The Research Of Gene Co-expression Network And The Mechanism Of Bones Around Implants In Hyperlipidemic Rats

ObjectiveHyperlipidemia could increase implant loss and decrease bone formation at implant-bone interface,but the mechanism is poorly understood.The purpose of this study is to build the gene co-expression network of bones around implants in hyperlipidemic rats and explore the possible mechanism.Methods1.A total of 20 adult male Wistar rats were equally divided into experimental and control group,respectively feed with high-fat and normal diet.8 weeks later,endocanthion vein of all rats was got to monitor serum lipid levels.After the successful induction of hyperlipidemic rats in experimental group,titanium implants were inserted into bilateral femoral metaphysis of rats.10 days later,1mm bone around implants were got.2.Some samples of two groups were fixed and embedded to make hard tissue sections.Haematoxylin-eosin staining was performed to observe osteoblasts,osteoclasts and collagen fibers in the implant-bone interface.3.RNA sequence was used to detect the expression of mRNAs,microRNAs(miRNAs),long non-coding RNAs(lncRNAs)and circularRNAs(circRNAs)of samples from two groups.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were used to predict their function.LncRNA-miRNA-mRNA and circRNA-miRNA-mRNA co-expression network of differentially expressed mRNAs and ncRNAs were constructed.Real Time Polymerase Chain Reaction(RT-PCR)was performed to verify the expression of some differentially expressed mRNAs and ncRNAs.4.A total of 8 adult male Wistar rats were fed with high-fat diet for 8 weeks to establish the model of hyperlipidemic rats.3 days before implantation,the experimental group was injected with the overexpression of miR-193a by lentiviral vector and the control group with the corresponding negative control.10 days after implantation,bones were got and RT-PCR was performed to detect the expression of miR-193a-3p,Sdccag3,Inc MSTRG97162.4,Inc MSTRG.78883.18,ALP and Runx2.Results1.The serum lipid levels of experimental group is significant higher than those in control group(P<0.05).2.In experimental group,there were less and disordered osteoblasts,and fewer new bone formation than those in control group.3.In total,389 mRNAs,70 miRNAs,770 IncRNAs and 7 circRNAs were differentially expressed(P<0.05,fold change>2).Functional analysis of the differential mRNA and ncRNAs revealed significant alterations of biological processes related to retromer complex,actin cytoskeleton and signal pathways related to osteogenesis and adipogenesis.4.The lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA network especially related to osteogenesis of these RNAs were constructed.Expression level of most RNAs by R.T-PCR was consist with that by RNA sequencing.5.After overexpression of miR-193,in experimental group,the expression of miR-193a-3p increased(P<0.05),and the expression of Sdccag3,Inc MSTRG97162.4,ALP and Runx2 decreased(P<0.05),but there was no significant difference of Inc MSTRG.78883.18 in two groups(P>0.05).Conclusions1.Hyperlipidemia could surpress bone formation around implants of rats.2.The co-expression network of differentially expressed mRNAs,miRNAs,IncRNAs and circRNAs regulate bone formation around implants of hyperlipidemic rats.3.MiR-193a-3p could inhibit bone formation around implants of rats by Inc MSTRG.97162.4-miR-193a-3p-Sdccag3.