The Effect Of MiRNA In The Resistance Of Breast Cancer To Adriamycin Mediated By Exosome

In recent years,durg-resistance in breast cancer is the major reason for chemotherapy failure and high recurrence rate.The mechanism of drug-resistance includes apoptosis inhibition,high expression of multi-drug resistance protein,abnormal expression of microRNAs,and so on.Exosome is a kind of nano-sized vesicles secreted by cells,which rich in a variety of RNAs,lipids and proteins.It can be used as information carrier,conveying material and information among cells.Exosomes secreted by tumor cells are closely related to tumor growth,metastasis,immune tolerance and drug resistance.In addition,studies have reported that multiple miRNAs are associated with tumor resistance.Such as mi R-302,miR-369,miR-200 c,mi R-34 a,miR-222 et al.Therefore,we studied exosomes secreted by breast cancer cells in this study,in order to explore the relationship between exosomes and their associated miRNAs and tumor resistance,and its possible mechanism,which may provide a new idea for multidrug resistance gene therapy of tumor.Part 1 Exosome can transmit adriamycin resistance between breastcancer cellsObjective: To investigate the exosome of breast cancer cells and to identify whether exosomes of MCF-7/ADR cells can transmit adriamycin resistance between cells.Methods:1 Extraction and identification of exosomes: Extract exosomes of human breast cancer cells MCF-7 and adriamycin-resistant human breast cancer cells MCF-7/ADR with the kit or ultra-high-speed centrifugation method.The morphology of isolated exosomes was observed by transmission electron microscopy(TEM).Western blot was used to detect the expression level ofexosomal protein CD9.2 Effect of exosomes on drug resistance of breast cancer cells: The exosome(ADR/exo)of MCF-7/ADR cells was extracted and was quant-ified by BCA method.ADR/exo was stained with DiO,laser confocal microscopy was used to observe whether ADR/exo could enter MCF-7 c ells by endocytosis.MTT assay was used to detect the proliferation rate and the resistance to adriamycin of MCF-7 cells,after which were cocultured with ADR/exo.Results:1 The exosome of human breast cancer cell MCF-7 and adriamycin resistant human breast cancer cells MCF-7/ADR can be obtained by kit and ultra-high speed centrifugation.Under the transmission electron microscope,the exosomes were 30-100 nm in size,showing a bilayer structure.Western Blot results showed that the transmembrane protein CD9 of exosome was highly expressed.2 The results of laser confocal microscopy showed that ADR/exo could enter MCF-7 cells when ADR/exo was co-cultured with sensitive cell MCF-7.MTT results showed that,the proliferation of MCF-7 cells that co-ultured with ADR/exosome was significantly decreased(P < 0.05).The exosomes content from more to less,the proliferation of MCF-7 cells was down 16%,24%,43%and 24%,respectively.MTT results showed that the resistance of MCF-7 cells to adriamycin was significantly improved after co-culture with ADR/exo,the difference was significant(P < 0.05).From high concentration to low concentration,the resistance to doxorubicin of MCF-7 cells increased by 5%,25%,40%,30%,29%,24%.Conclusions:1 MCF-7 cell and MCF-7/ADR cell can secrete exosome.It can be extracted by the kit and ultra-high-speed centrifugation,highly expressed in CD9 transmembrane proteins.2 ADR/exo can enter MCF-7 cells,and ADR/exo can transmit adriamycin resistance in MCF-7 cells through some mechanism.Part 2 The relationship among exosomes of breast cancer cells and theirassociated miRNAs and tumor resistanceObjective:To analyze the expression of multidrug-resistant microRNAs in breast cancer cells and their exosome.After selecting the target microRNA molecules,the possible target molecules were predicted by software.The expression changes of the target microRNA and target molecules were studied with microRNA