Research Of The Mechanism Of MiRNA And LncRNA In The Regulation Of Rapid Bone Formation In Distraction Osteogenesis And Their Differential Expression Profile Analyses

Objective Distraction Osteogenesis?DO?is an endogenous bone engineering,is through the mechanical force to let the bone break off,causing trauma,installing the traction device at both ends of the bone and pulling the bone ends at a certain speed,so that the new bone formed in the two ends of the bone.The quality of the new bone is close to the normal bone after DO.New bone formation is selflimiting and controllable.The rate of osteogenesis of DO is amazing,but the quality of bone formation is good,the mechanism of its rapid osteogenesis is unclear.The formation of blood vessels is throughout the process of rapid osteogenesis,the mechanism of rapid angiogenesis and vasculogenesis in DO is also not yet clear.Nowadays,the research on the mechanism of new bone formation and neovascularization of DO began to enter the era of gene network regulation.A class of genes that do not encode RNAs: non-coding RNAs?nc RNAs?regulate the biological process of the development of organisms.Nc RNAs not only involved in cell proliferation,differentiation,aging,apoptosis,but also in cancer,cardiovascular disease,neurodegenerative diseases,they play an important regulatory role.At present,the study of non-coding RNA is mainly focused on micro RNA?mi RNA?and long non-coding RNA?lnc RNA?.The role of mi RNA and lnc RNA in the regulation of new bone formation and neovascularization in DO has become one of the hot spots.In this study,we obtain mi RNA and lnc RNA expression profile of bone tissues from distraction gap,mandibles of dog's embryo,fracture tissue after fracture fixation,as well as bone tissue from mandibles of normal new born dogs and adult dogs through high throughput sequencing technique.To find the mi RNA and lnc RNA associated with new bone formation and neovascularization in distraction osteogenesis and to explore the mechanism of rapid osteogenesis in DO and find DO and embryonic development-related factors and signal pathways though analyzing gene expression differences of the mi RNA and lnc RNA of the mandibular bone tissues in the distraction groups and the non-distraction groups.Method Twelve healthy mongrel dogs of either gender were selected.The weight was 12.5kg ± 2.5kg?average 13.5kg?.Among them,three dogs were randomly selected into DO immediate?DO-I?group and three dogs were selected into DO2-week consolidation?DO-2?group,and three dogs were randomly selected into the fracture fixation 14 days group?BF1?and three dogs were randomly selected into the fracture fixation 28 days group?BF2?,respectively.We also selected three new born dogs,three dogs of 2 weeks old,three dogs of 4 weeks old and three healthy adult dogs into new born?NB?group,2 weeks old?PUP-2?group,4 weeks old?PUP-4?group and normal adult control?AC?group respectively.And we selected nine pregnant mongrel dogs into E1 group,pregnant 30 days;E2 group,pregnant 35 days;E3 group,pregnant 50 days.The unilateral mandibular DO was performed on the both DO-I group and DO-2 group,and they were sacrificed immediately after the completion of distraction and after 2weeks of consolidation period,respectively.the unilateral mandibular fracture operation was performed on the both fracture groups and they were sacrificed immediately after 14 days and 28 days of mandibular fracture fixation.All tissue samples in and around distraction gap and fracture gap were collected,and mandibular bone tissue samples of NB group,PUP-2 group,PUP-4 group and normal group were also collected.And embryonic dogs mandibular tissues of pregnant 30 days,35 days and 50 days were collected.The total RNA was extracted from the bone tissue samples of each group,and the c DNA library of mi RNA and lnc RNA were established.Illumina Hiseq4000 sequencing platform was carried out to achieve the mi RNA gene expression profile and lnc RNA gene expression profile.After mi RNA and lnc RNA sequencing,55 paired comparison groups were established based on 11 different samples.Then the differentially expressed mi RNA genes and lnc RNA genes were screened out and annotated by gene ontology?GO?and KEGG signaling pathways with specific softwares and databases.Result1.In DO-I 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.Abundant blood vessels and cells were observed in the neonatal tissues of the embryos groups,and there were many fibrous tissues in the fracture groups and osteogenesis was poor.2.354 mi RNAs,3967 lnc RNAs and 3813 novel-lnc RNAs were detected in all the samples.There were 279 mi RNAs with different expression and 12990 target genes.There were 161 lnc RNAs with different expression and 177 target genes.The downregulated numbers of differential mi RNAs and upregulated lnc RNAs in DO-I group were higher than embryonic groups.The upregulated numbers of differential mi RNAs and lnc RNAs in DO-I group were higher than the normal group.The downregulated numbers of differential mi RNAs and upregulated lnc RNAs in DO-2 group were higher than the normal group.The upregulated numbers of differential mi RNAs in DO-I group was higher than the fracture groups.Meanwhile,the upregulated numbers of differential mi RNAs and lnc RNAs in DO-2 groups was higher than fracture groups.The downregulated numbers of differential mi RNAs and upregulated lnc RNAs in DO-I group were higher than puppies groups,this results are the same as for DO-2 group.3.We selected the different expression of mi RNAs and lnc RNAs by comparing the DO groups & embryonic groups with the normal groups &fracture groups.We found that mi RNA-411,mi RNA-410,mi RNA-495,mi RNA-487 b,mi RNA-487 a,mi RNA-369,mi RNA-136,TCONS₀0000934,TCONS₀0170595 were most likely to regulate the formation of neovascularization and new bone formation in DO by regulating their target genes.Conclusion1.Based on the mi RNA and lnc RNA expression profile of the highthroughput sequencing results,we suggest that DO may activate potential embryonic development-related genes and these genes regulate neovascularization and new bone formation in DO.2.Mi RNA-411,mi RNA-410,mi RNA-495,mi RNA-487 b,mi RNA-487 a,mi RNA-369,mi RNA-136,TCONS₀0000934 and TCONS₀0170595 these genes are most likely to regulate their target genes to regulate the neovascularization and new bone formation in DO.But its specific function and mechanism still need to be further explored.