| Purpose: 1.Explore the effect of breast cancer cell exosomes on the resistance of breast cancer cells to tamoxifen;2.Explore the mechanism in miR-9-5p affecting TAM resistance of breast cancer cells.Method: In the previous work,we used microarray-based bioinformatics analysis to screen the differentially expressed breast cancer gene ADIPOQ and its regulatory miRNA-9-5p(miR-9-5p)for subsequent experiments.Prepare TAM-resistant MCF-7(MCF-7/TAM)cell lines and non-drug sensitive MCF-7 cell lines,and then isolate exosomes.After co-cultivation with TAM,the apoptosis rate of MCF-7 cells treated with exosomes was detected.Combining bioinformatics analysis and luciferase activity determination to identify the interaction between miR-9-5p and ADIPOQ.In order to verify the effects of miR-9-5p and ADIPOQ on TAM resistance of MCF-7 cells in vitro and in vivo,we introduced miR-9-5p mimics and inhibitors into MCF-7 cells and MCF-7/TAM cells respectively.The sensitive cell MCF-7 cells were co-cultured with exosomes in each group.RT-PCR was used to detect the expression of miR-9-5p in MCF-7 and MCF-7/TAM cell lines,and CCK-8 was used to detect the transfer of miR-9-5p.The cell viability and IC50 value of each group of miR-9-5p mimics and miR-9-5p inhibitors were stained,and the apoptosis of transfected cells was detected by flow cytometry to verify whether miR-9-5p can change the TAM of breast cancer cells Resistance.RT-PCR was used to detect the expression of miR-9-5p in each group of exosomes and the expression of miR-9-5p and ADIPOQ mRNA in MCF-7receptor cells co-cultured with each group of exosomes,and Wb to detect ADIPOQ The expression of protein in each group of cells verifies that exosomes can effectively carry miR-9-5p and affect the expression of ADIPOQ in breast cancer cells.Furthermore,CCK-8 was used to detect MCF-7 receptor cell viability and IC50 value after each group of exosomes co-cultured,and flow cytometry was used to detect MCF-7 receptor cell apoptosis after each group of exosomes co-cultured To verify the effect of miR-9-5p in exosomes on the drug resistance of breast cancer cells.Results: Through transmission electron microscope observation,nanoparticle tracking analyzer(Nano Sight NS300)to analyze the size of the extracted exosomes,and the WB experiment to verify the labeled protein in the exosomes confirmed that this experiment was successful in separating the exosomes.The exosomes of MCF-7 and drug-resistant MCF-7/TAM cells(labeled with PKH67 dye)were co-cultured with MCF-7 cells.The results suggest that the exosomes of drug-resistant cells can be taken up by sensitive cells.After treating MCF-7 cells with PBS,MCF-7 exosomes and MCF-7/TAM-exo,the results of CCK-8 experiment showed that under the treatment of different tamoxifen concentrations,it was compared with the control group PBS and MCF-7,the MCF-7/TAM-exo co-cultured the sensitive cell line MCF-7 cells to tamoxifen cell viability significantly increased,IC50 significantly increased(P <0.05).The cell cycle distribution of MCF7 incubated with three groups of exosomes and the apoptosis rate after 10 u M tamoxifen treatment were analyzed.The apoptosis rates of the three groups after 10 u M tamoxifen treatment were 28.33±3.21 in the PBS group.25.02±3.07 in MCF-7-exo group,9.81±1.09 in MCF-7/TAM-exo group.Compared with the control group PBS+MCF and MCF-7-exo group,MCF-7 cells increased in G1 phase and decreased in S phase after co-cultured with MCF-7/TAM-exo(P<0.05).The apoptosis rate was significantly reduced(P<0.05).The expression of miR-9-5p in MCF-7 and MCF-7/TAM cell lines detected by PCR was 0.99±0.10 and 2.39±0.20,respectively.The expression of miR-9-5p in the drug-resistant strain MCF-7/TAM was significant Improve(P<0.05).The results of transfection of miR-9-5p mimics and inhibitors and CCK-8experiments showed that the IC50(μM)values of the cells in each group were0.20±0.02 in the MCF-7+NC-mimic group,and MCF-7+miR-9-5p mimic group was 5.71±0.40,the MCF-7/TAM+NC-inhibitor group was 5.41±0.28,and the MCF-7/TAM+miR-9-5p inhibitor group was 