mimics and inhibitors.And to explore the relationship between exosomes and their associated mi RNAs and adriamycin resistance,and its possible mechanism.Methods:1 Resistance-related microRNA molecular screening: The expression of microRNAs in MCF-7,MCF-7/ADR,MCF-7/exo,ADR/exo were uantitatively analyzed with qRT-PCR technology.And then the target microRNA molecules with large differences in expression were screened for follow-up experiments.2 Screening of possible target microRNAs in breast cancer cells:Bioinformatics software was used to predict target genes that are partially associated with the 3’UTR region of the target microRNA associated with breast cancer resistance.The expression of target genes in MCF-7cells,MCF-7/ADRcells,MCF-7/exo and ADR/exo was quantitatively analyzed by qRT-PCR,and the most possible genes were screened out for follow up experiment.3 The relationship between target microRNA and intracellular target genes: The target microRNA mimics and inhibitors were synthesized and transfected into MCF-7 cells and MCF-7/ADR cells.q RT-PCR was used to detect the mRNA expression of target microRNAs and their target genes in transfected cells and exosomes.Western Blot was used to detect the expression of intracellular target protein.4 Effect of target micro RNA on drug resistance of breast cancer cells:Target microRNA mimics or inhibitors were transfected into MCF-7/ADR cells,and MTT assay was used to detect the sensitivity of cells to adriamycin.Results:1 Comparing with MCF-7cell and MCF-7cell exosome,the results of qRT-PCR showed that micro RNAs in MCF-7/ADRcell and ADR/exo,mi R-222-5p,miR-17-5p,miR-34a-5p,mi R-let-7a and miR-100-5p were significantly different(P < 0.05).There was no significant difference between Cells of miR-21-5p,miR-125b-5p and exosomes of miR-342-3p.Among them,the expression of miR-34a-5p in MCF-7/ADR cells was significantly decreased,while in ADR/exo expression was significantly increased.2 Four novel genes related to miR-34a-5p were obtained by software prediction,which were related to breast cancer resistance: NOTCH1,HDAC1,mTOR,HDAC7.The results of qRT-PCR showed that mRNAs in MCF-7/ADRcell and ADR/exo were significantly different(P < 0.05).The expression of NOTCH1,HDAC1,mTOR and HDAC7 in the cells was1.85±0.012,1.23±0.031,2.37±0.064,2.07±0.082 respectively.The expression in the exosomes was 0.14±0.007,0.07±0.019 respectively,and there is no expression of mTOR and HDAC7 in the exosome.3 After transfection of MCF-7/ADR cells with miR-34a-5p mimic,the expression of miR-34a-5p and NOTCH1 in MCF-7/ADR cells and their exosomes had significant difference compared with the control group(P <0.05).The expression of mi-34a-5p in MCF-7/ADR cells and exosomes was54.1±0.031 and 1.23±0.063.The expression of NOTCH1 was 0.51±0.017 and4.72±0.089.Western blot analysis showed that the expression of target protein NOTCH1 was significantly down regulated after mi R-34a-5p mimic was transfected in MCF-7/ADR cells(P < 0.05).4 MTT results showed that compared with the negative control group,MCF-7/ADR cells transfected with miR-34a-5p mimic had lower resistance to adriamycin(P < 0.05).Conclusions:1 miR-34a-5p was involved in the regulation of adriamycin resistance in breast cancer cells MCF-7,while the NOTCH1 gene may be one of the target genes of miR-34a-5p.2 Transfection of miR-34a-5p mimic can effectively up-regulate the expression of miR-34a-5p in MCF-7/ADR,and decrease the expression of NOTCH1 protein in cells significantly,and also can increase the sensitivity of adriamycin.3 The expression of miR-34a-5p in MCF-7/ADR cells was significantly lower than that in MCF-7 cells,and the expression of miR-34a-5p was significantly increased in ADR/exo,suggesting that one of the possible reasons for drug-resistance was miR-34a-5p can be excreted by exosome,resulting in a decrease in the effective concentration of intracellular cells.