The Role Of MiR122 And MiR185 On Hepatic Differentiation In HUC-MSCs

Objective:Genetic modification is involved in the regulation of various cell function and characteristics,as well as stem cell differentiation.The differentiation of Human Umbilical Cord Mesenchymal Stem Cells(hUC-MSCs)is modulated by various factors.In this study,miR122 and miR185 were over-expressed in hUC-MSCs by gene transfection technique.Then,hUC-MSCs were induced differentiation into hepatic like cells.After that,the expression of hepatocellular markers(AFP,ALB,CK18,CK19,HNF4a)by these cells were detected,in order to investigate the effect of miR122 and miR185 on the hepatic inducing differentiation of hUC-MSCs.Methods:1.hUC-MSCs' culture and identification:hUC-MSCs were isolated and cultured by tissue adherent method under aseptic conditions,the expression of cell specific markers,CD34,CD45,CD90,CD 105 and Oct-4,were detected by flow cytometry on hUC-MSCs at passage four.The hUC-MSCs copacity of multidirectional differentiation was identified by adipogenic and osteogenic differentiation in conditional media.2.The over-expression of miR122 and miR185 in hUC-MSCs experiment:Cells were transfected with miR122 and miR185 with different concentrations according to riboFECTTM CP transfection reagent.Cell growth status and their expression of target mRNA were evaluated after transfection.NC mimic transfection cells were used as blank and negative control group.hUC-MSCs transfected with different concentrations of miR122,miR185 or both were experimental groups.The expression of miR122 and miR185 by cells were detected by q-RT-PCR at D1,D7,D14,D21 and D28.Cell growth curves were also recorded in different groups at different time points after transfection.3.Effects of miR122 and miR185 on hUC-MSCs hepatic differentiation:Cells were devided into six groups:Blank control group,blank induction group,negative control induction group,miR122 over-expression induction group,miR185 over-expression induction group,miR122 and miR185 co-overexpression induction group.The blank control group was cultured by normal culture medium,and the induction group was cultured by hepatic inducing conditional medium.After 28 days,total RNAs and proteins were extracted respectively from each group of cells,q-RT-PCR and Western-Blotting were performed to test the hepatic markers:AFP,ALB,CK18,CK19,and HNF4?.PAS staining and ICG assay were used to detect cell function.Results:1.hUC-MSCs' culture and identification:hUC-MSCs obtained by tissue block adherent method were with long spindle and vortex form.At passage four,when cell was about 80%-90%confluence,flow cytometry was performed.The results showed that a high expression of CD90(99.16%±0.85%)and CD 105(99.26%±0.78%),but no expression of CD34(0.59%± 0.16%)and CD45(1.50%±2.04%)by these cells and 66.57%±4.61%cells expressed positively Oct-4,an embryonic stem cell marker.After 28 days of osteogenic induction,calcium salt deposition in hUC-MSCs were detected by Alizarin red staining which became dense red nodules.After 28 days of adipogenic induction,small lipid droplets in hUC-MSCs were observed after red oil O staining.The cells obtained from umbilical cord expressing specific MSC markers and had potential to be differentiated into osteoblast and adipocyte.2.Experiment on transfection of miR122 and miR185 into hUC-MSCs:The expression of miRNA122 and miRNA185 by hUC-MSCs was detected at different time points after transfection with different conditions and at different concentrations of miRNA.The results showed that 50 nM was the optimal transfection concentration.There was a stable and high level expression of miR122,miR1 85 and miRAB in corresponding groups on D1,D7,D14,D21 and D28 after transfection.Transfection of miRNA NC mimic had no effect on the expression of miR122 and miR185 by hUC-MSCs.During the 28 days after transfection,the cells in each group proliferated stably and grew well.3.Effects of miR122 and miR185 on hUC-MSCs hepatic differentiation:After transfection of miRNA 122 and miRNA185,hUC-MSCs were induced into hepatic differentiation.q-RT-PCR results showed that the ALB expression level in miR185 induction group(0.47 ±0.17)and the miRAB induction