumor cells is simple and easy for clinical implementation.Total RNAs acted as antigen to pulse DCs can strengthen the specific cytotoxicity of CIK cells, which will have good prospects for clinical application.