Snail、Slug基因RNA干扰对肾小管上皮细胞转分化的作用
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摘要
背景和目的:
肾间质纤维化是各种慢性肾脏疾病的最终共同途径。近来发现,肾小管上皮细胞间质转分化(epithelial to mesenchymal transition,EMT)是肾间质纤维化发生发展的重要机制。肾小管上皮细胞转分化是一个有序的发生过程,E-cadherin介导的细胞-细胞的粘附功能丧失被认为是EMT的关键步骤。EMT最早期改变就是E-cadherin的表达抑制,随着E-cadherin的丢失,细胞间的紧密连接遭到破坏,使上皮细胞失去粘附特性。Snail、Slug基因属于锌指转录因子超家族成员,可以通过与靶基因的E-box结构域结合从而抑制其转录。在多个细胞系统研究中发现,Snail通过与E-cadherin启动元件E-box结合,对E-cadherin的转录进行直接抑制,下调其表达,从而使上皮细胞间失去粘附,促进EMT的发生,参与胚胎发育、肿瘤转移及组织纤维化过程。
EMT信号系统中多个重要信号途径如转化生长因子-β1-Smad、Wnt/β-catenin/淋巴增强因子、Ras/MAPK/细胞外调节蛋白激酶通路等均可调节调节Snail与Slug基因在细胞内的表达表达。但Snail及Slug在人肾小管EMT过程中作用机制的研究尚未见报道。本研究拟采用siRNA技术,探讨slug与snail基因在EMT中的作用机制,为防治肾纤维化提供新的思路。
方法:
1.针对Snail、Slug基因mRNA的RNA干扰质粒的构建根据人Snail、Slug基因的mRNA,采用RNAi设计软件,进行全基因组扫描和序列同源性分析,设计针对Genbank中Snail、Slug基因的特异siRNA,同时设计序列相同的阴性对照组。采用siRNA表达载体的方法体外制备人类Snail基因与Slug基因的siRNA真核表达载体。用阳性脂质体LipofectamineTM2000进行HKCs的siRNA转染,倒置显微镜下观察细胞形态。
2.实验分组:将HKCs分组如下:①对照组(C组):用无血清培养基(free serum medium,FSM)培养48h;②TGF-β1培养处理HKCs组(T组):用含TGF-β1(20ng/ml)的FSM培养HKCs 48h;③TGF-β1培养处理HKCs-siRNA组(T+siR组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siRNA 48h为T+siRa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siRNA 48h为T+siRb亚组;④TGF-β1培养处理HKCs-siC组(T+siC组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siC 48h为T+siCa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siC 48h为T+siCb亚组。
3.RT-PCR方法各组细胞α-SMA、E-cadherin、Snail及Slug基因表达水平。
4.Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug在蛋白水平的表达。
5.免疫荧光检测各组HKCs的E-cadherin及α-SMA表达。
结果:
1.分别成功构建Snail、Slug基因的siRNA重组质粒,用阳性脂质体Lipofect amineTM2000转染细胞,转染效率达60%。
2.构建TGF-β1诱导的EMT体外细胞培养模型,倒置显微镜下观察细胞形态:C组呈铺路石样紧密排列生长;T组与siC组细胞均出现显著形态改变,类似于成纤维细胞呈长梭形,细胞间间距增大,多数细胞呈离散型生长;T+siRa组及T+siRb组细胞形态改变不明显,仅有少量细胞呈现长梭形;提示沉默Snail及Slug基因可抑制EMT的发生。
3.RT-PCR、Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug的表达:C组E-cadherin在基因和蛋白水平均有较强表达,α-SMA无表达,Snail与Slug表达均较弱;T组与siC组E-cadherin表达减弱,Snail、Slug表达明显增强,α-SMA表达明显增强;T+siRa组及T+siRb组E-cadherin表达增强,α-SMA表达较弱;提示抑制Snail及Slug基因可重建E-cadherin表达。
4.免疫荧光观察细胞E-cadherin、α-SMA的表达:C组HKCs细胞膜和胞浆E-cadherin表达均为阳性,而α-SMA表达为阴性;T组与siC组均可见E-cadherin表达显著下降,而α-SMA表达为明显增加;T+siRa组与T+siRb组E-cadherin表达增强,α-SMA表达较弱。
结论:
1.RNA干扰技术可沉默Snail、Slug基因的表达,从而阻断其生物学作用;
2.Snail、Slug基因与EMT密切相关,抑制Snail、Slug基因的表达可显著上调E-cadherin的表达,从而抑制EMT发生;
3.Snail、Slug基因可作为新靶点,在EMT防治中起重要作用。
Background and objective:
Renal interstitial fibrosis is the final common pathway of all chronic kidney disease. Recently, emerging evidences indicated that the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells were key mechanism in the development of renal interstitial fibrosis. The EMT of renal tubular epithelial cells is considered to be an orderly process, and the loss of E-cadherin-mediated intercellular adhesion function is considered to be a key step in EMT. The earliest change of EMT is the inhibition of E-cadherin expression, as the loss of E-cadherin, intercellular tight junctions would be destroyed which could ultimately lead to the lose of adhesion properties of epithelial cells. Snail and Slug gene are members of zinc-finger transcription factors family, and combined with E-box to inhibit transcription of target genes. Studies in multiple cell systems found that Snail could combine with E-cadherin starting element E-box to directly inhibit the transcription of E-cadherin, and down-regulate its expression, then lead to the loss of adhesion among epithelial cells, ultimately promote the occurrence of EMT, and participate in the process of embryonic development, tumor metastasis as well as tissue fibrosis.
