Study Of Screening Differentially Expressed Genes Before And After EGCG Decompression Of Odontogenic Keratocyst

Objective:Transcriptome expression profiles of paired samples of odontogenic keratocyst(OKC)before and after(-)-epigallocatechin-3-gallate(EGCG)decompression were analyzed using RNA sequencing(RNA-Seq),aiming to screen out differentially expressed genes(DEGs).Based on the results of high-throughput sequencing,in vitro experiments are conducted to further screen and verify biological functions of key candidate genes after EGCG decompression of OKC,thus finding potential targets for minimally invasive targeted therapy of OKC.Materials and Methods:Paired samples obtained from 3 OKC patients before decompression and 6 months after EGCG decompression were obtained for RNA-Seq.DEGs with significant differences(padj≤ 0.05 and |log2(FC)| > 1)were analyzed using edge R(3.12.1),which were further subjected to Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)for clustering analyses using DAVID.In addition,protein-protein interaction(PPI)score in STRING database was graded for predicting the potential PPI networks between DEGs encoded proteins,and those enriched in both EGCG-induced immune response of cystic wall of OKC,and PD-L1 interaction were further screened.These key DEGs were validated by real-time PCR(RT-PCR)and Western blot(WB).Protein level of PD-L1 in cystic wall tissues of OKC and normal oral mucosa tissues was examined by immunohistochemical staining.Moreover,m RNA and protein levels of PD-L1 in 3 paired OKC samples obtained before decompression and 6 months after EGCG decompression were detected by RT-PCR and WB,respectively.Results:A total of 403 DEGs were screened out from 3 paired OKC samples obtained before decompression and 6 months after EGCG decompression,including 119 upregulated and284 downregulated genes.GO analysis showed that immune and inflammatory responses were mainly enriched in EGCG decompression of OKC.Based on RNA-Seq data,PPI analysis was performed on 22 inflammatory factors annotated in immune response item and PD-L1,and 7 inflammatory factors were both involved in EGCG-induced immune response of cystic wall of OKC,and PD-L1 interaction.Later,m RNA and protein levels of the 7inflammatory factors were validated,and among them,IL-7R,CXCL1 and TLR2 were downregulated at 6 months after EGCG decompression(P < 0.05),which were consistent with RNA-Seq findings.Compared with normal oral mucosa tissues,PD-L1 protein was upregulated in cystic wall tissues of OKC(Z =-2.584,P < 0.05).There was no significant difference in the m RNA level of PD-L1 in OKC samples before decompression and 6months after EGCG decompression,but the protein level was significantly downregulated at6 months after EGCG decompression(P < 0.05).Conclusions:1.DEGs in 3 paired OKC samples before decompression and 6 months after EGCG decompression are screened using RNA-Seq,and further subjected to GO and KEGG.It is revealed that immune and inflammatory responses are of great significance during the process of EGCG marsupialization of OKC.2.EGCG decompression inhibits inflammatory osteoclastic reaction mediated by cyst wall cells and accelerates the shrinkage of OKC by downregulating inflammatory factors IL-7R,CXCL1 and TLR2 in cyst wall cells of OKC.3.PD-L1 may be a potential target of EGCG decompression of OKC.