| Renal interstitial fibrosis(RIF) is a common basic channel and major pathology of end-stage renal disease(ESRD). Regardless of etiology, all patients with chronic renal diseases show a progressive decline in renal function with time. And they need renal replacement therapy in the end. Know more about the reasons and mechanisms of renal interstitial fibrosis, find the ways to control fibrosis is important to patients with CKD.Epithelial-mesenchymal transition( EMT) plays an important role in renal interstitial fibrosis, transforming growth factor-β 1(TGF-β 1) is one of the key cytokines inducing fibrosis, which can independently initiate and complete the entire process of EMT. TGF-β 1 signaling pathway is an important target in treating renal fibrosis, know more about TGF-β 1 signaling pathway is very necessary.In recent years, with the rapid development of life science and technology, many thousands of regulatory non-protein-coding RNAs(nc RNAs), which regarded as junk, were found pervasively transcribed. And they have fulfilled critical roles as post-transcriptional regulators in physiology and pathphysiology; Micro RNAs(mi RNAs) are a class of small, endogenous RNAs of about 22 nucleotides(nts) in length, which functions in post-transcriptional regulation of gene expression. Aberrant expression of mi RNAs has been implicated in numerous disease states. Although mi RNA has been part of the research in EMT, it is minimal compared to the total number of mi RNA. And therefore we continue to explore miRNA function andmechanism of EMT, which will bring the great promising future in prevention and treatment of EMT and related diseases.In this study, we found that when m PTCs treated with TGF-β 1.The expression of mi R-30e* was decreased in a time- and does- dependent manner. To further demonstrate the role of mi R-30e* in EMT and its mechanism, we performed the following studies.Part1 The role of mi R-30e* in TGF-β1 induced mouse proximal tubular cells mesenchymal transitionObjective: To observe the role of mi R-30e* in TGF-β1 induced mouse proximal tubular cells mesenchymal transition.Methods:1. 10 ng/ml TGF-β1 was used to induce EMT in vitro, E-cadherin, vimentin and α-SMA were detected by q RT-PCR after TGF-β1 treated for 0,24 and 48 hours, which verified by western blot.2. Different concentrations of TGF-β1(0, 1, 5, 10, 20ng/ml) were used to stimulate m PTCs, the expression of mi R-30e* was detected in these cells by q RT-PCR. 3. To evaluate the time effect of TGF-β1 on mi R-30e* expression, m PTCs were treated with TGF-β1 at different time points of 0, 6, 12, 24, 48 hours.4. MPTCs stably over expression mi R-30e* were used, E-cadherin, vimentin and α-SMA were detected after treated with 10ng/ml TGF-β1 for 24 h.5. To examine the role of mi R-30e* over expression on TGF-β1-induced EMT, m PTCs were transfected with mi R-30e* mimic, the expression of mi R-30e* was measured by real-time PCR.Cells transfected with mi R-30e* mimic and negative control were the treated with 10ng/ml TGF-β1, E-cadherin, vimentin and α-SMA were detected by q RT-PCR and western blot.Results:1. TGF-β1 treatment reduced both m RNA and protein levels of E-cadherin and induced both m RNA and protein levels of vimentin and α-SMA in atime-dependent manner.2. TGF-β1 treatment reduced the expression of mi R-30e* significantly at 10ng/ml(by 27%, P<0.05).q RT-PCR shows TGF-β1 treatment reduced the expression of mi R-30e* in a time-dependent manner and begin to meaningful at 24h(by 23%,P<0.05).3. Cells stably over expression mi R-30e* exhibited anti-EMT effect compared with vehi, which have a higher level of E-cadherin(by 1.3 folds,P<0.05) and lower level of α-SMA and vimentin(by 35% and 