Study On The Effect Of MicroRNA21 To Renal Mesangial Cell Function By Means Of Biomaterials

BackgroundChronic kidney disease(CKD)can cause renal fibrosis and eventually lead to end-stage renal disease(ESRD).To explore the mechanism and prevent renal fibrosis is a hot spot and difficult problem,which has important social and economic significance.The understanding of renal fibrosis are mostly based on the two-dimensional(2D)cell models.2D cell culture models could not mimic microenvironment in vivo really well.Although the current three-dimensional(3D)cell culture models are closer to the physiological conditions,limitations inherent to such protocols remain a ubiquitous problem for biological applications.With emerging 3D printing technologies and more appropriate biomaterials,new in vitro methodologies can be established to better mimic the biological architectures in vivo renal systems.The change of gut microbiota metabolism in CKD patients has been suggested for some time.Trimethylamine-N-oxide(TMAO),a gut microbial-dependent metabolite is elevated in chronic kidney diseases.Possible effects of TMAO have been only partially explored so far.High level of microRNA 21(miR-21)was also reported in renal fibrosis.The pathogenesis for TMAO and miR-21 in RMCs are not fully elucidated.The anti-miR-21 may be an effective therapeutic tool in renal fibrosis.But it is difficult for constructing short chain miR-21i vectors.The non-viral vectors have advantages in biological safety,gene load capacity and synthesis,compared with viral vectors.However,the current non-viral vectors for miR-21i have low transfection efficiency and maybe synthesized through complex processes.Previous studies indicated that single-walled carbon nanotube(SWCNT)was an excellent delivery nanovector of small molecule drug,protein,plasmid DNA,small interfering RNA.To date,there are few reports describing the use of SWCNT as miRNA delivery vectors.Little is known about the ability of SWCNT to deliver miRNA sequences into renal mesangial cells for antifibrosis therapeutic purposes.Objective1.To construct 3D cell model of polylactic acid scaffolds(PLA)coated with biocompatible materials and to test the hypothesis that TMAO and miR-21 plays a direct effects on renal mesangial cells in 3D cell model.2.To develop a new effective SWCNT-based nanovetor for miR-21i delivery,which would be taken as a facile method for inhibition of renal fibrosis.Method and ResultsPart 1:Investigate the effect of TMAO and miR-21 to renal mesangial cells in 3D culture model based on 3D bio-printing polylactic acid scaffolds.1.The Fused deposition modelling method was used to print the polylactic acid scaffolds.3D-PLA-PBS scaffold was prepared after immersed in 75%alcohol.Then,3D-PLA-Gelatin scaffold and 3D-PLA-RGD scaffold were prepared after immersed in dopamine solution?gelatin solution or RGD solution,respectively.RMCs in 2D culture model were seeded in culture plate/bottle.RMCs in 3D culture models were seeded on the 3D scaffold,respectively.2.Morphology and cell adhesion of RMCs in 2D and 3D culture models were observed using an inverted optical microscope and scanning electron microscopy(SEM).It showed that the number of cells in 3D models began to increase rapidly and the cells were tightly attached to the scaffold and evenly distributed at all levels of the material which form the interconnected mesh structures.There was no significant difference between 3D-PLA-Gelatin group and 3D-PLA-RGD group.3.The survival status of RMCs was detected by LIVE/DEAD fluorescence staining.The results showed that there were no significant differences of cell viability in 2D group?3D-PLA-PBS group?3D-PLA-Gelatin group?3D-PLA-RGD group at 24h time point(F=0.815?P=0.521);There were significant differences at 72h time point(F=71.441?P<0.001).The cell viability in 3D-PLA-PBS group was lower than that in other groups at 72h time point(P<0.05).The results further confirmed that the 3D-PLA-Gelatin scaffold and 3D-PLA-RGD scaffold have good cell compatibility.4.Cytotoxicity of TMAO to RMCs were detected by CCK-8 kit.The results showed that the cytotoxicity was low within 72hrs when TMAO concentration was set below 200?M.5.RT-qPCR analysis was used to evaluate expression of miR-21 of RMCs in 2D group,2D+TMAO group,3D group,3D+TMAO group at 12h time point.The results showed that there