Sdccag3 Enhances Implant Osseointegration During Experimental Hyperlipidemia

PurposeSerologically defined colon cancer antigen-3(Sdccag3),as a novel endosomal protein,functions in actin cytoskeleton remodeling,ciliogenesis,protein trafficking and secretion,cytokinesis,and apoptosis,but its roles in implant osseointegration under hyperlipidemic conditions have not been uncovered.Hyperlipidemia is a metabolic disease that is characterized by the elevated level of serum lipid,often resulting in unfavorable osseointegration and implant loss.Genetic networks functioning in the peri-implant tissue might be another factor,as the loss of implants years after implantation might not be addressed by controlling infection/inflammation alone.Thus,in our previous study,we performed RNA sequencing analysis to identify the differential expression of the Sdccag3 gene and related noncoding RNAs(ncRNAs),including the long noncoding RNA(lncRNA)MSTRG.97162.4 and miR-193a-3p.Therefore,Sdccag3-enhancer/inhibitor,lncRNA-MSTRG.97162.4-enhancer?miR-193a-3p-enhancer/inhibitor recombinant lentiviral expression vectors were constructed,and micro-CT and BV/TV analysis?HE staining and BIC%analysis?qRT-PCR and Western blotting were used to further investigates the functional mechanism of Sdccag3 and its related lncRNA-MSTRG.97162.4 and miR-193a-3p in osseointegration during experimental hyperlipidemia,which might provide new therapeutic targets to osteoporosis and compromised osseointegration caused by hyperlipidemia.Methods(1)A total of 80 adult male Wistar rats were equally divided into 24 rats and 56 rats.24 4-week-old male Wistar rats were equally divided into hyperlipidemic and control groups and feed with high-fat and normal diet respectively,while 56 rats were only feed with high-fat diet.8 weeks later,the levels of serum lipid were detected by taking endocanthion vein of all rats to ensure the successful establishment of experimental hyperlipidemia models.(2)Threaded titanium implants(1.5 mm in diameter,2.5 mm in length)were inserted into the site 5 mm away from the bilateral distal femoral metaphysis of 24 rats in hyperlipidemic and control groups.Then 1mm bone around implant was dissected after 2 weeks and 4 weeks implantation respectively.(3)Recombinant lentiviral expression vectors were constructed to establish Sdccag3-enhancer?Sdccag3-inhibitor?lncRNA-MSTRG.97162.4-enhancer?miR-193a-3p-enhancer?miR-193a-3p-inhibitor,LV17-nc?LV16-nc vectors(LV17 was used as a negative control for Sdccag3-enhancer?lncRNA-MSTRG.97162.4-enhance;LV16 was used as a negative control for Sdccag3-inhibitor?miR-193a-3p-enhancer?miR-193a-3p-inhibitor).(4)56 experimental hyperlipidemia rats were equally divided into Sdccag3-enhancer?lncRNA-MSTRG.97162.4-enhancer?LV17-nc,Sdccag3-inhibitor?miR-193a-3p-enhancer?miR-193a-3p-inhibitor?LV16-nc groups and each group included 8 rats.The vectors were intramuscularly injected into the femoral metaphysis regions of hyperlipidemic rats at 3 and 0 days before implantation.At 2 weeks after implantation,1mm bone around implant was obtained.(5)Micro-CT analysis and HE staining were conducted to assess bone-implant osseointegration.(6)qRT-PCR and Western blotting were performed to assess the levels of genes and related proteins.(7)GraphPad Prism 6.0 software was used for date processing and statistical analysis.Results(1)Levels of serum lipid in the hyperlipidemic group were significantly higher than those in the control group(P?0.05).Hyperlipidemic experimental models were successfully established.(2)Compared with the control group,the BIC%and BV/TV were lower(P?0.05),the trabecular bone arrangement was disordered and the newly formed bone was less in hyperlipidemic rats.(3)In the Sdccag3-enhancer group,the BIC%and BV/TV were higher(P?0.05),the implant was surrounded by abundant newly formed trabecular bone,while the expression of Sdccag3?lncRNA-MSTRG.97162.4?Runx2 were dramatically upregulated(P?0.05)but miR-193a-3p was downregulated(P?0.05).Consistent with these results,Sdccag3?Runx2 protein levels were increased in the Sdccag3-enhancer group.By contrast,lower BIC%and BV/TV(P?0.05),less new bone tissue,downregulation of Sdccag3?lncRNA-MSTRG.97162.4?Runx2(P?0.05)and upregulation of miR-193a-3p(P?0.05)were observed in the Sdccag3-inhibitor group.Moreover,Sdccag3 and Runx2 protein levels were suppressed in the Sdccag3-inhibitor group.(4)In the lncRNA-MSTRG.97162.4-enhancer group,the BIC%and BV/TV were higher(P?0.05),the implant was bound by more newly formed trabecular bone,while the expression of IncRNA-MSTRG.97162.4?Sdccag3?Runx2 were dramatically upregulated(P?0.05)but miR-193a-3p was downregulated(P?0.05).As expected,Sdccag3?Runx2 protein levels were increased in the lncRNA-MSTRG.97162.4-enhancer group.(5)In the miR-193a-3p-enhancer group,the BIC%and BV/TV were decreased(P?0.05),the implant was surrounded by less newly formed trabecular bone,while the expression of miR-193a-3p was upregulated(P?0.05),but Sdccag3?lncRNA-MSTRG.97162.4?Runx2 were dramatically downregulated(P?0.05).In addition,Sdccag3?Runx2 protein levels were inhibited in the miR-193a-3p-enhancer group.In contrast,increased BIC%and BV/TV(P?0.05),more new bone tissue,downregulation of miR-193a-3p(P?0.05)and upregulation of Sdccag3?lncRNA-MSTRG.97162.4?Runx2(P?0.05)were observed in the miR-193a-3p-inhibitor group.Consistently,Sdccag3 and Runx2 protein levels were enhanced in the miR-193a-3p-inhibitor group.Conclusion(1)Hyperlipidemia could result in compromised implant osseointegration in rats.(2)Sdccag3 enhances implant osseointegration during experimental hyperlipidemia,and upregulates lncRNA-MSTRG.97162.4 but downregulates miR-193a-3p.LncRNA-MSTRG.97162.4 promotes implant osseointegration,and regulates the expression of Sdccag3 positively but modulates the expression of miR-193a-3p negatively in hyperlipidemia.While,miR-193a-3p inhibits implant osseointegration and downregulates the expression of Sdccag3 and lncRNA-MSTRG.97162.4.(3)The regulatory network of Sdccag3,lncRNA-MSTRG.97162.4 and miR-193a-3p plays a significant role in the osseointegration of experimental hyperlipidemic rats,which might be a promising target in improving the rate of implants of hyperlipidemia.