The Role And Mechanisms Of Aldo-Keto Reductase Family 1 Member C1 In The Osteogenic Differentiation Of Human Adipose-derived Stem Cells

Bone defect,caused by tumor,trauma,inflammation,congenital malformation and so on,affects not only physical function but also aesthetic and mental health of patients,bring heavy economic burden to the family and society.With the development of bone tissue engineering,tissue engineering bone is of great application prospect to repair large bone defects.Besides the potential of multi-directional differentiation,human adipose-derived stem cells(hASCs)have wide sources with little trauma to the body.How to effectively start the osteogenic differentiation of hASCs and inhibit their adipogenic differentiation is the key to the application of hASCs in bone tissue engineering.Aldo-keto reductase family 1 member C1(AKR1C1),a member of aldo-keto reductase family,is highly related to the occurrence and development of tumor,and is widely involved in the proliferation,migration and invasion of tumor cells.However,whether AKR1C1 can regulate the lineage commitment of stem cells is still not clear yet.In addition,AKR1C1 can participate in the regulation of steroid metabolism.By catalyzing sex hormones into inactive metabolites,it regulates the occupation and transactivation of sex hormone receptors,thus achieving pre-receptor regulation of sex hormones.However,whether AKR1C1 can directly regulate the expression of sex hormone receptors is rarely reported.Objective:This study aims to determine whether AKR1C1 is involved in the regulation of osteogenic differentiation of hASCs and explore the potential mechanisms how AKR1C1 regulates osteogenic differentiation of hASCs.Materials and Methods:1.We determined the expression of AKR1C1 by qRT-PCR and Western blot during the osteogenic and adipogenic differentiation of hASCs and in the BMMSCs of mice after ovariectomy.2.For in vitro experiment,AKR1C1 knockdown lentivirus was used to transfect hASCs,while the wild-type and mutant plasmids overexpressing AKR1C1 were used to transfect the AKR1C1 knocked-down hASCs cell line.The transfection efficiency was detected by qRT-PCR and Western blot.The cells were divided into control group and experimental group(AKR1C1 knockdown group and overexpressing group).After osteogenic induction,ALP and ARS staining and quantification were used to detect the effect of AKR1C1 on osteogenic differentiation;qRT-PCR and Western blot were used to detect the expression of osteogenesis-related genes,including ALP,RUNX2 and BGLAP.After adipogenic induction culture,oil red O staining was performed to evaluate the effect of AKR1C1 on adipogenic differentiation;qRT-PCR and Western blot were used to detect the expression of adipogenic-related genes,including PPARy and C/EBPa.3.For in vivo bone forming experiment,cells from experimental and control group were attached to ?-TCP and implanted subcutaneously on the back of nude mice.8 weeks later,the materials were taken for H&E staining and Masson staining.For in vivo fat formation experiments,after adipogenic induction culture,the cells were collected and mixed with collagen membrane scaffold materials for subcutaneous implantation on the back of nude mice 6 weeks later,the materials were taken for H&E staining and oil red O staining.4.For the mechanism study,the expression of progesterone receptor(PR)and androgen receptor(AR)were detected by qRT-PCR and Western blot.PR knockdown siRNA was used to transfect hASCs,and plasmids overexpressing PR were used to transfect the AKR1C1 knocked-down hASCs cell line.ALP staining and quantification,ARS staining and quantification,qRT-PCR and Western blot were used to evaluate the effect of PR on the osteogenic differentiation of hASCs.Results:1.The expression level of AKR1C1 in the process of osteogenic and adipogenic differentiation of hASCs first increased and then decreased;compared with the sham operation group,the protein expression level of AKR1C1 in the BMMSCs of mice after ovariectomy increased,suggesting that AKR1C1 may be related to the lineage commitment of stem cells.2.Knockdown of AKR1C1 significantly promoted the osteogenic differentiation and inhibited the adipogenic differentiation of hASCs in vitro and in vivo.Overexpression of wild-type AKR1C1 could weaken the osteogenic effect of AKR1C1 knocked-down hASCs and promote adipogenic differentiation,but overexpression of mutant AKR1C1 had no effect on osteogenic and adipogenic differentiation of hASCs,indicating that AKR1Cl's regulation of differentiation of hASCs depended on its enzyme activity.3.Mechanism studies showed that AKR1C1 regulated the expression of PR in an enzyme dependent manner.Knockdown of PR by siRNA affected the osteogenesis of hASCs in the middle and late stage;overexpression of PR by plasmid weakened the osteogenic effect of AKR1C1 knocked-down hASCs.Conclusions:Together,this study revealed that AKR1C1 was a negative regulator of osteogenesis as well as a positive factor of adipogenesis of hASCs.The regulation of AKR1C1 on the osteogenesis and adipogenesis of hASCs depended on its enzyme activity.Mechanistically,AKR1C1 could regulate the expression of PR independently of progesterone,and PR mediated the regulation of AKR1C1 on osteogenesis.Our study provided a new potential target for gene modification of seed cells in bone tissue engineering.