were significant differences between groups(F=58.643,P<0.001).The mRNA expression of miR-21 were upregulated in 3D model(p<0.05).TMAO had no influence on miR-21 expression in 2D model(P=0.068).The results showed that the conclusions obtained from 3D cell culture system and 2D cell culture system may not be consistent.6.Western blotting analysis was used to evaluate expression of TGF-?1?Collagen I and fibronectin(FN)in 2D group,2D+TMAO group,3D group,3D+TMAO group at 72h time point.The results showed that there were significant differences between groups(F=302.573,478.053,213.581,470.122;P values were all<0.001).TMAO could upregulate the proteins expression in 2D and 3D model(P values were all<0.05).Functions of RMCs were improved in 3D model than in 2D culture model(p<0.05).TMAO had a direct fibrotic effect on RMCs.Part 2:Study the intracellular delivery efficiency of SWCNT-based miR-21i nanovectors and their effects on renal mesangial cells.1.Preparation of miR-21i Hybrids:SWCNTs were synthesized according to a HiPCO method by Carbon Nanotechologies Co.The designed miR-21 i was synthesized according to the following sequence:5'-aaaaaaaa UCAACAUC AGUCUGAU AAGCU A-3'.The sequence of cell penetrating peptide(CPP)was synthesized as following:NH2-YGRK KRRQ RRRG GGLG ASWH RPDK GKKK KKK-COOH.The miR-21i adhered to the positively charged segments of CPP.The miR-21i-SWCNT and miR-21i-CPP-SWCNT hybrids was assembled via ultrasonic treatment and hydrophobic interaction to SWCNT.2.Atomic force microscopy(AFM)and Dynamic light scattering(DLS)were used to characterize the delivery system.AFM image showed that the miR-21i-SWCNT and miR-21i-CPP-SWCNT hybrids distributed dispersively with column-shaped.DLS data showed that the average length of miR-21i-SWCNT was 150.3nm,while that of miR-21i-CPP-SWCNT hybrid was 50.34nm.This implied that two hydrophilic end segments of CPP may form hydrogen bond to increase the stability of miR-21i-CPP-SWCNT delivery system.The zeta potential of miR-21i-SWCNT hybrid was about-17.1mV,while that of miR-21i-CPP-SWCNT hybrid was about 24.7mV.This implied that the CPP-binding miR-21i via electrostatic interactions endowed the positive-charge properties of the miR-21i-CPP-SWCNT hybrids.The positively charged miR-21i-CPP-SWCNT hybrids were easily binded to the natural negative cell surfaces.3.The cell toxicity of miR-21i hybrids were detected by CCK-8 kit.The results showed that the cytotoxicity was low within 48hrs when the miR-21i concentration was set below 300 nM.4.To verify the efficiency of miR-21i-carried vectors,green fluorescent FAM was labeled on the 5'end of the miR-21i and Confocal Laser Scanning Microscopy(CLSM)was used to track FAM-labeled miR-21i in RMCs.The results showed that the miR-21i-CPP-SWCNTs hybrids exhibited ability of penetrating into RMCs more effectively than the other hybrids(P<0.05).This implied that SWCNT alone was an effective miR-21i carrier,but CPP can increase the penetrating ability and stability of SWCNT.5.Western blotting analysis and immune-fluorescence staining assay were performed to explore the cellular expression of TGF-?1??-SMA,and Smad3 when treated with different hybrids.The results showed that miR-21i-CPP-SWCNTs hybrids exhibited inhibition of TGF-?1??-SMA expression more effectively than the other miR-21i-carried vectors(P<0.05).There appeared no inhibition of the smad3 expression in all miR-21i hybrids(P>0.05).This implied that the miR-21i could be released from the miR-21i-CPP-SWCNTs hybrids and play a role through TGF-?1 and ?-SMA pathway in RMCs.Conclusion1.3D cell models were constructed by 3D bio-printing PLA scaffolds coating with dopamine,Gelatin or RGD.The models had good compatibility and were superior to the 2D cell model in cell biology research of RMCs.2.TMAO could upregulate expression of miR-21 and promote secretion of TGF-?1 Collagen I and FN via directly targeting RMCs,which could cause renal fibrosis.miR-21 may participates in the pathogenesis of TMAO.3.A nano delivery system of miR-21 i with SWCNT and CPP was developed and had good compatibility.The method of construction is simple.4.The miR-21 i-CPP-SWCNT may be an effective target-binding delivery system.Inhibition of miR-21 prevents fibrogenic activation in RMCs by preventing TGF-?1 and a-SMA pathway,thus may represent a new therapeutic approach for kidney fibrosis.