Many important signaling pathway in the signaling systems of EMT affected the expressions of Snail and Slug genes in cells. For example, it has been demonstred that transforming growth factor-β1 (TGF-β1) - Smad, Wnt/β- catenin/LEF and Ras/MAPK/ERK pathways could regulate the transcription level of Snail and Slug genes although the mechanisms remain unclear. As two members in the same family, although there is a high degree of homology between Snail and Slug, their functions were different in different organs. Up to date, there was no report about the mechanisms of Snail and Slug in human renal tubular EMT. In this study siRNA technology was applied to examine the role and related mechanisms of Snail and Slug genes in EMT.
Methods:
1. Based on the sequence of human Snail and Slug mRNA, we designed Snail-siRNA and Slug-siRNA by using the software of RNAi Designer for genome wide scanning and sequence homology analysis. A negative control with identical sequence but not targeting at Snail and Slug was also included. The method of expression vector was used to construct the eukaryotic expression vector of Snail-siRNA and Slug-siRNA in vitro. LipofectamineTM2000 was used to transfected siRNA into HKCs. MorpHological characteristics were observed under the inverted microscope.
2. Human renel tubular epithelial cells(HKCs) were divided into four groups:①Control group(C):The HKCs were treated with FSM for 48 hours;②TGF-β1 treated HKCs group(T):The HKCs were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours;③TGF-β1 treated HKCs-siRNA group(T+siR): The HKCs-snail-siRNA were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours(T+siRa); The HKCs-slug-siRNA were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours (T+siRb);④TGF-β1 treated HKCs-siC group(T+siC): The HKCs-snail-siC were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours(T+siCa); The HKCs-slug-siC were co-incubated with TGF-β1 (20ng/ml) and FSM for 48 hours (T+siCb)
3. The mRNA expression of E-cadherin,α-SMA, Snail and Slug was determined by reverse transcription polymerase chain reaction (RT-PCR).
4. The protein expression of E-cadherin,α-SMA, Snail and Slug was determined by Western blot.
5. The expression of E-cadherin andα-SMA were observed by confocal laser scanning microscope after immunofluorescence.
Results:
1. The siRNA eukaryotic expression vector of Snail and Slug gene were constructed successfully which confered by Lipofectamine~(TM)2000 transfect HKCs with a high efficiency of transfection (60%).
2. The cell model of EMT was induced by TGF-β1 in vitro, MorpHological characteristics were observed under the inverted microscope: C group: the shape of cells showed a classic cobblestone morpHology and grew in a compact mode; T group and T+siC group cells becoming elongated in shape, and losing their cobblestone monolayer pattern; the shape of cells changes indistinctively in T+siRa and T+siRb group, cells showed a classic cobblestone morpHology, similar to Control group. The results showed that inhibition of the expression of Snail and Slug gene could prevent the EMT progress.
