Effect Of Porphyromonas Gingivalis On KYSE30 Cells By MiRNA

There may be a relationship between esophageal microbiota and esophageal squamous cell dysfunction.The infection of Porphyromonas gingivalis was detected in esophageal epithelial cells of patients with esophageal squamous cell carcinoma,and the infection of P.gingivalis was significantly negatively correlated with the survival of patients with esophageal squamous cell carcinoma.This suggests that P.gingivalis may play an important role in the development of esophageal squamous cell carcinoma.Microorganisms can also affect the function of host cells through mi RNAs.Objective:To study the alterations of mi RNAs in KYSE30 cells infected with Porphyromonas gingivalis,predict the target genes by bioinformatics analysis,and select relevant pathways to verify the role of mi RNAs.Method:(1)Screening and regulatory network analysis of mi RNA expression profiles in KYSE30 cells infected with Porphyromonas gingivalisKYSE30 cells were inoculated with P.gingivalis at a multiplicity of infection(MOI)ratio of 10:1.Differentially expressed mi RNAs were screened by mi RNA microarray,and top 20 most differentially expressed mi RNAs were selected,included 10 up-regulated and 10 down-regulated.The results of mi RNA microarray were verified by q RT-PCR,the results that consistent with the mi RNA microarray were selected and analyzed by bioinformatics,including target gene prediction(Target Scan,mi RDB and DIANA Tools database),KEGG analysis,GO analysis.(2)Validation of target genes for mi R-18a-5p and mi R-181a-5pThe expression of SMAD2,SMAD3 and SMAD4 protein effected by the mimics or inhibitors of mi R-18a-5p was detected by western blot;The expression of SMAD2,MAPK1 and IFNG protein effected by the mimics or inhibitors of mi R-181a-5p was detected by western blot;The regulation between mi R-18a-5p and mi R-181a-5p and their target genes respectively was verified by the dual-luciferase assay.(3)Effects of mi R-18a-5p and mi R-181a-5p on proliferation and migration of KYSE30The proliferation of KYSE30 were detected by CCK-8 assay under the effect of mimics or inhibitors of mi R-18a-5p and mi R-181a-5p;The migration of KYSE30 were detected by Wound Healing Test under the effect of mimics or inhibitors of mi R-18a-5p and mi R-181a-5p.Result:(1)The differentially expression mi RNAs were screened out by mi RNA microarray and were verified by q RT-PCR.Twelve differentially expression mi RNAs were identified which contains 8 up-regulated and 4 down-regulated mi RNAs.8 up-regulated mi RNAs: hsa-mi R-18a-5p,hsa-mi R-27a-3p,hsa-mi R-378 e,hsa-mi R-590-5p,hsa-mi R-181a-5p,hsa-mi R-19b-3p,hsa-mi R-30a-5p and hsa-mi R-424-5p;4 down-regulated mi RNAs: hsa-mi R-4645-3p,hsa-mi R-4421,hsa-mi R-4297 and hsa-mi R-3920.It is that Ras,MAPK,Fox O and TGF-β signaling pathways which twelve differentially expression mi RNAs has involved in by KEGG and GO analysis.TGF-β signaling pathway,hsa-mi R-18a-5p(mi R-18a-5p)and hsa-mi R-181a-5p(mi R-181a-5p)were selected as objects.By bioinformatic analyzing,SMAD2,SMAD3 and SMAD4 were target m RNAs for mi R-18a-5p and SMAD2,MAPK1 and IFNG were target m RNAs for mi R-181a-5p in TGF-β signaling pathway.(2)Western blot results showed that mi R-18a-5p mimic could significantly decrease the expression of SMAD2,SMAD3,SMAD4 protein,while mi R-18a-5p inhibitor had no significant effect on SMAD2,SMAD3,SMAD4 protein expression;Western blot results showed that mi R-181a-5p mimic could significantly decrease the expression of SMAD2,MAPK1 protein,while had no significant effect on the expression of IFNG.And mi R-181a-5p inhibitor had no significant effect on SMAD2,MAPK1 and IFNG protein expression;Dual-luciferase assay showed that mi R-18a-5p could bind to the 3’ UTR of SMAD2 directly.(3)CCK-8 assay showed that mi R-18a-5p mimic and mi R-181a-5p mimic had no significant effect on the proliferation of KYSE30,while mi R-18a-5p inhibitor and mi R-181a-5p inhibitor could significantly enhance the proliferation of KYSE30;Wound Healing Test showed that mi R-18a-5p mimic and mi R-181a-5p mimic significantly reduced the migration of KYSE30,while mi R-18a-5p inhibitor and mi R-181a-5p inhibitor had no significant effect on the migration of KYSE30.Conclusion:P.gingivalis could alter the expression of mi RNAs in KYSE30 cells,and differentially expressed mi RNAs may act on KYSE30 cells via Ras,MAPK,Fox O and TGF-β signaling pathways;mi R-18a-5p and mi R-181a-5p may inhibit the proliferation and migration of KYSE30 cells by inhibiting the activity of TGF-β signaling pathway,which may be related to mi R-18a-5p inhibition of SMAD2,SMAD3,SMAD4 expression and mi R-181a-5p inhibits SMAD2,and MAPK1 expression.