group(0.36±0.13)was significantly lower than that in the BC induction group(1.10±0.19)and NC induction group(1.41 ±0.14).There was no statistical significance between miR122 induction group(1.72 ± 0.43)and BC induction group or NC induction group in ALB expression level.In AFP expression level,there was no statistical significance among miR 122 induction group(2.04± 0.96),BC induction group(2.29 ±0.89)and the NC induction group(2.39± 0.94).There was a reduced expression in miR185 induction group(0.64 ±0.27).There was no statistical significance between miR185 induction group and miRAB induction group(0.79±0.53).The expression of CK18 mRNA was the highest in miR1 22 induction group(1.93±0.21).In miR185 induction group(0.21± 0.07)and the miRAB induction group(0.63±0.21)the expression of CK18 mRNA was significantly lower than that in the BC induction group and the expression in miRAB induction group was higher than that in miR185 induction group.Compared with the BC induction group(1.13±0.25),the expression of CK19 in the miR122 induced group was the highest(1.50±0.27).The expression levels of CK19 in miR 185 induction group(0.29 ±0.28)and miRAB induction group(0.23±0.15)were decreased.The results of HNF4? expression showed that the highest expression level was miR122 induction group(19.03±2.13).The miR185 induction group(8.84±2.19)was lower than that in the miR122 induction group,and the lowest expression level was miRAB induction group(0.58±0.33).Western Blotting results showed that the expressions of ALB(1.00 ± 0.05),AFP(0.85 ± 0.03),CK18(1.13 ± 0.02),CK19(1.09±0.03),HNF4?(1.41± 0.03)were increased in the miR122 induction group.The expressions of CK18(0.72 ± 0.03),CK19(0.45±0.02)and HNF4?(0.24 ± 0.02)were decreased in the miR 185 induction group.The expression level of ALB(0.62 ±0.04)and AFP(0.19± 0.02)in the miRAB induction group was higher than that in the BC induction group,and lower than miR122 induction group.The expression level of CK19(0.47 ± 0.03)and HNF4?(0.36 ± 0.01)in the miRAB induction group was lower than that in the BC induction group.And there was no statistical significance between the miRAB induction group(0.85 ± 0.05)and BC induction group in the expression of CK18.4.Cell function test after hepatic differentiation induction.The PAS staining showed no glycogen deposition in BC group.Comparing the five induction groups,we found that the amount of glycogen deposition in BC induction group and NC induction group is almost the same,while the amount of glycogen deposition in miR122 induction group is significantly higher than that of BC induction group and NC induction group.The glycogen content in miR185 induction group was significantly lower than that of the BC induction group and the NC induction group.The glycogen concentration of the miRAB induction group was similar to that of BC induction group and NC induction group.In the indocyanine(ICG)endocytosis and exocytosis experiment,there was no ICG in BC group.Comparing the other five groups,it was found that the quantity of ICG in BC induction group and NC induction group were similar,while a large number of ICG accumulation in the miR122 induction group,which was significantly higher than that of BC induction group and NC induction group.ICG accumulation was significantly lower in miR185 induction group than that of BC and NC induction group.ICG in miRAB induction group was slightly less than that of BC and NC induction group,but a little bit more than that in miR185 induction group.Conclusions:1.The cells abtained from umbilical cord have the same molecular markers characteristics as hUC-MSCs and they have as well the potential of multidirection differentiation.2.The optimal mRNA transfection concentrations was 50 nM in our experiment.Overexpression of miR122 and miR185 in hUC-MSCs is stable for 28 days after transfection.3.Overexpression of miR122 can promote hUC-MSCs hepatic differentiation.Overexpression of miR185 inhibited hepatic differentiation of hUC-MSCs.It seems miR122 and miR185 have an antagonistic effect on hUC-MSCs hepatic differentiation.Overexpression of certain kind of miRNA in cells by transfection or other gene modification skills could be ae effective way to modulate stem cell fate.