The Effect And Mechanism Of Exosomes Transmitting MiR-200c On Paclitaxel-resistant Breast Cancer Cells

Objective: In this paper,we analyse the possibility of whether exosome miR-200 c can be a biomarker for early diagnosis of BC.Next,we also conducted a preliminary study on the effects of miR-200 c on the malignant behaviour of BC cells.Finally,we also evaluated the impact and possible mechanisms of exosome miR-200 c in BC cells during resistance to paclitaxel.We expect that this study will provide a fresh perspective and ideas for the early diagnosis and treatment of BC.Methods:(1)Bioinformatics analysis of the pre-sequencing results to find differentially expressed miRNAs;(2)Analysis of miR-200 c expression levels and survival of BC patients using the Onco Lnc database;(3)Clinical specimens were collected,the expression of exosome miR-200 c was obtained by q PCR and analysed for its clinical diagnostic value;(4)q PCR to detect miR-200 c expression in MCF-10 A,MDA-MB-231,MCF-7 and MCF-7 Taxol cells and exosomes;(5)Transfection of miR-200 c minic/miR-NC,miR-200 c inhibitor/inhibitor-NC to assess the effect of miR-200 c on MCF-7 malignant behaviour.(proliferation analysis: EDU,CCK-8;invasive motility analysis: Transwell;migratory motility analysis: scratch,Transwell;colony formation analysis: colony formation assay);(6)q PCR and GW4869 to detect the presence of extracellular miR-200 c forms,q PCR and PKH-26 exosome markers to detect coculture of MCF-7 after Taxol uptake of MCF-10A-derived exosomes;(7)CCK-8 and q PCR to compare the resistance of MCF-7 and MCF-7 Taxol to Taxol,and the change in resistance after MCF-7 Taxol uptake of exosome miR-200c;(8)Transfection of miR-200 c minic/miR-NC to assess the effect of miR 200 c on the malignant behaviour of MCF-7 Taxol(Study method as above);(9)q PCR assays for apoptosis-related genes within MCF-7 Taxol being affected by alterations in miR-200 c.Results:(1)After analyzing the sequencing results,we identified several differentially expressed miRNAs.miR-200 c was one of the miRNAs with the most decreased expression levels in BC cell exosomes,so miR-200 c was chosen as the subject of study.(2)In this study,we downloaded survival data of BC patients from the Onco Lnc database and found that low expression of miR 200 c was indeed associated with a high risk of death in patients;(3)Clinical specimens were collected and serum exosome miR-200 c levels were found to be significantly lower in BC patients compared to controls,indicating that serum exosome miR-200 c has some diagnostic efficacy for BC and may be a promising biomarker for early diagnosis of BC;(4)In cellular level studies,miR-200 c was found to be lower in MDA-MB-231,MCF-7,MCF-7 Taxol intracellularly and exocytically compared to MCF-10 A,and was expressed at the lowest level in MCF-7 Taxol exocytosis;(5)Up-regulation of miR-200 c expression level inhibited the proliferation,invasion and migratory movement,colony formation ability of MCF-7 cells formation ability,while down-regulation of miR-200 c had the opposite result;(6)Extracellular miR-200 c existed mainly as encapsulated by exosomes,and MCF-7 Taxol could increase its intracellular miR-200 c expression level by uptake of MCF-10A-derived exosomes;(7)Uptake of exosome miR-200 c by MCF-7 Taxol could reduce its resistance to Taxol;(8)Up-regulation of miR-200 c expression levels could inhibit the proliferation,invasion and migratory movement,and colony formation ability of MCF-7 Taxol cells;(9)Up-regulation of miR-200 c expression levels of MCF-7 Taxol could promote P53,Bax,Caspase 3,Caspase 9 expression,at the gene level,and inhibit Bcl-2 expression.Conclusion: In this study,we found that serum exosome miR-200c had some diagnostic efficacy in this group of BC samples and has the potential to be a biomarker for early diagnosis of BC.Regulation of the intracellular miR-200 c expression level of MCF-7 could significantly suppress its malignant behaviour.Further studies revealed that MCF-7 Taxol could phagocytose miR-200c-rich exosomes,thereby reversing their drug resistance and inhibiting malignant behaviour.Finally,it was found that exosome miR-200 c may enhance drug sensitivity and suppress malignant behaviour of Taxol-resistant BC cells by activating apoptosis-related genes.