16% respectively,P<0.05).4. Mptcs transfected with mi R-30e* mimic shows an anti-EMT properties:higher level of E-cadherin(by 1.8 folds,P<0.05) and lower level of α-SMA and vimentin(by 39% and 52% respectively,P<0.05) than negative control.Conclusion: TGF-β1 can induce mPTCs –mesenchymal transition and reduced the expression of mi R-30e* in a time and dose-dependent manner. Over expression of mi R-30e* could block EMT triggered by TGF-β1, which implies that mi R-30e* could participate in renal interstitial fibrosis as a negative regulator.Part2 Snail1 is a direct target of mi R-30e*Objective: To explore the target genes of mi R-30e*, and verify if Snail1 is a direct target of mi R-30e*.Methods: 1. Target gene predict programmes were used to predict the probably target genes of mi R-30e* in EMT. 2. m PTCs which can over expression or knock down mi R-30e* were cultured. Protein and m RNA levels of predicted genes were detected by western blot and q RT-PCR respectively in these cells.3. TGF-β1 induced expression of Snail1 was detected by q RT-PCR and western blot.4. Expression of Snail1 was detected by q RT-PCR and western blot in cells transfected with mi R-30e* mimic or mi R-30e* and their controls, then treated with TGF-β1.5. Luciferase assaywas constructed to verify if Snail1 is a direct target of mi R-30e*Results:1. Three target gene predict programmes all predicted that Snail1 is a direct target of mi R-30e*.And the combination site of mi R-30e* in the 3’UTR of Snail1 is number 720-726.2. The expression of mi R-30e* and Snail1 has a negative correlation.3. TGF-β1 induced the expression of Snail1 sigenificantely(by 1.9 folds) at an early time, and ectopic mi R-30e* decreased TGF-β1 induced expression of Snail1.4. Luciferase assay shows Snail1 is a direct target of mi R-30e*: wild type 3’UTR of snail1, when con-transfected with mi R-30e* mimic, the luciferase activity significantly decreased(by 47%, P<0.05).While when wild type 3’UTR of snail1 con-transfected with mi R-30e* inhibitor, he luciferase activity significantly increased(by 1.77 folds, P<0.05).Conclusion:Snail1 is a direct target of mi R-30e*Part3 The role of mi R-30e*/Snail1 in TGF-β1 induced EMT in m PTCsObjective: To investigate the role of mi R-30e*/Snail1 in TGF-β1 induced EMT in m PTCs.Methods: 1. Cultured m PTCs were divided into four groups: pc DNA3.1, p Snail1,pc DNA3.1 and 10ng/ml TGF- β 1, p Snail1 and 10ng/ml TGF- β 1.The expression of Snail1、E-cadherin、α-SMA and vimentin were measured by q RT-PCR and western blot.2. mi R-30e* mimic and p Snail1 were con-transfected to m PTCs, pc DNA3.1 and p Snail1 as control, or NC and mi R-30e* mimic as control. After these cells treated with 10ng/ml TGF-β 1, m RNA levels of E-cadherin、α-SMA and vimentin were detected by q RT-PCR at 24 hours, and protein levels were detected by western blot at 24 hours.3. When mi R-30e* inhibitor was transfected to m PTCs, the cells exert EMT phnotype.While Snail1 si RNA could partly block the effect of knockdown miR-30e*.Results:1. Snail1 facilitates EMT induced by TGF-β 1: when treated with TGF-β 1, the snail1 over expression group has a lower expression of E-cadherin(by 38%, P<0.05) and higher expression of α-SMA and vimentin(by 1.4 folds and 1.3 folds respectively, P<0.05) compared with pc DNA3.1 group. 2. mi R-30e* can blunt EMT facilitated by snail1, which induced by TGF-β 1. And over expression of snail1 reducing the effect of mi R-30e*. 3. Block of Snail1 with snail1 si RNA attenuates EMT induced by knock down of mi R-30e*.Conclusion: Ectopic mi R-30e* blunts EMT induced by TGF-β 1 via targeting Snail1, knock down of mi R-30e* induced EMT partly via Snail1. |
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