Research On The Mechanism Of LncRNA SERPINB9P1 Regulating SIRT6 Mediated Osteogenic Diferentiation Of BMSC Via MiR?545?3p

Background:Bone marrow stromal cells(BMSC)are a group of heterogeneous stem cells that can differentiate along multiple lineages.They also have the biological potential of easy separation,inducing proliferation,low immunogenicity and secreting a variety of cell regulatory factors.Compared with embryonic stem cells,BMSC will not cause related ethical problems.Now as a kind of biomaterials,they play a significant role in gene cell therapy and regenerative medicine.In the field of bone tissue engineering,the basic principle of BMSC treatment is to replace or regenerate the damaged tissue by direct intravenous injection or combined with absorbable biological stent transplantation at the site of bone injury or bone defect.Existing experimental studies have found that it has good therapeutic effects in repairing craniomaxillofacial bone defects,promoting the repair of tendon and bone interface damage,fracture healing and osteoporosis.However,the number of stem cells in human bone marrow is limited,which can not be directly extracted for treatment.So it needs proliferation in vitro to obtain an effective number of cells before transplantation.Due to the influence of different donors(gender,age,health status,etc.),acquisition methods,in vitro amplification conditions,and the differences of the receptor itself,the differentiation ability of the transplanted cells is reduced,and there are also genetic instability,cell apoptosis and other problems.The treatment prognosis are seriously affected by these factors mentioned above.Meanwhile,they lead to the facts that most of the reports on stem cells are still in the stage of animal experiments.In order to improve the osteogenic ability of transplanted BMSC,some researchers have considered to modify them with important osteogenic genes before transplantation.By overexpressing or knocking down specific genes,they found that they could improve the osteogenic differentiation ability of BMSC and enhance their therapeutic effect.Therefore,it has great influence to research the important factors and specific molecular mechanisms that determine the direction of osteogenic differentiation of BMSC,which will help to accurately regulate the direction of stem cell differentiation in the future,solve the practical problems faced above,and take an important step forward for BMSC cell therapy from laboratory to clinical.In recent years,reports of various non-coding RNAs(Nc RNAs)participating in the osteogenic differentiation of BMSC have gradually increased,which has become a hot spot in orthopedic research.Nc RNA is transcribed from the genome,not directly translated into protein,but participates in the protein translation process of coding m RNA,such as mi RNA,lncRNA,Circ RNA,which are the key nc RNAs that regulate the function of BMSC cells.However,the relevant research is still shallow and needs to be deepened.Objective:To study the role of lncRNA SERPINB9P1 in the osteogenic differentiation of BMSC,and to investigate whether lncRNA SERPINB9P1 can regulate SIRT6 mediated osteogenic differentiation of BMSC via mi R-545-3p.This study will reveal a new regulatory pathway of osteogenic differentiation of BMSC,and provide new ideas and potential therapeutic targets for stem cell therapy of related diseases.Methods:Bone marrow stromal cells(bmscs)were isolated from bone marrow of patients who undergoing orthopaedic surgery by adherent method.And flow cytometry was used for phenotype identification of BMSC.Cells expressed CD44,CD90 and CD105,but not CD45 and CD34.Osteogenic medium(OM)induced osteogenic differentiation of BMSC.The expression level of key osteogenic markers ALP,RUNX2 and OCN protein during differentiation was detected by Western blot and gray scale analysis;Alizarin red S staining,ALP staining and ALP activity determination were used to check the osteogenic