| Diabetic nephropathy(DN) is a pressing public health challenge, and it is the most common cause of chronic kidney disease, which lead to end-stage renal disease(ESRD) and premature death. In recent years, many researchers have proposed that the inflammatory pathways play central roles in the progression of DN. Various inflammatory molecules, including Interleukin-18(IL-18), tumor necrosis factor-α(TNF-α), myeloid differentiation factor88(MYD88), and intercellular adhesion molecule-1(ICAM-1), have been found to be abnormally expressed and closely related to the progression of DN in animal experiments and clinical studies. These inflammatory molecules, which are transcribed by nuclear factor-kappa B(NF-κB), contribute to the progression of diabetic renal damage, include alteration of the extracellular matrix, fibrosis and glomerulosclerosis.Recently, increasing evidence has suggested that micro RNA(mi RNA) are involved in DN. In our previous research, it was reported that mi R-451 suppresses mesangial hypertrophy in early DN. Recent evidence suggests that mi R-451 is widely dysregulated in human disease and plays a critical role in inflammation. However, the exact mechanism of mi R-451 and NF-κB activation remain unclear in DN.NF-κB inhibitor alpha(IκBα) regulates activation of NF-κB, which translocates to the nucleus when IκBα is degraded by the immunoproteasome. Large multifunctional protease 7(LMP7) is one catalytic core of immunoproteasome. Overexpression of LMP7 has been shown to promote inflammatory responses, due to improved NF-κB activation. Hence, Overexpression of LMP7 might contribute to the development of DN via the NF-κB signaling pathway.In our earlier research of lung cancer, LMP7 has been found to be a direct target of mi R-451. So we speculate whether mi R-451 targets LMP7 in DN; however, this phenomenon has not been reported. We provide an experimental evidenc to show that mi R-451 suppresses the NF-κB-mediated inflammatory molecules expression through inhibiting LMP7, and attenuates glomerular injury in DN. Part I The expression of mi R-451 in diabetic nephropathy and validation of target geneObjective: To observe the expression of mi R-451 in mouse mesangial cells cultured with high glucose conditions and in the renal cortices of db/db mice. To predict and identify mi R-451 regulates directly to LMP7 in mouse mesangial cells.Methods: Quantitative real-time PCR(q RT-PCR) experiments demonstrated the expression of mi R-451 in the renal cortices of db/db mice and mouse mesangial cells cultured in high glucose conditions. Bioinformatics analysis predicts the target gene of mi R-451. q RT-PCR and western blot demonstrated the relative expression of endogenous LMP7 in db/db mice and mouse mesangial cells cultured in high glucose conditions. The relative expression levels of mi R-451 and LMP7 were detected by q RT-PCR and western blot experiments when mi R-451 mimics and inhibitor transfected into mouse mesangial cells. Luciferase reporter plasmid was constructed which contained sites of the LMP7 3’ UTR wild-type(LMP7 3’ UTR WT) or seed-sequence mutant(LMP7 3’ UTR Mut), and the interaction between mi R-451 and LMP7 3’ UTR performed dual luciferase assays in 293 T cells.Results: In mouse mesangial cells cultured with high glucose conditions and in the renal cortices of db/db mice, mi R-451 levels were significantly decreased and the relative expression levels of endogenous LMP7 were significantly elevated(p<0.05). In mouse mesangial cells, the relative expression levels of endogenous mi R-451 were significantly elevated with mi R-451 mimics and significantly decreased with mi R-451 inhibitor(p<0.05). q RT-PCR, western blots and luciferase assays demonstrated mi R-451 directly targeted the relative expression levels of LMP7(p<0.05).Conclusions: It may be involved in the pathogensis of diabetic neuropathy mice when the relative expression level of mi R-451 was significantly decreased. Mi R-451 could directly recognize and bind to LMP7 3’ UTR, and regulate the relative expression levels of LMP7.Part II mi R-451 regulates proinflammatory moleculesexpression via LMP7/NF-κB signaling pathway in mousemesangial cellsObjective: To study the effect of LMP7/NF-κB signaling pathway when the relative expression level of mi R-451 was regulated. To observe the effect of mi R-451 in renal fibrosis.Methods: Western blot examined the relative protein