| Objective:The purpose of this study was to determine the sequence of microRNA in Chinese hamster's oral mucosa carcinoma by nest generation sequencing,to construct the differential expression profiles of miRNA,predict their target genes,and screen molecular markers of oral mucosa carcinoma by bioinformatics analysis,to explore the regulation mechanism of mi RNA in oral mucosal carcinogenesis,invasion,metastasis and prognosis,and to provide a theoretical basis for the prevention and treatment of oral mucosa carcinoma from the point of view of comparative medicine.Methods:We established Chinese hamster oral mucosa carcinoma model by smearing DMBA(9,10-Dimethy1-1,2-Benzanthracence).The differentially expressed miRNA profiles were constructed by microRNA sequencing and bioinformatics analysis in Chinese hamster's oral mucosa carcinoma tissue and normal tissue.We predicted the target genes and analyzed the GO enrichment and KEGG Pathway.The differential expression of miRNAs were validated by RT-qPCR technique.The expression levels of PTEN,p-AKT in PTEN/PI3K/AKT pathway regulated by miR-21 and related apoptosis proteins Bax,Bcl-2,Caspase-3 and Caspase-9 were detected by immunohistochemistry and RT-qPCR.Results:Based on the successful construction of Chinese hamster's oral mucosa carcinoma model,we obtained 268 known differentially expressed miRNAs,of which 137 were up-regulated and 131 were down-regulated expression.Further,we got 11 significantly differentially expressed miRNAs,including 8 up-regulated miRNAs(miR-542-3p,miR-130b-3p,miR-142-5p,miR-34c-3p,mi R-34c-5p,miR-34b-5p miR-21-3p,and miR-21-5p)and 3 down-regulated miRNAs(miR-504,miR-499-5p,miR-486-3p).Target gene prediction and functional analysis revealed by GO entries,including 340 biological processes,47 cell components,and 46 molecular functions.Meanwhile,we obtained 15 KEGG pathway associated with OSCC.The enriched GO entries are mainly in anatomical structure,signal regulation,cell communication regulation,cell protuberance,cytoskeleton,neuronal part,anion coordination,transferase activity and ATP binding.The extremely significant KEGG Pathway(q<0.01)included notch signaling pathway,phosphatidylinositol signaling system and chronic myeloid leukemia.At the same time,we predicted 208 novel miRNAs between normal and cancer groups(112 novel up-regulated miRNAs and 96 novel down-regulated miRNAs),including 3 significantly differentially expressed novel miRNAs with p-value < 0.05(1 miRNAs up-regulated and 2 miRNAs down-regulated).1GO entry enriched by the target genes of novel miRNAs related to cellular component was obtained by bioinformatics analysis.The results of significantly differentially expressed miRNA verified by RT-qPCR were basically consistent with the mi RNA-Seq results.In PTEN/PI3K/AKT pathway,the expression of PTEN was down-regulated and the expression of p-AKT was up-regulated.The expression of related apoptotic proteins Bax,Caspase-3 and Caspase-9 was down-regulated,and the expression of Bcl-2 was up-regulated.Conclusions:In this study,we identified 11 significantly differentially expressed miRNA and predicted 3 new significantly differentially expressed miRNAs,which lead to activation of15 pathways associated with oral mucosal cancer including Notch signaling pathway andphosphatidylinositol signal transduction pathway and chronic myeloid leukemia.The expression of two mature variants of miR-21(miR-21-3p and miR-21-5p)increased significantly.Its direct target gene PTEN that was down-regulated and the expression of p-AKT was up-regulated affected the activation of the PI3K/AKT pathway,thereby affecting the expression of related apoptosis proteins,which leads to the occurrence and development of oral squamous cell carcinoma.mi R-21 can be used as a molecular marker to guide the clinical practice of OSCC. |
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