| Background and Objective:Due to the high incidence of chest cancer,especially lung cancer,oncologists think that the majority of patients with this kind of disease need radiotherapy.However,all managements for tumors are a double-edged sword,radiotherapy is no exception.Good results always accompany by side effects.DNA double-strand break(DSB)is the most serious type of damage.In normal lung tissues,the unfavorable repair of DSB is closely related to the occurrence and severity of radiation damage.Until now,there is still no particularly effective treatment for radiation-induced lung injury,so it is urgent to develop new therapeutic agents to improve radiation-induced lung injury.The previous studies of our group have found that mesenchymal stem cells(MSCs)have a good effect on the repair of radiation-induced lung injury.Neverthless,it is still unclear that whether MSCs can participate in the repair of DSB through paracrine effect.Therefore,we decided continually to study the repair mechanism of MSCs on DSB,and explore the potential mechanism of MSC-derived Exosome(MSC-exo)on DSB repair.Methods:(1)MSC-exo isolated by total exosomes isolation reagent.The exosome marker proteins were detected by Western blot analysis.Nanoparticle tracking analysis(NTA)and transmission election microscope detect exosome's shape and diameter.(2)Different cell lines(A549/Beas-2b)incubated with DMEM,MSCM and MSC-exo,after irradiation by 10 Gy X-rays,which detect the expressions of key proteins(?H2AX,BRCA1,LIG4,53BP1 and Rad51)in DSB repair by Western blot analysis.The apoptosis was detected by flow cytometry.(3)The MSCs were radiated with 2 and 10 Gy,non-irradiation and post-irradiationMSC-exo were collected,then isolated by total exosomes isolation reagent.The target cells were incubated by MSC-exo without and with 2 or 10 Gy X-ray irradiation,which detect the key protein ?H2AX.Then the changes in miRNA expression profiles were analyzed by high-throughput sequencing.The downstream targets of miRNA were identified though literature review and target gene analysis.The miRNA target genes were identified by miRNA mimics.(4)The miRNA target genes were overexpressed and nominated,the target proteins were identified,then their target protein functions were further validated.Results:(1)The exosomes were isolated by total exosomes isolation reagent from MSC culture medium.The results detected with NTA and the transmission election microscope showed that exosomes were round or ovoid with the diameter of 50-200 nm.The Western blot showed that the exosomes expressed the markers CD9,CD63 and CD81.The exosomes were stained with PKH26 dye,the target cells could stain in a short time,which suggest that exosomes have membrane fusion ability.(2)The results in vitro showed that MSCs participated in repair of DSB by exosome-mediated paracrine effects.Flow cytometry showed that MSC-exo effectively reduced the apoptosis.(3)High-throughput sequencing revealed a change in the expression profile of miRNA after MSCs received ionizing radiation.Ionizing radiation could up-regulate the expression of miR-1246,especially 2 Gy irradiation.(4)Ionizing radiation could up-regulate the expression of miR-1246.MiR-1246 could promote the repare in non-homologous end joining(NHEJ)pathway by targeting LIG4,then alleviate radiation damage.Conclusion:The MSC-exo-mediated paracrine effect has a clear repair effect on DSB.Ionizing radiation induces MSC-exo to up-regulate miR-1246,and target LIG4,and promot the NHEJ pathway. |
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