3. The levels of E-cadherin,α-SMA, Snail and Slug were determined by RT-PCR and Western blot. The results showed that the expression of Snail, Slug andα-SMA was significantly increased and the expression of E-cadherin was significantly decreased in T group and T+siC group vs C group; while the expression of Snail, Slug andα-SMA were significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group. The results also conviced that expression of Snail and Slug were negative to the expression of E-cadherin.
4. The expression of E-cadherin andα-SMA were further detected by immunofluorescence:α-SMA was significantly increased and the expression of E-cadherin was significantly decreased in the T group vs C group and T+siC group; whileα-SMA expression was significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group.
Conclusion:
1. The expression of Snail and Slug gene can be silenced by RNA intereference, also their biological function were bloked.
2. Snail and Slug gene were highly associated with EMT; the silencence of Snail and Slug gene could prevent the EMT progress and its downstream effects.
3. Snail and Slug gene could be a new target for the prevention and treatment of renal fibrosis.
肾间质纤维化是各种慢性肾脏疾病的最终共同途径。近来发现,肾小管上皮细胞间质转分化(epithelial to mesenchymal transition,EMT)是肾间质纤维化发生发展的重要机制。肾小管上皮细胞转分化是一个有序的发生过程,E-cadherin介导的细胞-细胞的粘附功能丧失被认为是EMT的关键步骤。EMT最早期改变就是E-cadherin的表达抑制,随着E-cadherin的丢失,细胞间的紧密连接遭到破坏,使上皮细胞失去粘附特性。Snail、Slug基因属于锌指转录因子超家族成员,可以通过与靶基因的E-box结构域结合从而抑制其转录。在多个细胞系统研究中发现,Snail通过与E-cadherin启动元件E-box结合,对E-cadherin的转录进行直接抑制,下调其表达,从而使上皮细胞间失去粘附,促进EMT的发生,参与胚胎发育、肿瘤转移及组织纤维化过程。
EMT信号系统中多个重要信号途径如转化生长因子-β1-Smad、Wnt/β-catenin/淋巴增强因子、Ras/MAPK/细胞外调节蛋白激酶通路等均可调节调节Snail与Slug基因在细胞内的表达表达。但Snail及Slug在人肾小管EMT过程中作用机制的研究尚未见报道。本研究拟采用siRNA技术,探讨slug与snail基因在EMT中的作用机制,为防治肾纤维化提供新的思路。
方法:
1.针对Snail、Slug基因mRNA的RNA干扰质粒的构建根据人Snail、Slug基因的mRNA,采用RNAi设计软件,进行全基因组扫描和序列同源性分析,设计针对Genbank中Snail、Slug基因的特异siRNA,同时设计序列相同的阴性对照组。采用siRNA表达载体的方法体外制备人类Snail基因与Slug基因的siRNA真核表达载体。用阳性脂质体LipofectamineTM2000进行HKCs的siRNA转染,倒置显微镜下观察细胞形态。
2.实验分组:将HKCs分组如下:①对照组(C组):用无血清培养基(free serum medium,FSM)培养48h;②TGF-β1培养处理HKCs组(T组):用含TGF-β1(20ng/ml)的FSM培养HKCs 48h;③TGF-β1培养处理HKCs-siRNA组(T+siR组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siRNA 48h为T+siRa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siRNA 48h为T+siRb亚组;④TGF-β1培养处理HKCs-siC组(T+siC组):用含TGF-β1(20ng/ml)的FSM培养HKCs-snail-siC 48h为T+siCa亚组,用含TGF-β1(20ng/ml)的FSM培养HKCs-slug-siC 48h为T+siCb亚组。
3.RT-PCR方法各组细胞α-SMA、E-cadherin、Snail及Slug基因表达水平。
4.Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug在蛋白水平的表达。
5.免疫荧光检测各组HKCs的E-cadherin及α-SMA表达。
结果:
1.分别成功构建Snail、Slug基因的siRNA重组质粒,用阳性脂质体Lipofect amineTM2000转染细胞,转染效率达60%。
2.构建TGF-β1诱导的EMT体外细胞培养模型,倒置显微镜下观察细胞形态:C组呈铺路石样紧密排列生长;T组与siC组细胞均出现显著形态改变,类似于成纤维细胞呈长梭形,细胞间间距增大,多数细胞呈离散型生长;T+siRa组及T+siRb组细胞形态改变不明显,仅有少量细胞呈现长梭形;提示沉默Snail及Slug基因可抑制EMT的发生。