0.21±0.03.After transiently transfecting miR-9-5p mimic in the MCF-7 cell line,the cell inhibition rate was significantly decreased(P<0.05),IC50 increased(P<0.05),and the inhibition rate was dose-effect relationship.Similarly,after transiently transfected with miR-9-5p inhibitor,compared with the inhibitor blank group,the cell inhibition rate increased significantly(P<0.05),IC50 decreased(P<0.05),and the inhibition rate also showed a dose-effect relationship.Flow cytometry was used to detect the apoptosis of the transfected cells.The results showed that after transiently transfecting miR-9-5p mimic in the MCF-7 cell line,compared with the mimic blank group,the apoptosis rate of the cells decreased significantly.After transiently transfecting miR-9-5p inhibitor,compared with the inhibitor blank control group,the apoptosis rate increased significantly.The relative miR-9-5p expression level of exosomes transfected with miR-9-5p mimic MCF-7 cells was significantly higher than that of miR-9-5p control transfected exosomes(P<0.05).The relative miR-9-5p expression level of MCF-7/TAM exosomes transfected with miR-9-5p inhibitor was lower than that of cells in the blank control group.The exosomes of each group after transfection were co-cultured with MCF-7,and the expression levels of miR-9-5p and ADIPOQ in the recipient cell MCF-7 were detected.The results showed that the MCF-7 cell line was compatible with the transfected miR-9-5p mimics after co-cultivation with MCF-7-derived exosomes,the expression of miR-9-5p was significantly higher than that of the mimic control group(P<0.05);ADIPOQ mRNA and protein expressions were significantly reduced(P <0.05).After the MCF-7 cell line was co-cultured with MCF-7/TAM-derived exosomes transfected with miR-9-5 inhibitor,the expression of miR-9-5p was reduced by about 70% compared with the control group(P<0.05);ADIPOQ mRNA and protein expression increased significantly(P<0.05).Different groups of transfected cell lines were tested for drug sensitivity.CCK-8 was used to detect the viability of each group and calculate the IC50value(μM)of each group of cells.The results indicate that the MCF-7 cell line is similar to the transfected miR-9-5p after co-cultivation of MCF-7-derived exosomes of the drug substance,compared with the blank control group,the cell inhibition rate was significantly decreased(P<0.05),IC50 was increased(P<0.05),and the inhibition rate was in a dose-effect relationship.The same method was used to detect the effect of down-regulation of miR-9-5p on the tamoxifen sensitivity of MCF-7.The results showed that the MCF-7 cell line and the source of MCF-7/TAM transfected with miR-9-5p inhibitor after exosomes co-culture,compared with the inhibitor blank control group,the cell inhibition rate increased significantly(P<0.05),IC50 decreased(P<0.05),and the inhibition rate also showed a dose-effect relationship.Flow cytometry was used to detect the apoptosis of the transfected cells.The results showed that the MCF-7 cell line was co-cultured with miR-9-5p miR-9-5p mimic-transfected MCF-7-derived exosomes,and compared with miR-9-5p mimic blank control group.The apoptosis rate of cells decreased significantly(P <0.05).After the MCF-7 cell line was co-cultured with MCF-7/TAM-derived exosomes transfected with miR-9-5 inhibitor,the apoptosis rate was significantly increased compared with the inhibitor blank control group(P <0.05).Conclusion: The exosomes derived from MCF-7/TAM cells can be taken up by the sensitive cells MCF-7 cells and enhance the drug resistance of the sensitive cells MCF-7 cells to TAM.miR-9-5p changes the sensitivity of BC cells to TAM by negatively regulating the expression of ADIPOQ target genes.In addition,MCF-7/TAM cell-derived miR-9-5p inhibits MCF-7 cell apoptosis and enhances cell resistance to TAM.miR-9-5p enhances the resistance of BC cells to TAM by down-regulating the expression of ADIPOQ target genes,suggesting that ADIPOQ target genes can be used as candidate molecular targets for BC therapy. |
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