Analysis For Time Series Geneexpression Profiles And Candidate Genes During Recovery Phase Of Severed Rat Medial Collateral Ligament

Background:The medial collateral ligament(MCL)is the main stable structure inside the knee joint.The MCL injury is a common soft tissue injury in physical labor and physical exercise.The repairing mechanism of injured MCL,however,has not been extensively studied,which results in the limited the understanding of key factors involving in the recovery phase.This article was intended to depict the dynamic changes of gene expression profiles during the recovery phase of severed rat MCL.Furthermore,the candidate genes in healing of MCL injury were excavated,and the gene and protein expression level were verified in a rat MCL healing model to better understand the healing mechanism after MCL injury.Methods and materials: The GSE10720 gene expression profiles of severed rat MCL after 1,2,4,7,10 and 14 day(s)of injury were downloaded from Gene Expression Omnibus(GEO)database.STEM(Short Time-series Expression Miner)software was conducted to cluster genes with similar expression patterns,followed by identification of differentially expressed genes(DEGs)between injured group and sham control group,Gene Ontology(GO)annotation and pathway enrichment analysis.Subsequently,gene functional interaction(FI)network and protein-protein interaction(PPI)network were respectively constructed using Reactome FI and STRING.Transcription factors(TFs)were predicted by Animal TFDB.In the second part of the study,a rat MCL healing model was constructed: the rats were divided into two groups,the model group and the sham operation group.After general anesthesia in the model group,the MCL was cut off;the sham control group was exposed to the same analgesia/anesthesia protocol,and bilateral sham surgery was performed,but the ligament was not cut.Three sham-operated rats and three MCL model rats were sacrificed on days 1 and 7 after modeling,respectively.Tissues of MCL were collected and frozen in liquid nitrogen prior to RNA isolation or fixed in 4% paraformaldehyde.Subsequently,the expression levels of DEGs were detected by q PCR.Next,the expression levels of proteins encoded by DEGs were verified by Western blot.Finally,immunohistochemical staining was used to further verify the expression levels of these proteins.Results: Three significant temporal expression patterns were uncovered after MCL injury,among which the most significant cluster consisted of 3854 DEGs.Of these DEGs,102 constructed the gene FI network consisting of 9 significant modules.GO analysis revealed that cell differentiation was the most significant GO term enriched by 240 DEGs,and among these DEGs,33 genes,such as EGFR,VEGFC,ITGAV,ITGA1,significantly enriched in both focal adhesion and ECM-receptor interaction pathways.PPI network demonstrated 10 genes with degree > 20.Totally 22 TFs were predicted to target to these 240 DEGs.By verifying in the rat MCL injury model,the m RNA expression levels of EGFR,ITGAV,LAMB2 and VEGFC were found significantly down-regulated on the first day of injury compared with the sham-operated group,and their expression levels were significantly higher than the sham-operated group on days 7.In addition,Western blot results showed that the protein expression levels of EGFR,ITGAV and LAMB2 were consistent with the m RNA expression results,and immunohistochemistry analysis showed similar results for ITGAV and EGFR.Conclusion: The expression profiles of severed rat MCL significantly changed during the recovery phase.DEGs in the early 14 days of MCL healing,including VEGFC,EGFR,ITGAV and LAMB2,are more likely to play essential roles during this process.