| Acute kidney injury(acute kidney injury, AKI), a clinical common critical disease, is a variety of causes of acute kidney function decline, with or without oliguria or anuria of a group of clinical syndromes. The case fatality rate is higher and childhood AKI has become the important factors of chronic renal insufficiency(ESRD), a serious threat to patient safety. Therefore, the study of pathology and physiology during AKI is of great significance for early prevention and treatment of AKI and ESRD. In the process of AKI, renal tubular epithelial cells is the main damaged cells, including early differentiation, broken of the integrity of the cytoskeleton, loss of polarity, disappearance of marker protein, cell apoptosis and necrosis, etc.Micro RNAs(mi RNAs) are a class of endogenous non-coding small RNAs that modulate gene expression via binding to the 3’-untranslated region(3’-UTR) of target m RNAs and thereby induce m RNA degradation or block protein translation at the post-transcriptional level.mi RNAs are widely involved in diverse physiological and pathological processes such as development, cell proliferation, differentiation and apoptosis. There is emerging evidence exhibited that many mi RNAs are tightly related to the pathology of AKI or ESRD. Furthermore there is evidence showed that mi R-709 was upregulated in liver as aging. In our previous study, the high throughput microarray assays found that mi R-709 was up-regulated in our mouse aldosterone model. However, very little is known about the role of mi R-709 in AKI induced by cisplatin.Mitochondria is the power plant of cells, where ATP synthesis, electron transfer, oxidative phosphorylation are happened. Recently, more and more attentions have been paid on the mitochondrial dysfunction in renal disease. In multiple kidney disease models, abnormalities of mitochondrial ultramicrostructure, mutations of mitochondrial DNA and downregulation of mitochondrial DNA copy numbers may beassociated with podocyte injury. Our previous study found that mitochondrial dysfunction is an early event in aldosterone and cisplatin induced kidney injury model. In view of the above findings and previous research results, our research aimed to observe the variation of mi R-709 in m PTC treated with cisplatin. The study illustrated the effects of mi R-709 on the apotosis, the expression of E-cadherin and KIM-1 and mitochondrial functions of m PTC, which is divided into three parts in total. The research discussed the expression and role of mi R-709 in m PTC injury induced by cisplatin and the underlying mechanism, providing a new sight for the physiopathologic basis and theraputic targets of AKI.Part I The expression and role of mi R-709 in m PTC injury induced by cisplatinObjective: To investigate the expression and the role of mi R-709 in the m PTC injury induced by cisplatin.Methods: Mouse renal proximal tubular cells(m PTC) were cultured in vitro, and treated with cisplatin of 0, 1, 5, 10, 20μmol/L respectively and 10μmol/L with different time points of 0, 2, 6, 12, 24 h respectively. The expression of micro RNA-709 was analyzed by quantitative real-time PCR. m PTCs were cultured in vitro, transfected transiently with negative control(NC), mi R-709 mimic, inhibitor negative control(INNC),mi R-709 inhibitor, and were divided into six groups: NC group, mi R-709 mimic group, INNC group, mi R-709 inhibitor group, Cisplatin + INNC or mi R-709 inhibitor group. Apoptosis of renal proximal tubular cells was assessed by annexin V/flow cytometry detection. Caspase-3 activities in renal proximal tubular cells were assessed by caspase activity assay kits. The expressions of E-cadherin, KIM-1 and NGAL were examined by real-time PCR and western blot.Results:(1) Cisplatin dose-dependently and time-dependently increased the level of mi R-709. The level of mi R-709 was raised to approximately 2.15 times, 5.35 and 5.7 times as against control group at 24 h in 5, 10, 20μmol/L cisplatin-treated m PTCs. In addition, the level of mi R-709 was increased to 1.63 times at 12 h in 10μmol/L cisplatin-treated m PTCs and 24 h later increased to 5.3 times as against control group.(2) Upregulation of mi R-709 increased cell apoptosis and the activation of caspase-3(by 0.54、0.31 times,all P<0.05), decreased the expression of E-cadherin(by 59.3%) and increased the expression of KIM-1 and NGAL(by 0.4 and 0.62 times, all P<0.05) when compared with NC group.