Exploration Of Therapeutic MiRNA Loaded By Exosomes From Human Colostrum

MicroRNA(miRNA)is closely related to tumor genesis and development.For they are commonly referred to as tumor suppressors and oncogenes.Previous studies of our group found that miR-140-3p expression was significantly higher in breast fibroadenoma than in lobar tumor.Overexpression of miR-140-3p in human fibrosarcoma cell line HT1080 can inhibit the proliferation and migration of tumor cells.However,the free miR-140-3p is easily decomposed by RNase,which is widely available in the environment.Therefore,the stability of miR-140-3p should be maintained in order to apply it to the treatment of tumors.Extracellular vesicles are bilayer membranous cystules that fall off the cell membrane.These include exosomes,microvesicles,and apoptotic bodies.Exosomes are extracellular vesicles with diameters between 30nm and 200nm,and their double lipid membrane structure protects miRNA from RNase degradation.Human colostrum is rich in vesicle structures,and there are no reports on treating tumors with human colostrum exosomes as miRNA carriers.Study objectives1.Exosomes of human colostrum were extracted and verified.2.To explore the feasibility of using exosomes from human colostrum as therapeutic miR-140-3p vector.Material and methods1.The milk of healthy mothers within 1 week after delivery was collected and exosomes were isolated from colostrum by supercentrifugation.2.Identification of extracted exosomes:①Expression of membrane protein CD9,membrane binding protein TSG101,nuclear protein TFIIB、Lamin A/C in colostrum exosomes was detected by Western blot.②Transmission Electron Microscope(TEM)was used to observe the morphology and membrane integrity of exosomes.③The concentration and particle size of exosomes were detected by Nanoparticle Tracking Assay(NTA).3.Fluorescent labeling of exosomes:The membrane proteins of exosomes were labeled with PKH67 green fluorescent dye,and miR-140-3p was synthesized and labeled with red fluorescent Cy3.The fluorescentially labeled miR-140-3p was transferred into exosomes by repeated freeze-thaw,and then co-incubated with HT1080 cells for 24h.Whether miR-140-3p was transferred into exosomes and entered cells with exosomes was observed under fluorescence microscope.4.After the exosomes transformed into miR-140-3p and then were incubated with HT1080 cells for 24h.RNA in the cells was extracted and the expression of miR-140-3p was verified by qRT-PCR,and compared with the empty exosome group.Results1.A total of 14 samples of colostrum from healthy parturients were collected and centrifuged,respectively.The concentration of total protein in colostrum extract was high by BCA method.Western blot result showed that the expression of membrane protein CD9 and membrane binding protein TSG101 in colostrum extract was positive,while the expression of nuclear protein TFIIB and Lamin A/C was negative,which was consistent with the proteins expression of exosomes.2.Transmission Electron Microscopy showed that "coaster-shaped" vesicles could be seen in the colostrum extract.The membrane structure was intact despite the freeze-thaw process and consistent with the normal size of exosomes.3.Detection by Nanoparticle Tracking Assay showed that the average diameter of vesicles in colostrum was 197.7±27.6nm,and the concentration was 1.47±0.89×1012 Particles/ml,which was in line with the diameter range of exosomes.Compared with the exosomes of mature milk and nipple discharge previously measured by our research group,the size of colostrum exosomes was larger and the concentration was higher.4.Fluorescence detection results showed that Cy3-labeled miR-140-3p could enter human colostrum exosoms through repeated freeze-thaw and could enter HT1080 cells through co-incubation process.5.After the miR-140-3p was transferred into exosomes and co-incubated with HT1080 cells in the group,the expression of miR-140-3p was significantly increased.ConclusionThe extraction efficiency of human colostrum exosome by supercentrifugation is high and exosome enrichment can be achieved.Colostrum exosomes can be transferred into miR-140-3p by repeated freeze-thaw method and transferred into human fibrosarcoma cell line by incubation method.Human colostrum is rich in exosomes,which can be used as the carrier of miR-140-3p for tumor treatment.