mineralization.The knockout/overexpression target gene was constructed by cell transfection technology,RNA binding protein immunoprecipitation(RIP),double luciferase reporter assay and q RT-PCR were used to detect the relative expression of lncRNA SERPINB9P1,mi R-545-3p and SIRT6 m RNA to confirm the interaction between lncRNA SERPINB9P1 and mi R-545-3p,mi R-545-3p and SIRT6.Finally,all data were statistically processed.Results:1.lncRNA SERPINB9P1 was increased and mi R-545-3p was decreased during BMSC osteogenic diferentiationFirst,we examined the expression levels of lncRNA SERPINB9P1 and mi R-545-3p in isolated BMSC.The classifcation of the isolated BMSC is determined according to the characteristics of the cell surface antigens.The relative specifc molecules of surface antigens detected by fow cytometry for BMSC phenotypic identifcation,includ ing CD44,CD90,CD105,CD45,and CD34.The protein levels of ALP,RUNX2,and OCN in BMSC on days0,1,5,10,and 15 were also tested.As the increase of culture time,that all three markers increased at the same time.The results also showed that the induction of osteogenic diferentiation by OM was successful(Supplemental Fig.1B).We then tested the rela tive levels of lncRNA SERPINB9P1 and mi R-545-3p during osteoblast diferentiation.During osteoblast diferentiation,lncRNA SERPINB9P1 was overexpressed and mi R-545-3p was inhibited with the prolongation of culture time,suggesting that lncRNA SERPINB9P1 and mi R-545-3p may be linked with the diferentiation of BMSC.2.Lnc RNA SERPINB9P1 knockdown inhibits osteogenic differentiation of BMSCTo clarify whether lncRNA SERPINB9P1 participates in BMSC osteogenic diferentiation,the lncRNA SERPINB9P1 expression was knocked down by sh-SERPINB9P1 and overexpressed by SERPINB9P1 lentiviruses in BMSC.After infection of sh-SERPINB9P1 or oe-SERPINB9P1 into BMSC cells for about 48 h,cell samples were collected and the knockdown and overexpression efciency was tested.Compared with control cells,the level of lncRNA SERPINB9P1 in BMSC infected with sh-SERPINB9P1 decreased signifcantly and the expression of lncRNA SERPINB9P1 dramatically increased with oe-SERPINB9P1 lentiviruses.After 21 days of incubation,ALP and alizarin red S staining were performed in all five groups(control group,sh vector group,sh-SERPINB9P1 group,oe vector group,oe lncRNA SERPINB9P1 group).It was found that compared with control cells,the staining area of ALP and alizarin red S decreased after knocking out lncRNA SERPINB9P1,indicating that the expression of ALP decreased,and the number of calcium salt nodules in cells were significantly weaker than that in control group;When the lncRNA SERPINB9P1 was overexpressed,the decreased staining area was relieved,the expression of ALP was increased,and the number of calcium salt nodules in cells were significantly greater than that in the control group.The determination of ALP activity showed that compared with the control cells,the knockdown of lncRNA SERPINB9P1 significantly reduced ALP activity,while the oe-lncRNA SERPINB9P1 group significantly increased ALP activity.WB experiment also showed that the expression level of ALP,OCN and RUNX2 were reduced after knocking down lncRNA SERPINB9P1.Meanwhile,overexpression of lncRNA SERPINB9P1 increased the expression level of these markers.These results showed that the knockdown of lncRNA SERPINB9P1 inhibited the osteogenic differentiation of BMSC,while overexpression promoted its differentiation.3.Lnc RNA SERPINB9P1 can sponged mi R-545-3pThe relative mi R-545-3p level was increased or decreased after lncRNA SERPINB9P1 knockdown or overexpression,respectively.Bioinformation predicts the existence of binding sites between the two.Ago2 RIP assay was used to test whether lncRNA SERPINB9P1 and mi R-545-3p occupied the same RNA-induced silencing complex in BMSC.Our results indicated that lncRNA SERPINB9P1 and mi R-545-3p were enriched in the Ago2 group compared to that in the Ig G group.The assay