| Objective: To investigate changes of microRNA(miRNA)expression in hypoxia-induced human kidney 2(HK2)cell-derived extracellular vesicles(EVs),and explore the specific mechanism by which the miRNA secreted by the hypoxia-induced HK2 cell-derived EVs repairs renal ischemia-reperfusion injury(IRI).Methods:1.The HK2 cell-derived EVs were extracted by ultracentrifugation.The EVs were identified by nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM)and Western blot(WB);Moreover,C57BL/6 mice were divided into 4 groups: sham surgery(SHAM)group,bilateral renal IRI injected with PBS(IRI+PBS)group,or IRI+EVs from HK2 cells cultured under normoxic conditions(NOR-EVs)group,or IRI+EVs from hypoxia-induced HK2 cells(HYP-EVs)group.The degree of injury to kidney tissue in different treatment groups was assessed by PAS staining,renal tubule injury score,and serum creatinine(Scr)assay.2.Combined with high-throughput sequencing results,miRNAs in EVs(EV-miRNAs)under normoxia and hypoxia were extracted and verified by RT-PCR.The elevated miRNAs under hypoxic conditions were selected and analyzed by bioinformatics analysis to find targeted genes.3.HK2 cells were transfected with miRNA-125-5p mimics(miR-MI)and microRNA negative control(miR-NC)respectively,and the expression of miRNA-125b-5p in HK2 cells and HK2 cell-derived EVs was validated by RT-PCR;In addition,the content of NLRC5 in HK2 cells transfected with miR-MI and miR-NC was detected by RT-PCR and WB respectively,and the dual luciferase reporter gene technology was used to verify the targetingrelationship between miRNA-125b-5p and NLRC5.4.C57BL/6 mice were divided into 4 groups: SHAM group,IRI+PBS group,or IRI+EVs from HK2 transfected with miR-NC(NC-EVs),or IRI+EVs from HK2 transfected with miR-MI(MI-EVs);The PAS staining,renal tubule injury score,and Scr assay were used to evaluate the degree of injury of kidney tissues,and the NLRC5 in each kidney tissues group was identified by immunohistochemistry.Results:1.The TEM,NTA and WB results were consistent with the phenotypic characteristics of EVs;By PAS staining,renal tubule injury score and SCr assay were found that compared with the IRI+PBS group,EVs groups can significantly alleviated the degree of renal IRI,and the protective effect of IRI+HYP-EVs group is the most obvious.2.In combination with high-throughput sequencing results,RT-PCR results showed that four EV-miRNAs were significantly up-regulated under hypoxic conditions compared with the normoxic conditions;The bioinformatics analysis found that both miRNA-125-5p and NLRC5 are closely related to the PI3K-AKT pathway of renal IRI,and miRNA-125-5p has a binding site with NLRC5 3' untranslated regions(UTR).3.Compared HK2 cells transfected with miR-NC,the RT-PCR results of HK2 cells transfected with miR-MI showed that miRNA-125-5p was up-regulated in HK2 cells and HK2 cell-derived EVs;At the same time,RT-PCR and WB results showed that the expression of NLRC5 was also decreased in the HK2 cells.Dual luciferase reporter gene demonstrated that miRNA-125-5p can target NLRC5 in a targeted manner.4.Compared with IRI+PBS group,PAS staining,renal tubule injury score and SCr assay found that EVs can significantly renovate the degree of renal IRI,and IRI+MI-EVs group effect is significantly obvious;Immunohistochemistry results found that compared with the IRI+PBS group,the NLRC5 content in the kidney tissue was significantly decreased after EVs treatment and the protective effect of IRI+MI-EVs group is the most obvious.Conclusion: miRNA-125b-5p was significantly up-regulated in hypoxia-induced HK2cell-derived EVs and miRNA-125b-5p can exert its protective effect to renal IRI by targeting negative regulation of NLRC5. |
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