Essential Role For MicroRNA-222 Containing Exosomes In Breast Cancer With Chemotherapeutic Resistance

Background:To date,although many significant advances have been made in chemotherapy,chemo-resistance still remains a major obstacle to successful treatment of breast cancer.For poor understanding of the mechanisms underlying chemo-resistance,it is necessary to identify the possible biomarkers and pathways in breast cancer resistant to chemotherapy.On the basis of our previous finding that drug-sensitive breast cancer cells could partly increased their resistance capacity by the uptake of drug-resistant cell-derived exosomes,which contain some higher levels of miRNAs(miR-100,miR-222,and miR-30a).However,we cannot eliminate interferences to indentify the unique role of miRNAs due to many contents of drug-resistant cell-derived exosomes may alter drug-resistance.Here,we established the miRNA-222-mimic-and miRNA-222-inhibitor-exosomes complexes to explore the functions of miR-222-pathway in transfer of chemo-resistance and its diagnostic and prognosis implications in breast cancer patients with neoadjuvant chemotherapy.Materials and methods:1.We established adriamycin(ADR)-resistant human breast sublines(MCF-7/ADR)derived from the human breast cells(MCF-7/S)at 500nM ADR.The expression levels of P-gp(P-glycoprotein)and TSG101(tumor susceptibility gene 101)were identified by Immunofluorescence.The amount of exosomes derived from MCF-7/ADR(A-exos)or MCF-7/S(S-exos)in the same volume of culture medium were assessed by Bradford assay.2.Different amounts of ADR-exos(50/100/200?g A-exos)were added into the culture medium of MCF-7/S,then the amount of exosomes(PKH26 labelled)that absorbed in recipient cells was assessed and visualized by confocal microscope.Moreover,the level of miR-222 was caculated by qRT-PCR;the apoptosis rates of all groups were identified with a flow cytometer.3.Characterization of exosomes derived from HBL-100 cell line(H-exos),then optimizing loading conditions of exosomes with miR-222 mimic(mimic-exos).Visualizing the uptake of FAM-mimic-exos in recipient cells by confocal microscope and assessed the miR-222 level.4.Co-cultured inhibitor-exos with MCF-7/ADR,then compared the level of miR-222 and PTEN with negative control group.Furthermore,we assessed the apoptosis rates of all groups.5.NCG mice bearing xenograft tumors were injected with 50?g inhibitor-exos at several intratumor sites,NC-exo(NC-exosomes)and H-exos.Monitoring the size of tumors in three groups,and explored the expression level of miR-222 and PTEN in tumors and serum-exosomes.6.Idengtified the expression level of miR-222,PTEN and TSG101 in chemo-resistant paired-tissues(before and after neoadjuvant chemotherapy).Results:1.MCF-7/ADR expressed a great higher P-gp and TSG101.Results showed that the amount of A-exos was much more than S-exos in the same volume of culture medium.2.The images showed that fluorescence intensity was related to the amount of exosomes added.Moreover,cells co-incubated with more A-exos showed a higher miR-222 expression and a lower apoptosis rate.3.Electroporation at 180V with 50?g/10?l H-exos could maximized the efficiency of loading miR-222 mimic into H-exos.4.After co-cultured with inhibitor-exos,MCF-7/ADR was displayed a lower resistance to ADR,along with an increase of miR-222 level and an inhibition of PTEN expression.5.The size of tumors in group with inhibitor-exos were significantly decreased compared with negative and blank group.qRT-PCR assay showed that the expression level of miR-222 was down-regulated and PTEN was up-regulated in tumor tissues and serum-exosomes.6.The significantly higher level of miR-222 in chemo-resistant tissues and a corresponding inhibition of PTEN expression.Intersetingly,we found that TSG101,an exosome marker,were remarkably up-regulated in chemo-resistant tissues.Conclusions:1.A great enhancement of the amount of exosomes in adriamycin-resistant cells compare with sensitive cells.2.Exosomes derived from adriamycin-resistant cells can transmit drug-resistance to sensitive cells and modulate miR-222 pathway.3.The mimic-exos and inhibitor-exos complexes could used to study the roles of miRNAs in drug-resistance mediated by exosomes,and actually affected the resistance to ADR in MCF-7/S or MCF-7/ADR cells.4.The inhibitor-exos could decrease the drug-resistance of breast cancer in vivo.5.Exosomes involved in the progress of chemo-resistance by modulating miR-222/PTEN pathway in breast cancer patients with neoadjuvant chemotherapy.