Study of the Mechanism for Yak Milk Exosomal miRNA in Alleviating Hypoxia Injury of Intestinal Epithelial Cells

Author:Gao Hai Na

Supervisor:ren fa zheng yu qun li

Database:Doctor

Degree Year:2019

Download:34

Pages:110

Size:5951K

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Intestinal epithelial cells(IECs)are an important part of intestinal barrier.The barrier function was disrupted in hypoxia,but milk-derived exosomes can regulate the intestinal barrier function.However,the mechanisms underlying the association between yak milk exosomes and hypoxia in IECs remain poorly understood.This study was carried out to investigate:Milk-derived exosomes are rich in functional components such as nucleic acid(miRNA,etc).This stability allows contents to reach intestinal intact and be absorbed,and playing an important role in regulating the gastrointestinal development.However,it is still difficult to isolate pure exosomes from bovine milk owing to the complexity of raw materials(casein).Furthermore,the quantitative standard of exosomes is still unclear.In this follow-up study,we proposed an effective optimization method for purifying yak milk-derived exosomes.We isolated yak milk-derived exosomes using a differential method.Method 1 is ultracentrifugation,Method 2 is ultracentrifugation with Rennet precipitation.The exosomes were purified by ultracentrifugation combined with rent.Compared with the previously reported method,our extraction procedure is simpler,the isolated exosomes are of higher purity and the exosomes are lost less.TEM showed that the yak milk-derived exosomes were 50-200 nm vesicles surrounded by a cup-shaped lipid layer.Nanoparticle size distribution analysis(NSD)showed a peaking diameter of 109.2 ± 57.1 nm by isolation method 1 compared to a peaking diameter of 112.4 ± 48.6 nm by isolation method 2.The results of the NSD analysis were consistent with the TEM results.The presence of the known exosome markers TSG101,CD63,and Hsp70,and the absence of endoplasmic reticulum(ER)marker calnexin were analyzed by the western blot(WB).Method 2 resulted in slightly higher expression of the exosome-specific surface markers than Method 1.We then confirmed the exosome count,which analyzed by flow cytometry.Compared to the Method 1,Method 2 showed a higher exosome number(3 times compared to Method 1;P < 0.001).Our results showed that Method 2 is more suitable than Method 1 for the efficient purification of milk-derived exosomes.Intestinal epithelial cells(IECs)are an important part of intestinal barrier.The barrier function was disrupted in hypoxia,but milk-derived exosomes can regulate the intestinal barrier function.However,the mechanisms underlying the association between yak milk exosomes and hypoxia in IECs remain poorly understood.In this follow-up study,we proposed an effective optimization method for purifying yak milk-derived exosomes.The western blot(WB)analyses indicated that the expression of the TSG101,CD63 and heat stress protein 70(Hsp-70)proteins from yak milkderived exosomes were significantly higher than those in cow milk-derived exosomes.Flow cytometry analysis showed yak milk were 3.7 times more number of exosomes compared to cow milk.Moreover,we explored whether yak milk exosomes could facilitate intestinal cell survival under hypoxic condition in vitro.The MTT results showed that yak milk-derived exosomes was significantly increased IEC-6 cells survival with rates of up to 29% for cells incubated in hypoxic conditions for 12 h,compared to those of cow milk derived exosomes post-treatment(rates of up to22% for cells incubated in hypoxic conditions for 12 h).The confocal microscopy revealed that the IEC-6 cells uptake more yak milk-derived exosomes than cow milk in hypoxic conditions.Furthermore,the WB analyses indicated that yak milk-derived exosomes significantly promote PHD-1 expression and decrease the expression of HIF-α and its downstream target VEGF in the IEC-6 cells.Further,yak milk-derived exosomes significantly inhibited p53 levels.In conclusion,our findings demonstrate that yak milk-derived exosomes more effectively activate the HIF signaling pathway,thus promoting IEC-6 cells survival,which may result in higher hypoxia tolerance than cow milk-derived exosomes.Two mi RNA libraries were constructed using RNA in exosomes isolated from Cow-Exo and Yak-Exo using high-throughput sequencing technology,they involved the pathways and biological processes were also be researched.10 556 766 and 15 954 320 clean reads were obtained respectively which accounted for 67.18% and 71.81% of their raw reads respectively.130 differential expressed mi RNAs were screened out from 2 samples.There were 51 significantly up-regulated mi RNAs and 79 significantly down-regulated mi RNAs at Cow-Exo and Yak-Exo.The results of q RT-PCR verification of 12 mi RNAs with significant differential expression were consistent with the results of high-throughput sequencing.Finally the GO enrichment showed that the target genes of differentially expressed mi RNAs mainly involved in a variety of biological processes such as intracellular signal transduction and vacuolar transport.KEGG enrichment showed that the Lysosome pathway was a highly significant enrichment term(P<0.01),enriching to 79 candidate target genes.TNF signaling pathway、MAPK signaling pathway、Long-term potentiation、Central carbon metabolism in cancer、Sphingolipid signaling pathway、AGERAGE signaling pathway in diabetic complications、Phosphatidylinositol signaling system、neurotrophin signaling pathway was a significant enrichment term(P<0.05).The top 20 of the most significant differences in expression were related to the HIF-α regulation.HIF-1 signaling pathway enriching to 58 candidate target genes(P=0.052).The effect of yak milk-exosomal miRNAs on the proliferation of intestinal epithelial cell line IEC-6 was investigated.We explored whether bta-miR-31/34 a could facilitate intestinal cell survival under hypoxic condition in vitro.The MTT and IF results showed that bta-miR-31/34 a were significantly increased IEC-6 cells survival,compared to in hypoxic conditions.Furthermore,the WB analyses indicated that bta-miR-31/34 a significantly promote PHD-1 expression and decrease the expression of HIF-α and its downstream target VEGFA in the IEC-6 cells.Further,bta-miR-31/34 a exosomes significantly inhibited p53、Bax、Caspase-9 and Caspase-3 levels.The dual-luciferase reporter gene identification showed that the reported gene activity of Caspase-9-3 ’-UTR region was inhibited by bta-miR-34 a significantly.It indicates that bta-miR-34 a have a targeted relationship with Caspase-9 gene and inhibits its expression.In conclusion,our findings demonstrate that bta-miR-31/34 a effectively activate the HIF signaling pathway and Apoptosis signaling pathway,thus promoting IEC-6 cells survival.