High-throughput Sequencing Of Bone Tissues From Mandibular Distraction Gap And Its Differential Expression Profile Analysis

BACKGROUNDDistraction osteogenesis(DO)technique involves creating an osteotomy in an area adjacent to an area of bone deficiency.Applying slow tension force separates the bony edges,which creates a regenrated chamber from which the new bone and soft tissues are formed.With decades of development,DO has been become a useful technique to generate bone and soft tissue,can be applied to craniofacial reconstruction,including orthognathic surgery,cleft lip and palate reconstruction,dentoalveolar unit reconstruction for dental implants and transport DO for discontinuity defects,etc,and has gained satisfactory clinical outcome.However,the specific biological mechanisms of DO,especially of bone regeneration during DO are very complicated,which still remain unclear.Many researches have demonstrated that,during the process of DO,a series of related cytokines may have played a important and complicated role during this procedure;yet interactions among all these cytokines can also be observed,which regulate the process of DO.Such as BMPs,VEGF,FGF,TGF-?,etc.MicroRNA are endogenous,approximately 22 nucleotide-long,non-codingRNAs,which participate in most of pivotal biological process.They functions in transcriptional and post-transcriptional regulation of gene expression.However,few reports can be found about miRNA regulation during DO,especially during maxillofacial DO.Hence,the revelation of the regulatory function of miRNAs during maxillofacial DO can allow us to understand and interpret the biological mechanisms of DO from a brand new perspective.Moreover,we also hypothesize that the distracting force and local microtrauma caused by DO may activating certain cytokines and signaling pathways which are normally activated during fast growth period and/or embryonic development period.And a ossification pattern different from normal bone tissue repair and regeneration may occur during the process of DO.OBJECTIVESTo establish surgical model of canine mandibular distraction osteogenesis,and to obtain mRNA and miRNA expression profile of bone tissue from distraction gap,as well as bone tissue from mandibles of normal new born dogs and adult dogs through high throughput sequencing technique.By analyzing differential expression of mRNAs and miRNAs among each groups to understand the expression of related mRNAs and miRNAs and participated signaling pathways during the process of DO.And to anticipate to screen out possible target genes of related miRNAs.We also anticipate to find out possible factors and signaling pathways of fast growth period and/or embryonic development period during the process of DO METHODS1.Six healthy mongrel dogs of either gender were selected(age: 1.5-2.0y,average weight: 13.5kg).Among them,three dogs were randomly selected into DO immediate(DO-0)group and another three dogs were selected into DO 2-week consolidation(DO-2)group.We also selected three new born dogs(born within an hour),three 2 weeks old dogs,three 4weeks old dogs,three 8 weeks old dogs and three healthy adult dogs into new born(P-0)group,2 weeks old(P-2)group,4 weeks old(P-4)group,8 weeks old(P-8)group and normal adult control(NC)group respectively.Unilateral mandibular DO was performed on dogs of both DO-0 group and DO-2 group,and they were sacrificed immediately after the completion of distraction and after 2 weeks of consolidation period respectively.Bone tissue samples in and around distraction gap were collected,and mandibular bone tissue samples of P-0 group,P-2 group,P-4 group,P-8 group and NC group were also collected.2.TotalRNAs of all 7 groups were extracted,and c DNA libraries of mRNA and miRNA were then built.Illumina Hiseq4000 high throughput sequencing platform was applied to achieve the mRNA gene expression profile and miRNA gene expression profile.3.After mRNA sequencing,12 paired comparison groups were established based on 7 different samples.Then the differentially expressed mRNA genes were screened out and annotated by gene ontology(GO)and KEGG signaling pathways with specific softwares and databases.4.After miRNA sequencing,12 paired comparison groups were established based on 7 different samples.Then the differentially expressed miRNA genes were screened out and target genes of all screened miRNAs were achieved.GO and KEGG annotation of these miRNAs were achieved as well.RESULTS1.All subjects of DO-0 group and DO-2 group had successfully undergone DO and the subsequent follow-up observation.Postoperative infection of incision occurred in 2 cases,which were healed through thorough debridement.And no severe complications were observed in other cases.Obvious mesial advancement of right mandibular canine was observed of all dogs,indicating that the right mandible had been successfully elongated by DO.2.Adequate totalRNAs was extracted from all 7 samples,and c DNA libraries of mRNA and miRNA for subsequent sequencing were successfully built respectively.High-throughput sequencing of bone tissues from distraction gap and from normal mandibles was completed successfully.3.Based on the results of all 12 paired comparison groups,we found3598 differentially expressed mRNA genes,of which 1491 were up-regulated and 2107 were down-regulated.By analyzing them,we found several development-related mRNAs were up-regulated in both DO-0 group and P-0 group,including LOXL2,LGALS1,PTK7,etc;through GO annotation,we found that some of the differentially expressed mRNA genes were enriched in body development,cell metabolism,osteogenic differentiation,and other biological process.4.We also found 1299 differentially expressed miRNAs,in which 800 were up-regulated and 499 were down-regulated.GO annotation found that these miRNAs were mainly enriched in body metabolism,organ formation,cell differentiation,post embryonic development.CONCLUSION1.Based on the sequencing results,we suggest that DO may activate the potential related factors and signaling pathways during fast growth period and/or embryonic development period.However,further studies should be conducted to verify the existence of this particular activation and to clarify its mechanisms.2.miRNA may play important roles during the process of DO.However,further studies should be conducted to clarify the exact regulatory mechanisms.