MicroRNA Let-7f-5p Promotes Bone Marrow Mesenchymal Stem Cells Survival In Alzheimer Disease Model

Background and PurposeBone marrow-derived mesenchymal stem cells?BM-MSCs?can differentiate into neurons and glial cells.They are capable of homing to lesion tissues,secreting different neurotrophic growth factors?NTFs?to promote regeneration and neuroprotection and holding great potential in stem cell-based therapies for neurodegenerative diseases.Alzheimer disease?AD?is common form of dementia.MSCs can cross the blood-brain barrier,stimulate neurogenesis and significantly rescue learning and memory deficits of animals.But the low survival rate after transplantion hampered the efficiency of MSCs.MicroRNAs?miRNAs?is reported to be functionally linked with cell survival.Let-7 family are regulators of cell proliferation and apoptotic genes.Let-7 inhibits doxorubicin-induced and paclitaxel-induced apoptosis,as well as ultraviolet B-induced apoptosis.Let-7f-5p exerts a vital role in MSCs differentiation,but its influence on MSCs apoptosis has not been studied yet.Accordingly,the present study was designed to test the hypothesis that let-7f-5p could regulate the survival of implanted MSCs in AD models.Methods:1.BMSCs were isolated from SPF level C57BL/6J mice?aged 8 weeks,25-35g?and cultured by whole bone marrow culture,and then subcultured to 6generations for the following experiments.2.MSCs were treated by A?_25-355-35 for 24 hours,compared to untreated group.Cell viability was tested by MTT assay and cell apoptosis was examined by Annexin V/PI staining.Western blot analysis compared the expression of caspase-3,cleaved caspase-3 in two groups of cells.The expression of let-7f-5p was detected by PCR.3.Mmu-let-7f-5p-LV,mmu-let-7f-5p-inhibition-LV and mmu-let-7f-NC-LV were constructed and transfected into three groups of MSCs:let-7f-5p upregulation group,let-7f-5p downregulation group and negative control?NC?group.After A?_25-355-35 treatment,Annexin V/PI staining flow cytometry was used to analyze cell apoptosis.4.The complementary binding between let-7f-5p and caspase-3 genes was predicted and verified by dual luciferase reporter assay.The differences of caspase-3and cleaved caspase-3 expression between two groups were detected by Western blot.5.MSCs from let-7f-5p upregulation group,let-7f-5p downregulation group and negative control?NC?group were injected into AD mice respectively,with wild type?WT?group mice as blank control.After 5 days of transplantation,the apoptotic rate of MSCs was confirmed by histology experiments:hematoxylin and eosin staining,TUNEL and immunofluorescence.6.Bioluminescent imaging was used to observe the survival time of transplanted MSCs in AD mice.Results1.A?_25-355-35 could induce cell apoptosis of MSCs,mainly early apoptosis,with caspase-3 activation and let-7f-5p suppression.2.Let-7f-5p exerts an anti-apoptosis effect.3.Caspase-3 was a target gene of let-7f-5p and there is a negative correlation between caspase-3 and let-7f-5p.4.Let-7f-5p could inhibit apoptosis of MSCs in vivo.5.Let-7f-5p could prolong the retention of transplanted xenograft MSCs.Conclusions1.A?_25-355-35 is cytotoxic to MSCs and could induce apoptosis of MSCs,especially early apoptosis.2.Let-7f-5p can inhibit A?_25-35-induced apoptosis of MSCs and promote cell survival by targeting caspase-3.3.Let-7f-5p can promote the survival of MSCs after transplantation in AD animals.