3.RT-PCR、Western blot方法检测各组细胞α-SMA、E-cadherin、Snail及Slug的表达:C组E-cadherin在基因和蛋白水平均有较强表达,α-SMA无表达,Snail与Slug表达均较弱;T组与siC组E-cadherin表达减弱,Snail、Slug表达明显增强,α-SMA表达明显增强;T+siRa组及T+siRb组E-cadherin表达增强,α-SMA表达较弱;提示抑制Snail及Slug基因可重建E-cadherin表达。
4.免疫荧光观察细胞E-cadherin、α-SMA的表达:C组HKCs细胞膜和胞浆E-cadherin表达均为阳性,而α-SMA表达为阴性;T组与siC组均可见E-cadherin表达显著下降,而α-SMA表达为明显增加;T+siRa组与T+siRb组E-cadherin表达增强,α-SMA表达较弱。
结论:
1.RNA干扰技术可沉默Snail、Slug基因的表达,从而阻断其生物学作用;
2.Snail、Slug基因与EMT密切相关,抑制Snail、Slug基因的表达可显著上调E-cadherin的表达,从而抑制EMT发生;
3.Snail、Slug基因可作为新靶点,在EMT防治中起重要作用。
Background and objective:
Renal interstitial fibrosis is the final common pathway of all chronic kidney disease. Recently, emerging evidences indicated that the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells were key mechanism in the development of renal interstitial fibrosis. The EMT of renal tubular epithelial cells is considered to be an orderly process, and the loss of E-cadherin-mediated intercellular adhesion function is considered to be a key step in EMT. The earliest change of EMT is the inhibition of E-cadherin expression, as the loss of E-cadherin, intercellular tight junctions would be destroyed which could ultimately lead to the lose of adhesion properties of epithelial cells. Snail and Slug gene are members of zinc-finger transcription factors family, and combined with E-box to inhibit transcription of target genes. Studies in multiple cell systems found that Snail could combine with E-cadherin starting element E-box to directly inhibit the transcription of E-cadherin, and down-regulate its expression, then lead to the loss of adhesion among epithelial cells, ultimately promote the occurrence of EMT, and participate in the process of embryonic development, tumor metastasis as well as tissue fibrosis.
Many important signaling pathway in the signaling systems of EMT affected the expressions of Snail and Slug genes in cells. For example, it has been demonstred that transforming growth factor-β1 (TGF-β1) - Smad, Wnt/β- catenin/LEF and Ras/MAPK/ERK pathways could regulate the transcription level of Snail and Slug genes although the mechanisms remain unclear. As two members in the same family, although there is a high degree of homology between Snail and Slug, their functions were different in different organs. Up to date, there was no report about the mechanisms of Snail and Slug in human renal tubular EMT. In this study siRNA technology was applied to examine the role and related mechanisms of Snail and Slug genes in EMT.
Methods:
1. Based on the sequence of human Snail and Slug mRNA, we designed Snail-siRNA and Slug-siRNA by using the software of RNAi Designer for genome wide scanning and sequence homology analysis. A negative control with identical sequence but not targeting at Snail and Slug was also included. The method of expression vector was used to construct the eukaryotic expression vector of Snail-siRNA and Slug-siRNA in vitro. LipofectamineTM2000 was used to transfected siRNA into HKCs. MorpHological characteristics were observed under the inverted microscope.
