The Study On The Mechanism Of MiR-146b/Merlin/YAP Signaling Pathway In Hucmsc-derived Exosomes Delaying Diabetic Kidney Disease

Objective: Diabetic kidney disease(DKD)is a serious diabetic microvascular complication and the leading cause of end-stage renal disease.Early research by the research group found that miR-146 b may be a biomarker for early diagnosis and prognosis of acute kidney injury.Here we further explore the mechanism of miR-146 b in human umbilical cord mesenchymal stem cell-derived exosomes(hucMSC-Ex)in delaying DKD,and provides new experimental evidence for clinical applications.Methods: In vivo experiments,high-fat diet and STZ-induced SD rats were used to construct a DKD model,normal SD rats(Normal group)were used as controls.Renal tissues were taken at 8,16,and 24 weeks,and HE staining and Masson staining were used to verify the success of the injury model.Construct a high glucose environment in vitro and culture three kinds of kidney cells: rat mesangial cells(HBZY-1),rat renal tubular epithelial cells(NRK-52E),and rat podocytes;RNA extraction from rat kidney tissue and kidney cells cultured in vitro to detect miR-146 b expression,,and determine the target cells for research.Through a lot of literature reading and TargetScan software to predict the target gene of miR-146 b as NF2(neurofibromatosis type 2),encoding the Merlin protein,which regulates the Hippo/YAP signaling pathway,the double luciferase reporter assay to verify;Then overexpress/inhibition of miR-146b’s expression in HBZY-1 cells,Western Blot,qRT-PCR and cellular immunofluorescence were used to verify the downstream pathways.HucMSC-Ex was used to intervene in vivo and in vitro models.QRT-PCR was used to detect the expression of miR-146 b in each group,Western Blot and qRT-PCR were used to detect the expressions of Merlin and YAP.The expression of Merlin in renal tissue was detected by immunohistochemistry,and the Merlin/YAP fluorescence intensity was detected by cell immunofluorescence.The upstream molecules of miR-146 b were screened by StarBaseV2.0 software and combined with literature reading,and the circ-0002940 molecule was identified as the further research object.QRT-PCR was used to detect its expression in hucMSC and hucMSC-Ex.The double luciferase reporter gene experiment and siRNA transfection experiment were performed to validate the effect of circ-0002940 on the miR-146b/Merlin/YAP pathway.Results: The results of HE staining and Masson staining showed that the DKD model was successfully constructed.The expression of miR-146 b increased significantly in the DKD group at 16 weeks,and gradually increased with the progress of the disease.The results of in vitro experiments showed that the expression of miR-146 b was mainly increased in HBZY-1.And the Merlin is a target gene of miR-146 b by double luciferase reporter assay.In addition,transfection of miR-146 b mimics and inhibitors verified downstream pathways,and the results showed that Merlin expression decreased after miR-146 b overexpression,and YAP expression increased,while the opposite was observed in the inhibitor group.Both in vitro and in vivo experiments showed that miR-146 b expression decreased after hucMSC-Ex intervention;Western Blot and qRT-PCR results showed that Merlin expression was down-regulated and YAP and α-SMA expression were up-regulated in the DKD group;The expression of YAP and α-SMA was down-regulated;the results of immunohistochemistry showed that Merlin expression in DKD kidney tissue was significantly reduced,while the expression in hucMSCEx group was enhanced,and the cell immunofluorescence results showed that the fluorescence intensity of Merlin protein in the 30 mM high glucose group decreased,and the fluorescence intensity of YAP protein increased and the nucleation increased;the hucMSC-Ex group did the opposite.By screening,the upstream molecule of miR-146 b was circ-0002940.The double luciferase reporter assay verified that there was a targeting relationship between the miR-146 b and circ-0002940.After transfection of siRNA,the expression of circ-0002940 were decreased significantly,while Merlin was down regulated,YAP and α-SMA were upregulated,which indicates that hucMSC-Ex can regulate the Hippo/YAP signaling pathway through the sponge effect of circ-0002940 and miR-146 b.Conclusions: The expression of miR-146 b is elevated in in vivo and in vitro models of DKD.The downstream target gene of miR-146 b is Merlin,which acts on DKD through the Hippo/YAP signaling pathway.HucMSC-Ex may inhibit the effect of miR-146 b and improve renal function through the circ-0002940 molecule.