HUC-MSC Enhanced Autophagy In AOPP-treated HK-2 Cells Through MicroRNA-145 And The Investigation Of Underlying Mechanism

ObjectivesIn this study,we cultured HK-2 cells(a human proximal tubular epithelial cell line)in vitro.We first investigated the effect of HK-2 cell autophagy by miR-145.Furthermore,we investigated whether hUC-MSCs enhanced-autophagy in AOPP-treated HK-2 cells.Lastly,we examined whether miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by inhibiting the PI3K/AKT/mTOR signaling pathway.Methods1.AOPPs preparation and content determinationHOC1(200 mmol/L)was mixed with BSA solution for 30 min at room temperature,and then dialyzed against PBS at 4? to remove free HOCl for 24 h.Native BSA was dissolved in PBS alone as the control.The endotoxin levels were measured and were required to be<0.025 EU/mL.The AOPP content was measured at 340 nm to obtain theabsorbance under acidic conditions and calibrated using chloramine-T in the presence of potassium iodide.2.HK-2 Cell Culture and Treatment3.hUC-MSCs Isolation and Co-culture with HK-2 cellsBriefly,a 10 cm umbilical cord from a full-term healthy newborn was washed with PBS(containing 1%penicillin-streptomycin double-resistant solution)3 times.Then,we cut the cord into small pieces,dislodged the umbilical vein and umbilical artery,and left Wharton's jelly at last.Wharton's jelly was then cut into 1 mm × 1 mm × 1 mm and cultured in DMEM/F12 medium containing 5%FBS;3-6 passages(P3-6)were selected for the following experiments.We used a co-culture chamber to block the immediate contact between the hUC-MSCs and HK-2 cells to explore the paracrine action of hUC-MSCs in the co-culture system.4.Flow Cytometry and hUC-MSCs IdentificationhUC-MSC were trypsinized,and the cell concentration was adjusted to 1×106/mL in PBS.Then,200 ?1 of the suspension was incubated with 5 ?l of antibodies against CD90,CD105,CD73,CD34,CD45,CD11b,CD14,and HLA-DR without light for 30 min.Primary antibodies were directly conjugated with FITC and phycoerythrin.For isotype control,non-specific FITC-conjugated IgG was substituted for the primary antibodies.Lastly,the samples were analyzed using flow cytometry.5.RNA Extraction and Quantitative Real-time PCR(RT-qPCR)AnalysisTotal RNA was extracted from treated HK-2 cells using TRIzol reagent(TaKaRa,Japan).1 ?g RNA was reverse transcribed using the MMLV reverse transcriptase kit according to the manufacturer'^instructions(TaKaRa,Japan).The U6 and miR-145 primers were provided directly by RiboBio Company and the sequences were kept secreted by RiboBio Company.All data were normalized using the internal control U6.6.Western Blotting AnalysisWe extracted total protein using RIPA lysis buffer.Then,20-50 ?g of protein was separated using SDS-PAGE and transferred to PVDF membranes.Subsequently,the membranes were blocked in 5%nonfat milk or BSA at room temperature for 2 h,followed by incubation of the primary antibodies at 4?overnight.The primary antibodies were as follows:anti-LC3B,anti-Beclin 1,anti-p62,anti-p-mTOR,anti-mTOR,anti-p-AKT,anti-AKT,anti-p-PI3K(dilution,1:1000)and anti-GAPDH(dilution,1:5000).The membranes were incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 2 h and were finally detected using an enhanced chemiluminescence(ECL)system.GAPDH was used as the internal control and semiquantitative analysis was performed using the ImageJ system.7.Immunofluorescence StainingCells plated in 96-well plates were fixed with 4%paraformaldehyde,permeabilized with 0.5%Triton X-100 for 10 minutes and incubated in a blocking buffer containing 5%BSA for 30 min at room temperature.Then,cells were incubated with LC3B antibody(1:50 dilution)overnight at 4?.Finally,we incubated cells with:fluorescently labeled secondary antibodies(Alexa Fluor 488,1:400,Gongsi)for 1 h at room temperature in the dark,followed by incubation with 0.1%DAPR for 10 min.We used an inverted fluorescence microscope to observe and record the LC3B-positive staining.8.Transmission Electron Microscopy(TEM)HK-2 cells were gently harvested using trypsin with EDTA and were centrifuged at a speed of 1000 r/m for 5 min.Then,the sediment was washed twice with cold PBS and fixed in 2.5%glutaraldehyde for 2 h at 4?.Cells were conventionally dehydrated,embedded,sliced into 60 nm sections and stained with uranyl acetate for 15 min at room temperature,followed by lead citrate for 15 min at room temperature.Autophagosome(AP)and autolysosome(AL)formation were observed using TEM at a magnification of low-power field(x6000,x8000,x10000)or high-power field(x40000)operating at 60 kV.9.HK-2 Cell Transfection And hUC-MSC TransfectionHK-2 cells were transfected with miR-145 siRNA,miR-145 mimics,or negative siRNA,and hUC-MSCs was transfected with n:miR-145 siRNA,using Lipofectamine 2000(Invitrogen,CA,USA)according to the manufacturer's instructions.Targeting sequences were as follows:miR-145 siRNA:sense(5'-AGGGAUUCCUGGGAAAACUGGAC-3');miR-145 mimics:sense(5'-GUCCAGUUUUCCCAGGAAUCCCU-3'),antisense(5'-GGAUUCCUGGGAAAACUGGACUU-3');negative siRNA:sense(5'-UUCUCCGAACGUGUCACGUTT-3'),antisense(5'-ACGUGACACGUUCGGAGAATT-3').10.Statistical AnalysisStatistical analyses were performed using SPSS 20.0 software.Continuous variables are presented as the mean± SD.One-way