(3) Downregulation of mi R-709 decreased cell apoptosis, the activation of caspase-3, attenuated the loss of E-cadherin and the enhanced expression of KIM-1 and NGAL induced by cisplatin when compared with INNC+Cisplatin group.Conclusion: mi R-709 was increased in m PTC in a dose and time dependent way treated with cisplatin; overexpression of mi R-709 induced m PTC injury and downregulation of mi R-709 attenuated m PTC injury induced by cisplatin.Part II The role of mi R-709 in mitochondrial dysfunction in m PTC induced by cisplatinObjective: To investigate the role of mi R-709 in mitochondrial dysfunction induced by cisplatin.Methods: Mouse renal proximal tubular cells were cultured in vitro, transfected transiently with negative control(NC), mi R-709 mimic, inhibitor negative control(INNC), mi R-709 inhibitor, and were divided into six groups: NC group, mi R-709 mimic group, INNC group, mi R-709 inhibitor group, Cisplatin + INNC or mi R-709 inhibitor group. Mitochondrial membrane potential(MMP) was determined by JC-1 fluorescence. The production of cellular mitochondrial superoxide was examined by mito SOX fluorescence. mt DNA copy numbers were determined by real-time PCR. Caspase-9 activities in renal proximal tubular cells were assessed by caspase activity assay kits.Results:(1) Upregulation of mi R-709 decreased mitochondrial membrane potential(MMP) and mt DNA copy number(by 34.2%、41.8%,all P<0.05),increased the production of mito SOX and the activation of caspase-9(by 0.31 and 0.33 times,all P<0.05) when compared with NC group.(2) Downregulation of mi R-709 ameliorated mitochondrial dysfunction induced by cisplatin: including the downregulation of MMP and mt DNA copy number, and blocked the production of mito SOX and the activation of caspase-9 when compared with INNC+Cisplatin group.Conclusion: Overexpression of mi R-709 induced mPTC mitochondrial dysfunction and downregulation of mi R-709 attenuated mitochondrial dysfunction induced by cisplatin.Part III The identification of mi R-709 target gene in m PTCObjective: To investigate the mi R-709 target gene in mPTC.Methods: Mouse renal proximal tubular cells(mPTC) were cultured in vitro, and treated with cisplatin of 0, 1, 5, 10, 20μmol/L respectively and 10μmol/L at different time points of 0, 2, 6, 12, 24 h respectively. The expression of TFAM was analyzed by quantitative real-time PCR and western blot. m PTC were cultured in vitro, transfected transiently with pc DNA3.1-vehicle or pc DNA3.1-TFAM, and were divided into three groups: pc DNA3.1-vehicle group, cisplatin+pc DNA3.1-vehicle group, cisplatin+ pc DNA3.1-TFAM. We detect the apoptosis of m PTC and mitochondrial function with methods as described before. RNA Hybrid program was used to predict the secondary structure of the TFAM 3’-UTR/mi R-709 duplex.TFAM luciferase reporter constructs were generated, and luciferase assays were performed to identify that mi R-709 can negatively regulate TFAM gene by targeting its 3’-UTR sequence.We determined the m RNA and protein expression levels of TFAM in m PTC after transfected with mi R-709 mimic. To further verify whether mi R-709 negatively regulate the expression of TFAM leading to mitochondrial dysfunction, we cotransfected mi R-709 mimic and pc DNA3.1-TFAM. The m PTC were divided into four groups: NC + pc DNA3.1-vehicle, mi R-709 mimic + pc DNA3.1-vehicle, NC + pc DNA3.1-TFAM,mi R-709 + pc DNA3.1-TFAM. The expression of TFAM were detected by RT-PCR and western blot.The markers related to m PTC injury and mitochondrial function were detected with methods as described before.Results:(1) The m RNA level and protein expression of TFAM was decreased in cisplatin 5 μmol/l 、10μmol/L、20μmol/L stimulation group(by 34.2%, 66.3%,67.9% respectively,all P<0.05),and also downregulated 12 h and 24 h later(by 23.1%, 52.3%) after cultured in 10μmol/l cisplatin compared with control group.(2) Overexpresion of TFAM ameliorated mitochondrial dysfunction induced by cisplatin: including the downregulation of MMP and mt DNA copy number, and blocked the production of mito SOX and the activation of caspase-9 when compared with pc DNA3.1-vehicle + Cisplatin group.(3) TFAM was verified to be the target gene of mi R-709 in m PTC. As determined by RNA Hybrid analysis, we found that mi R-709 and its binding site in TFAM 3’-UTR could potentially form a very stable secondary structure. The minimum free energy predicted for hybridization with the TFAM 3’-UTR and mi R-709 at this site was △ G=-27.1 kcal/mol, consistent with an authentic mi RNA targeting; Overexpressing mi R-709 decreased TFAM reporter gene activity by approximately 32% compared with NC control; The m RNA level and protein expression of TFAM were both decreased(by 68.1%, 40%, all P<0.05) in m PTC after transfected with mi R-709 mimic.(4) Ovexpression of TFAM could block the m PTC injury and mitochondrial dysfunction induced by mi R-709.Conclusion: TFAM was verified to be the target gene of mi R-709 in m PTC and could block the mitochondrial dysfunction and m PTC injury induced by mi R-709. |
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