Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell- Exosomal MiR-340 In Treating Radiation-induced Lung Injury

BackgroundRadiation-induced lung injury(RILI)frequently occurs in patients with lung,breast cancer after ionizing irradiation treatment.Patients with severe radiation pneumonitis have an almost 50% mortality.Natural medicines,biological reagents,chemical medicines,and small molecules have been used to prevent and treat acute radiation injury by reducing free radicals and stabilizing the DNA structure.however,their disadvantages include a short half-life,an easily degradable nature,and the ability to cause serious adverse events.To date,although lung transplantation is the most useful intervention for treating radiation-induced pulmonary fibrosis,it is limited by the lack of available donated lungs and transplantation-related complications.Increasing evidence has suggested that MSCs release exosomal miRNAs as vital extracellular communicators to mediate the regenerative and immunomodulatory effects that prevent inflammatory and fibrogenic activity in injured tissue.ObjectiveIn order to study the mechanism of human umbilical cord mesenchymal stem cell delievers exogenous miR-340 via exosome to attenuate radiation induced lung injury.To improve the ability of anti-inflammation of MSCs-exosome therapy and use a safe,effective cell-free therapeutic strategy for RILI.MethodFirst,the possibility of miR-340 binding to the 3-'UTR sequence of TLR4 gene was predicted using bioinformatics software Target Scan and miRanda database.The ability of miR-340 to bind to the 3-'UTR of TLR4 was detected by double luciferase assay.RT-PCR,Western blot detected that the expression levels of TLR4 and its downstream target protein phosphorylation(p-p65)was regulated miR-340 by targeting the 3'-UTR of TLR4.Identification of hUCMSCs adipogenesis,osteogenesis and chondrogenesis by differentiation induction.The Umibio exosome extraction kit extracted stem cell exosomes and identified surface markers.Then the radio active lung injury mice model(IR)was established,and PBS,MEX-miR-NC,MEX-miR-340 were injected into the tail vein respectively.The mice were divided into four groups:(1.Normal,IR+PBS,IR+MEX-miR-NC,IR+ MEX-miR-340).The inflammatory cytokines in plasma of RILI mice with MEX-miR-340 treatment was detected via enzyme-linked immunosorbent assay(ELISA).TLR4 expression and phosphorylation level of its downstream target proteins NF-?B/p65 were detected by western blot in lung tissue samples of RILI mice after 16 weeks.H&E,IHC staining were performed to observe the degree of reduction in lung tissue of RILI mice after treatment with MEX-miR-340.To observe the 20-week survival rate of RILI mice with MEX-miR-340 treatment.ResultThe dual-luciferase assay results showed that dual luciferase assay showed that miR-340 had the ability to bind to the 3'-UTR of TLR4.Meanwhile,Western blot showed the expression levels of TLR4 and its downstream target protein phosphorylation(p-p65)were significant decreased in RAW264.7 and 293 T cells after transfection with miR-340mimic(50 nM)or mimic NC for 48 hours.This suggests that miR-340 can inhibit the expression of TLR4 and its downstream inflammatory pathway by binding to the 3'-UTR of TLR4.hUCMSCs had the potential of osteogenesis and lipogenesis.Flow cytometry analysis showed that the surface marker CD29?CD73?CD90?CD105 was positive and CD14?CD19?CD45?CD34 was negative.The transmission electronmicroscopy images showed hUCMSC-derived exosomes were round vesicles.The markers of hUCMSC-exosomes were CD9 and CD63 by Western blot.The relative content of miR-340 was the highest in MEX-miR-340 than others by RT-PCR.The results of ELISAshowed that TNF-?,IL-1? and IL-6 in MEX-miR-340+IR decreased,while IL-10 increased.Western blot showed that the TLR4 and phosphorylation of NF-kB were significantly reduced by MEX-miR-340.H&E,Masson' s trichrome staining,IHC staining showed that can significantly attenuate lung injury.hUCMSC-exosomes overexpressing miR-340 treatment improved the survival rate of RILI mice.MiR-340 was down-regulated in plasma of patients with RILI.ConclusionMiR-340 suppresses TLR4 expression by targeting 3-'UTR of TLR4.Compared with hucmsc exosomes,over expression of miR-340 in hUCMSC exosomes is more effective in inhibiting TLR4 signaling pathway and alleviating inflammatory response.Compared with MEX,MEX-miR-340 can improve the anti-inflammatory ability more effectively,which can be used as a safe and effective cell-free treatment strategy for RILI.