ANOVA was carried out to determine differences among groups.For the comparnson of 2 groups,the LSD method was used when the assumption of variance was equal.Otherwise,the Dunnett T3 method was used.P values<0.05 indicated a statistically significant difference.Results1.Identification results of hUC-MSCsWe chose P3 hUC-MSCs to identify the cell phenotype by flow cytometry.Flow cytometry analysis of the cell phenotype showed that the hUC-MSCs were positive for CD90,CD105,and CD73,but were negative for CD34,CD45,CDllb,CD14,and HLA-DR.2.miR-145 was upregulated in HK-2 cells under hUC-MSCs co-cultured systemThe result showed that when HK-2 cells were co-cultured with hUC-MSCs,the mRNA level of miR-145 was significantly upregulated.Next,we knocked down miR-145 in the hUC-MSCs and then co-cultured it with HK-2 cells.We found that in the later co-culture system,miR-145 did not increased in HK-2 cells.The data indicated that hUC-MSCs secreted miR-145 and transferred it to HK-2 cells through paracrine action.3.miR-145 affected HK-2 cell autophagyWe first downregulated or upregulated the expression of miR-145 in HK-2 cells and examined the mRNA level of miR-145.The RT-qPCR results showed that miR-145 siRNA downregulated miR-145;however,miR-145 mimics upregulated miR-145 in HK-2 cells.At the same time,the expression of miR-145 showed no difference between negative siRNA and the normal control group.We then assayed the autophagy-related proteins LC3B,Beclin 1,and p62 by western blotting.The results showed that when we inhibited the expression of miR-145 by siRNA,the protein levels of LC3 II and Beclin 1 decreased and the protein level of p62 increased.However,when we upregulated the expression of miR-145,the protein levels of those markers mentioned above had the opposite effect.In addition,there was no difference between negative siRNA and the normal control group.Furthermore,the autophagic activity of HK-2 cells was assessed using immunofluorescence technology and TEM.LC3B-positive staining was significantly decreased in cells transfected with miR-145 siRNA but increased in cells transfected with miR-145 mimics compared with that of the normal control group.Similarly,the TEM results demonstrated a decrease in the number of typical Aps(double membrane vacuoles engulfing cytoplasmic structures)and ALs(s:ingle membrane vacuole structures containing high density materials)in miR-145 siRNA transfected cells but revealed an increase in the number of Aps and ALs in miR-145 transfected cells compared with those in the normal control group.Collectively,these results suggested that overexpression of miR-145 activated HK-2 cell autophagy.4.miR-145 mediated hUC-MSCs enhanced-autophagy in AOPP-treated HK-2 cellsWe transfected HK-2 cells with miR-145 siRNA after AOPP-treated HK-2 cells were co-cultured with hUC-MSCs to investigate the effect of hUC-MSCs on HK-2 cells.We first measured the miR-145 expression in transfected and co-culture system.RT-qPCR analysis showed that the level of miR-145 was increased in the hUC-MSCs and AOPP co-culture group compared with that in the normal control group,but the miR-145 level was decreased in the transfected and co-culture system compared with the hUC-MSCs and AOPP co-culture group.Next,we detected the autophagous activity after transfection with miR-145 siRNA in the hUC-MSCs and AOPP co-culture system.The western blotting results showed that when we decreased the expression of miR-145 by siRNA,the protein levels of LC3 ? and Beclin 1 decreased and the p62 protein level increased compared with those in the hUC-MSCs co-culture system.Furthermore,LC3B-positive staining was significantly decreased in cells transfected with miR-145 siRNA compared with that in the hUC-MSCs co-culture system.Similarly,the TEM results demonstrated a decrease in the numbers of typical Aps and ALs in HK-2 cells in siRNA transfected with the hUC-MSCs co-culture system compared with those in the hUC-MSCs co-culture system alone.Taken together,these results indicated that miR-145 secreted from hUC-MSCs enhanced HK-2 cell autophagy5.miR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells by inhibiting the PI3K/AKT/mTOR signaling pathwayTo verify the underlying pathway,we detected PI3K/AKT/mTOR signaling pathway related proteins after regulating miR-145 expression in HK-2 cells or transfection with miR-145 siRNA in the hUC-MSCs co-culture system.The western blotting results showed that when we decreased the expression of miR-145 by siRNA,the phosphorylation of PI3K,AKT,and mTOR was induced.However,when we upregulated the expression of miR-145 by mimics,the phosphorylation of PI3K,AKT,and mTOR was inhibited.In addition,there was no difference between negative siRNA and the normal control group.Finally,transfection with miR-145 siRNA in the hUC-MSCs co-culture system promoted the phosphorylation of PI3K,AKT,and mTOR,which was inhibited by hUC-MSCs.We concluded thatmiR-145 mediated hUC-MSCs enhanced-autophagy in HK-2 cells via inhibiting the PI3K/AKT/mTOR signaling pathway.ConclusionhUC-MSC played an autophagy enhancement role via overexpression of miR-145 by Inhibiting the PI3K/AKT/mTOR Signaling Pathway,which provided a novel mechanism for the investigation of hUC-MSCs in AKI treatment.