2. Human renel tubular epithelial cells(HKCs) were divided into four groups:①Control group(C):The HKCs were treated with FSM for 48 hours;②TGF-β1 treated HKCs group(T):The HKCs were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours;③TGF-β1 treated HKCs-siRNA group(T+siR): The HKCs-snail-siRNA were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours(T+siRa); The HKCs-slug-siRNA were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours (T+siRb);④TGF-β1 treated HKCs-siC group(T+siC): The HKCs-snail-siC were co-incubated with TGF-β1(20ng/ml) and FSM for 48 hours(T+siCa); The HKCs-slug-siC were co-incubated with TGF-β1 (20ng/ml) and FSM for 48 hours (T+siCb)
3. The mRNA expression of E-cadherin,α-SMA, Snail and Slug was determined by reverse transcription polymerase chain reaction (RT-PCR).
4. The protein expression of E-cadherin,α-SMA, Snail and Slug was determined by Western blot.
5. The expression of E-cadherin andα-SMA were observed by confocal laser scanning microscope after immunofluorescence.
Results:
1. The siRNA eukaryotic expression vector of Snail and Slug gene were constructed successfully which confered by Lipofectamine~(TM)2000 transfect HKCs with a high efficiency of transfection (60%).
2. The cell model of EMT was induced by TGF-β1 in vitro, MorpHological characteristics were observed under the inverted microscope: C group: the shape of cells showed a classic cobblestone morpHology and grew in a compact mode; T group and T+siC group cells becoming elongated in shape, and losing their cobblestone monolayer pattern; the shape of cells changes indistinctively in T+siRa and T+siRb group, cells showed a classic cobblestone morpHology, similar to Control group. The results showed that inhibition of the expression of Snail and Slug gene could prevent the EMT progress.
3. The levels of E-cadherin,α-SMA, Snail and Slug were determined by RT-PCR and Western blot. The results showed that the expression of Snail, Slug andα-SMA was significantly increased and the expression of E-cadherin was significantly decreased in T group and T+siC group vs C group; while the expression of Snail, Slug andα-SMA were significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group. The results also conviced that expression of Snail and Slug were negative to the expression of E-cadherin.
4. The expression of E-cadherin andα-SMA were further detected by immunofluorescence:α-SMA was significantly increased and the expression of E-cadherin was significantly decreased in the T group vs C group and T+siC group; whileα-SMA expression was significantly decreased and E-cadherin expression was significantly increased in T+siRa and T+siRb group.
Conclusion:
1. The expression of Snail and Slug gene can be silenced by RNA intereference, also their biological function were bloked.
2. Snail and Slug gene were highly associated with EMT; the silencence of Snail and Slug gene could prevent the EMT progress and its downstream effects.
3. Snail and Slug gene could be a new target for the prevention and treatment of renal fibrosis.
引文
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21. Bolos V, Peinado H, Perez-Moreno MA, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. [J]. Cell Sci, 2003, 116(3): 499-511.
22. Radisky DC. Epithelial - mesenchymal transition. [J]. Cell Sci, 2005, 118 (19): 4325-4326.
23. Kallui R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Ivest, 2003, 112: 1776-1784.
24. Carver EA, Jiang R, Lan Y, et al. The mouse Snail gene encodes a key regulator of the epithelial-mesenchymal transition. Mol Cell Biol, 2001, 21:8184-8188.
25. Nieto MA, Sargent MG, Wilkinson DG, et al. Control of cell behavior during vertebrate development by Slug, a zinc fnger gene. Science, 1994, 264: 835-839.
26. Masszi A, Fan L, Rosivall L, et al. Integrity of cell-cell contacts is a critical regulator of TGF-beta1 induced epithelial-to-myofibroblast transition: role for beta-catenin. Am J Pathol, 2004, 165: 1955–1967.
27. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, et al. Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK. J Cell Biol, 2003, 163: 847-857.
28. Barrallo-Gimeno, M.A. Nieto, A. The Snail genes as inducers of cell movement and survival: implications in development and cancer. [J]. Development, 2005, 132: 3151–3161.
29. Jun Y, Toshiaki M, Mihoko T, et al. Snail1 is involved in the renal epithelial- mesenchymal transition. [J]. BBRC, 2007, 362: 63–68.
