| Objective: The anterior cruciate ligament(ACL)is a crucial component in maintaining knee joint stability.Repeated ACL sprains can result in a range of complications and severe decline in athletic ability.It is difficult for ACL injuries to heal endogenously and they have a high recurrence rate.Similar to the ACL,the medial collateral ligament(MCL)has a greater capacity to mend itself.Circular RNAs(Circ RNA)are non-coding RNAs produced through reverse splicing.Circ RNAs are involved in numerous physiological processes in the body via the ce RNA network.Circ RNA have been proven to play an inadequate role in the repair of a variety of tissues,but their significance in ACL repair remains understudied.In this study,we cross-referenced the variations in circ RNA expression before and after partial damage in rabbit ACL and MCL and performed a functional analysis to investigate the possible molecular processes underlying the failure of endogenous healing in ACL.Methods:(1)Human ACL-injured tissues and normal ACL tissues were collected.Six healthy 3-month-old male randomly selected Oryctolagus cuniculus served as the control group,which received sham surgery without ACL and MCL injury.The remaining three rabbits were used to create partial damage models of the ACL and MCL.One week after modeling,ligaments were excised from rabbits in both groups.For preservation,all ex vivo ligaments were flash-frozen in liquid nitrogen.Two samples were randomly taken from each of the normal and injured rabbit ACL and MCL groups for sequencing The remaining rabbit tissue samples from the same group were validated using quantitative reverse transcription polymerase chain reaction(q RT-PCR),whereas the remaining human tissue samples were sequenced.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)were used to identify the functions of the source genes of the differentially expressed Circ RNA.We predicted the downstream genes of DECs and mapped the circ RNA-miRNA-m RNA network.(2)Chose the common DECs between humans and rabbits.q RT-PCR was used to determine the expression trends in both humans and rabbits.Agarose electrophoresis were used to confirm that the RNA formed loops.Using MTS,Calcein-AM/PI cell immunofluorescence,TUNEL cell immunofluorescence,q RT-PCR,and Western Blotting,this study analyzed the impact of changing the expression of circ＿EPB41L2 and its downstream miR-133a-3p on the survival rate and apoptosis of rabbit primary ACL fibroblasts.In this study,q RT-PCR,Western Blotting,and dual-luciferase reporter assays were used to examine the targeting relationship between circ＿EPB41L2/miR-133a-3p/ collagen,type I,alpha 1 gene(COL1AI)and the effect on the expression of type I collagen.Results:(1)All groups of human and rabbits were screened for 517 DECs,and the q RT-PCR results of the DECs matched the trend of high-throughput sequencing.The ce RNA networks after ACL and MCL injury were quite different.GO and KEGG analyses showed that different circ RNA after ACL and MCL injuries were significantly different in three ways,including angiogenesis,inflammatory response,cell proliferation and apoptosis.NC 013686.1:35078653|351,NW003159656.1:66918|127907,and 6:31271073|31355592 had the highest correlation coefficients in the circ RNA-miRNA-m RNA network graph after injury to rabbit ACL,rabbit MCL,and human ACL.(2)(1)The circ＿EPB41L2 q PCR results were consistent with the trend of high-throughput sequencing in humans and rabbits.Agarose electrophoresis result compared that the circ＿EPB41L2 splice site formed a loop.(2)Calcein-AM/PI cell immunofluorescence and MTS results showed that circ＿EPB41L2 increased the survival rate of rabbit primary ACL fibroblasts through miR-133a-3p.TUNEL cell immunofluorescence,q RT-PCR,and Western Blotting results showed that circ＿EPB41L2 inhibited apoptosis of rabbit primary ACL fibroblasts through miR-133a-3p.(3)Dual-luciferase reporter assay,q RT-PCR,and Western Blotting results showed that circ＿EPB41L2 increased COL1 AI expression level and the expression of type I collagen of rabbit primary ACL fibroblasts through miR-133a-3p.Conclusion:(1)GO and KEGG analyses showed that DECs of ACL and MCL injuries were significantly different in three ways,including angiogenesis,inflammatory response,cell proliferation and apoptosis.NC 013686.1:35078653|351,NW 003159656.1:66918|127907,and 6:31271073|31355592 may pretended important roles after injury to rabbit ACL,rabbit MCL,and human ACL.(2)The expression of circ＿EPB41L2 was reduced in partially wounded ACLs of both humans and rabbits,but there was no significant difference in the rabbit MCL injury model.In rabbit anterior cruciate ligament fibroblasts,circ＿EPB41L2 suppressed apoptosis.It also stimulated the formation of type I collagen in these cells via the miR-133a-3p/COL1A1 pathway. |
PDF Full Text Request