levels of role factors involved in NF-κB signaling, including IκBα, IκBα phosphorylation(p-IκBα) and p65 when mi R-451 mimics transfected into mouse mesangial cells cultured with high glucose conditions. Western blot examined the relative protein levels of IκBα, p-IκBα and p65 when mi R-451 inhibitor transfected into mouse mesangial cells cultured with low glucose conditions. Nuclear translocation of p65 was confirmed by immunofluorescence cytochemistry when mi R-451 mimics and inhibitor transfected into mouse mesangial cells. Western blot examined the relative protein levels of IκBα, p-IκBαand p65 were regulated by mi R-451 when LMP7 was inbited by PR-957. Chromatin immunoprecipitation(Ch IP) assays examined the relative binding levels of p65 to the promoters of TNF-α, IL-18, MYD88, and ICAM-1 via regulation of mi R-451 levels, and q RT-PCR demonstrated the m RNA levels of the proinflammatory molecules TNF-α, IL-18, MYD88, and ICAM-1. The effect of mi R-451 in renal fibrosis was examined by q RT-PCR.Resulte: mi R-451 mimics resulted in decreased phosphorylation and accelerated accumulation of IκBα, which was accompanied by decreased nuclear translocation of p65(p<0.05). mi R-451 inhibitor significantly increased phosphorylation and down regulation of IκBα, which corresponded to increased nuclear accumulation of p65(p<0.05). Immunofluorescence cytochemistry assays showed mi R-451 mimics decreased nuclear translocation of p65 and mi R-451 inhibitor increased nuclear accumulation of p65. From Ch IP and q RT-PCR studies showed that mi R-451 regulated with the DNA-binding activity of the p65 element located on the promoters of TNF-α, IL-18, MYD88, and ICAM-1, then lead to change genetic transcription of these proinflammatory molecules(p<0.05). Profibrotic factors TGF-β1, FN, collagen I and collagen IV were regulate with mi R-451 in mouse mesangial cells(p<0.05).Conclusions: mi R-451 directly targeted LMP7 expression to inhibit NF-κB activity, leading to mi R-451 interfered with the DNA-binding activity of the p65 element located on the promoters of TNF-α, IL-18, MYD88, and ICAM-1, at last reduced genetic transcription of these proinflammatory molecules. mi R-451 also reduced genetic transcription of profibrotic factors TGF-β1, FN, collagen I and collagen IV. Part III mi R-451 regulates LMP7/NF-κB signaling pathway indiabetic nephropathy miceObjective: To study overexpression of mi R-451 regulates LMP7/NF-κB signal pathway and proinflammatory molecules expression in db/db mice, and affects urinary microalbumin levels, blood glucose, glomerular hypertrophy, mesenteria extension and renal fibrosis. To provide the theoretical evidence for mi R-451 regulates the molecular mechanism in DN.Methods: Via the tail vein, mi R-451 agomir were injected into db/db mice which had high urinary microalbumin levels, until the urinary microalbumin level of the db/db-mi R-451 group was lower than that of the untreated db/db mice group. The relative expression levels of mi R-451 and LMP7 were detected by q RT-PCR in the four groups. Western blot examined the relative protein levels of LMP7, IκBα, p-IκBα and p65. q RT-PCR demonstrated the m RNA levels of the proinflammatory molecules TNF-α, IL-18, MYD88, and ICAM-1. The kidneys of db/db mice were stained by H&E and Masson. The glomerular hypertrophy and mesenteria area were measured by the software. Profibrotic factors TGF-β1, FN, collagen I and collagen IV were demonstrated by q RT-PCR.Resulte: The relative expression level of mi R-451 was significantly elevated in db/db-mi R-451 group(p<0.05). In db/db-mi R-451 group, the relative expression level of LMP7 and the activation of NF-κB were inhibited by mi R-451 agomir(p<0.05), and the m RNA levels of the proinflammatory molecules TNF-α, IL-18, MYD88, and ICAM-1 were decreased(p<0.05). In db/db mice, over-expression of mi R-451 could decreasd urinary microalbumin levels, blood glucose, glomerular hypertrophy, mesenteria extension and inhibited the relative expression levels of profibrotic factors TGF-β1, FN, collagen I and collagen IV(p<0.05).Conclusions: In db/db DN mice, mi R-451 directly inhibits LMP7 and suppresses the NF-κB-mediated inflammatory molecular transcription. Meanwhile, mi R-451 improves blood glucose, urinary microalbumin levels and fibrosis. mi R-451 may be a protective role on the development of DN. |
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