showed that the luciferase activity of 293 T cells transfected with lncRNA SERPINB9P1-WT was reduced by mi R-545-3p analog.These results indicated that lncRNA SERPINB9P1 sponged mi R-545-3p.4.lncRNA SERPINB9P1 regulated BMSC osteogenic diferentiation via mi R-545-3pOn the basis of knocking down lncRNA SERPINB9P1,we added the inhibitor of mi R-545-3p to test the functional relationship between lncRNA SERPINB9P1 and mi R-545-3p in BMSC osteogenic differentiation.Aspredicted,the sh-SERPINB9P1 infection led to an elevation of mi R-545-3p in BMSC,which was abrogated by introducing the mi R-545-3p inhibitor.Subsequently,the ability of osteogenic diferentiation was evaluated.Compared with the control cells,when the lncRNA SERPINB9P1 was knocked down,the staining area of ALP and alizarin red s reduced,ALP content and calcified nodules decreased;When mi R-545-3p inhibitor was added to BMSC infected with sh-SERPINB9P1,the decrease of ALP content and calcified nodules were reversed.ALP activity assay showed that lncRNA SERPINB9P1 knockout significantly reduced ALP activity compared with control cells,whereas inhibition of mi R-545-3p expression attenuated the reduction in ALP activity.After knocking down lncRNA SERPINB9P1,levels of ALP,OCN,and RUNX2 were decreased and this phenomenon was blocked after mi R-545-3p inhibitor treatment in WB experiment.Lnc RNA SERPINB9P1 overexpression or inhibition of mi R-545-3p were also found to decrease the expression of RANKL and increase the expression of OPG.The evidence above proved that lncRNA SERPINB9P1 regulated BMSC osteogenic differentiation via mi R-545-3p.5.Mi R-545-3p regulates osteogenic differentiation of BMSC through binding with SIRT6 directlyTo determine the interaction between mi R-545-3p and SIRT6,the relative expression of SIRT6 was evaluated after transfection with mi R-545-3p mimic or inhibitor.We found that mi R-545-3p overexpression or knockdown led to a downregulation or upregulation of SIRT6,respectively.We also found that mi R-545-3p mimic sharply attenuated the luciferase activity driven by SIRT6 WT.To determine if the impact of mi R-545-3p on BMSC osteogenic diferentiation was mediated by SIRT6,BMSC were treated with mi R-545-3p mimic and oe-SIRT6.Overexpression mi R-545-3p caused a reduction of SIRT6,and the decreased expression of SIRT6 were blocked by overexpres sion of SIRT6.Subsequently,the ability of osteogenic diferentiation was evaluated.Compared with control cells,mi R-545-3p overexpression led to reductions of ALP and Alizarin Red S staining area,and this phenomenon was blocked after oe-SIRT6 infection.ALP activity assay showed that mi R-545-3p mimic decreased ALP activity,and SIRT6 overexpression could rescue the decrease of ALP activity.Western blot results revealed that SIRT6,ALP,OCN,and RUNX2 were decreased after mi R-545-3p overexpression,and SIRT6 overexpression abrogated the reduction of ALP,OCN,and RUNX2 of BMSC.In conclusion,mi R-545-3p regulates osteogenesis and differentiation of BMSC through SIRT6.6.lncRNA SERPINB9P1 regulates SIRT6 mediated osteogenic diferentiation of BMSC via mi R-545-3pRT-PCR showed that after knocking down Lnc RNA SERPINB9P1,the expression level of SIRT6 was down-regulated,and inhibition of mi R-545-3p expression could reverse this effect;Western blot showed that knockdown of Lnc RNA SERPINB9P1 caused down-regulation of SIRT6 protein expression,while in sh-Lnc RNA SERPINB9P1 + mi R-545-3p inhibitor co-transfection group,mi R-545-3p inhibitor can save the down-regulation of SIRT6 protein expression.These results further confirmed lncRNA SERPINB9P1 regulates SIRT6 mediated osteogenic diferentiation of BMSC via mi R-545-3p.Conclusion:1.During the osteogenic differentiation of BMSC,Lnc RNA SERPINB9P1 was over-expressed,while mi R-545-3p was inhibited.2.Lnc RNA SERPINB9P1 knockdown inhibits osteogenic differentiation of BMSC.3.lncRNA SERPINB9P1 regulates SIRT6 mediated osteogenic diferentiation of BMSCs via mi R-545-3p.