The Regulation And Mechanism Research Of MicroRNA-21 On Inflammation Of Human Corneal Keratocytes

Objective The aim of this study is to investigate the effects of miR-21 on LPS-induced inflammation in corneal keratocytes.In addition,we examined the potential target genes of miR-21 and its regulatory roles.Methods 1.The human corneal keratocytes were purchased from Sciencell Biotechnology,USA.Corneal keratocytes were treated with LPS.The release of inflammatory cytokines and miR-21 was determined by real-time PCR.2.The inflammatory changes of human corneal keratocytes were observed by enhancing or inhibiting the expression of miRNA-21.2.1 The expression of miRNA-21 mRNA was detected by qPCR.The gene expression of inflammatory cytokines,for example interleukelin-6(IL-6)and Interleukelin-8(IL-8)mRNA and inflammatory protein secretion were detected by qPCR and Elisa kits after transfected with hsa-miR-21 agomir.CCK8 kit and cell migration experiments were conducted to detect the cell proliferation activity and migration rate of corneal keratocytes after transfected with hsa-miR-21 agomir.2.2 The expression of miRNA-21 mRNA was detected by qPCR.The mRNA and inflammatory protein secretion of IL-6 and IL-8 were detected by qPCR and Elisa kits after hsa-miR-21 antagomir.CCK8 kit experiment was conducted to detect the cell proliferation activity of corneal keratocytes with hsa-miR-21 antagomir.3.Gene software was used to identify the target gene of miR-21 in corneal keratocytes.Real-time quantitative PCR was used to study the effect of miR-21 on its target gene.Result 1.The results of CCK8 experiment showed that the survival rate of cells in 0.5μg/ml LPS group,1μg/ml LPS group,5μg/ml LPS group was(90.7±3.84)%,(82.6±4.25)%,(39.6±1.53)% respectively,with the survival rate in control group set 100%.The differences were statistically significant(F=82.8,P<0.001).After the multiple comparisons among groups,1μg/ml LPS was chosen to build the inflamation models.2.The expression of miR-21 was significantly increased with LPS(P<0.001).3.1)The expression of miR-21 was significantly increased with hsa-miR-21 agomir in corneal keratocytes(P<0.001).The results of realtime PCR and Elisa indicated that the relative expression levels of IL-6 mRNA and IL-8 mRNA in hsa-miR-21 agomir group were significantly decreased compared to untransfected hsa-miR-21 agomir(P<0.001).The results of CCK8 and migration experiments showed that the cell proliferation activity and migration rate were significantly higher than those in the negative control group(P<0.001).3.2)The expression of miR-21 was significantly reduced with hsa-miR-21 antagomir in corneal stromal cells(P<0.001).The results of realtime PCR and Elisa indicated that the relative expression levels of IL-6 mRNA and IL-8 mRNA in hsa-miR-21 antagomir group were significantly increased(P<0.001).The results of CCK8 experiments showed that the cell proliferation activity was significantly lower than those in the negative control group(P<0.001)4.Gene-related software predicts that PDCD4 and PTEN are potential target genes for miR-21,and the expression of PDCD4 and PTEN were down-regulated with hsa-miR-21 agomir by qPCR Conclusions 1.MiRNA-21 is significantly elevated in LPS-induced corneal stromal cells 2.Over-expression of miRNA-21 can significantly inhibit the high expression of inflammatory cytokines of human corneal keratocytes induced by LPS,while overexpression of miRNA-21 can promote cell proliferation and migration.3.The target gene of miRNA-21 PTEN and PDCD4 decreased in the inflammatory response of human corneal keratocytes induced by LPS.