Expression And Differential Analysis Of Angiogenesis Related Non Coding RNA In Distraction Osteogenesis

ObjectiveDistraction osteogenesis(DO)is an endogenous bone tissue engineering technology and the mechanical and biological properties of the regenerated bone is close to nativeones.DO is without implantation of autogenous bone or artificial bone,effecting of autologous tissue caused donor defect,immunological rejection caused by allograft.There is no significant difference in the quality of bone between the regenerated one in DO and the native one,but its rapid angiogenesis mechanism has not been fully clarified.The mechanism of angiogenesis has been studied through the analysis of tissue morphology,specific protein expression,and the regulation of specific m RNA.However,the study of angiogenesis mechanism entered a new era---the regulation analysis of genome network.There is only a small amount of DNA sequence in the genome sequence coding to produce protein.The other large gene sequence does not encode protein,but is in the form of non-coding RNA(nc RNA)to regulate the life process of the organism.Nc RNA not only plays an important role in cell proliferation,differentiation,aging and apoptosis,but also in cancer,cardiovascular diseases and neurodegenerative diseases.Now,the study on nc RNA is mainly focused on the micro RNA(mi RNA)and long non-coding RNA(lnc RNA).But the effest of these two nc RNA in distraction osteogenesis is unknowable.Therefore,the expression of angiogenesis related micro RNA and lnc RNA in regenerated tissue in DO was analyzed.The regulatory role of mi RNA and lnc RNA in angiogenesis process in DO is studied to explore the mechanism of the rapid angiogenesis in distraction osteogenesis.Methods1.The regenerated tissues in DO group,which was with 0(DO-0 group)and 2(DO-2 group)weeks' consolidation,and mandibles of 0(P-0 group),2(P-2 group),4(P-4 group)and 8-week-old(P-8 group)puppy dogs and adulut dogs were harvested respectively for HE staining.The changes of the vascular and cellular components were observed and analyzed.2.Immunohistochemistry staining was used for detection the expression of CD31 and VEGF in all the samples.The micro vascular density in the samples was calculated according the expression of CD31.3.Total RNA was extracted from the mandible of DO group,puppy group and normal group.mi RNAs and lnc RNAs c DNA library was constructed and then gene sequencing was performed using the Illumina hiseq 2500 platform.The numbers,expression,target genes and relative KEGG pathways of mi RNAs and lnc RNAs were measured and predicted.Differential gene expression among the groups was studies and the vascularization relatived genes were found.Results1.In DO-0 group,abundant blood vessels and cells were observed in the regenerated tissue.The blood vessels were further developed and matured in the DO-2 group,which provided nutrition for the proliferation and differentiation of various kinds of cells.The number of blood vessels,the quantity and activity of osteoblasts showed a single peak trend and the peak appeared in P-2 and P-4 group.2.The micro vascular density and the expression of VEGF in DO-0 group was highest in all groups and that in normal group was lowest.In puppy dogs group,micro vascular density and the expression of VEGF in P-2 group was highest.3.317 mi RNAs and 1845 lnc RNAs were detected in all samples.The downregulated numbers of the significantly differential lnc RNAs and mi RNAs in DO-0 groups was higher than P groups.The downregulated numbers of differential mi RNAs and upregulated lnc RNAs in DO-0 groups was higher than P groups.The upregulated numbers of differential mi RNAs and downregulated lnc RNAs in DO-0 groups was higher than N groups.The downregulated numbers of differential mi RNAs and upregulated lnc RNAs in DO-2 groups was higher than N groups.There are 227 differential expression mi RNAs was detected and 145 target genes was predicted.All the target genes of differential mi RNAs was related to metabolism pathway,etc.and activation of adenylate cyclase activity,etc.1172 target genes were predicted for lnc RNAs,which were related to proteasome pathway and 263 functions such as blood vessel development.4.The upregulation of mi R-21?mi R-451?mi R-486 and downregulated of mi R-99 a was closely related to vascularization in DO group.The target gnen of mi R-451 was PSEN1.37 lnc RNAs was found relative to blood vessel development.Lnc RNAs,including TCONS₀0221463,TCONS₀0221470 and TCONS₀0045543 was significantly upregulated in DO group than the puppy group and normal group,and the target gene was JMJD6 protein(jumonji domain containing protein 6).TCONS₀0196446 was significantly downregulated and the target gene was RBM15(RNA binding motif protein15).Conclusions1.The expression of VEGF was significantly increased in DO group and fast development period group,which indicating that the angiogenesis ability in these two group was similar.2.mi R-21 upregulated PTEN,and then activated AKT and ERK signaling pathway,promoted angiogenesis in DO.3.TCONS₀0221463 can upregulate JMJD6 and TCONS₀0196446 can downregulate RMB15,promoting angiogenesis in DO.