30. Ohkubo T, Ozawa M. The transcription factor Snail down-regulates the tight junction components independently of E-cadherin downregulation. J Cell Sci, 2004, 117: 1675-1685.
31. Cho HJ, Baek KE, Saika S, et al. Snail is required for transforming growth factor-β-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway. BBRC, 2007; 353: 337-343.
32. Sato M, Muragaki Y, Saika S, et al. Targeted disruption of TGF-b1/Smad3 signaling protects against renal tubulointerstitial fbrosis induced by unilateral ureteral obstruction. J Clin Invest, 2003, 112: 1486-1494.
33. Morita T, Mayanagi T, Sobue K. Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via Slug induction and actin remodeling. J Cell Biol, 2007, 179: 1027-1042.
34. Vallin J, Thuret R, Giacomello E, et al. Cloning and characterization of three Xenopus Slug promoters reveal direct regulation by Lef/beta-catenin signaling. J Biol Chem, 2001, 276: 30350–30358.
35. Hoot KE, Lighthall J, Han G, et al. Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J Clin Invest, 2008, 118: 2722-2732.
36. Agnes B, Cristina A D,Patrick H M, et al. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J, 2006, 25:5603–5613.
37. Zhou BP, Deng J, Xia W, et al. Dual regulation of Snail by GSK-3beta-mediated pHospHorylation in control of epithelial-mesenchymal transition. [J]. Nat Cell Biol, 2004, 6 (10): 913-915.
38.郑颖,张璟,卓文磊等. TGF-β1对人肾小管上皮细胞表达糖原合成激酶-3β的影响.重庆医学. 2007, 36(5): 414-416.
39. Come C, Magnino F, Bibeau F, et al. Snail and slug play distinct roles during breast carcinoma progression. [J]. Clin Cancer Res, 2006, 12 (18): 5395-5402.
40. Cano, A, Perez-Moreno, M.A., Rodrigo, I., et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2000, 2: 76–83.
41. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial pHenotype? [J] Nat Rev Cancer, 2000, 7 (6): 415-428.
42. Batlle E, Sancho E, Franci C, et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000, 2: 84-89.
43. Hajra KM, Chen DY, Fearon ER. The SLUG zinc-fnger protein represses E-cadherin in breast cancer. Cancer Res, 2002, 62: 1613-1618.
44. Damian M, Eliszbeth D, Bjorn R. Snail and Slug Promote Epithelial-Mesenchymal Transition Throughβ-Catenin-T-Cell Factor-4-dependent Expression of Transforming Growth Factor-β3. MBoC, 2008, 19(11): 4875-4887.
45. Maralice CS, Inbal S, Tamar BY, et al. Autoregulation of E-cadherin expression by cadherin–cadherin interactions: the roles ofβ-catenin signaling, Slug, and MAPK. J Cell Biol, 2003, 4: 847-857.
1. Vongwiwatana A, Tasanarong A, Rayner DC, et al. Epithelial to mesenchymal transition during late deterioration of human kidney transplants: the role of tubular cells in fibrogenesis. Am J Transplant 2005, 5: 1367–1374.
2. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005, 132: 3151–3161.
3. Manzanares M, Locascio A, Nieto M A. The increasing complexity of the Snail superfamily in metazoan evolution. [J]. Trends Genet, 2001, 17: 178-181.
4. Kataoka H, Murayama T, Kita T, et al. A novel Snail related transcription factor Smuc regulates basic helix–loop-helix transcription factor activities via specific E-box motifs. [J]. Nucleic Acids Res. 2000, 28: 626-633.
5. Nakayama H, Scott IC, Cross JC, et al. The transition to endore-duplication in tropHoblast giant cells is regulated by the mSNA zinc finger transcription factor. Dev. Biol. 1998, 199: 150–163.
6. Hemavathy K, Ashraf SI, Ip YT. Snail/Slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000, 257: 1-12.
7. Nieto MA. The Snail superfamily of zinc finger transcription factors. Nat Rev Mol Cell Biol. 2002, 3:155-166.
8. Battle E, Sancho E, Franci C, et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumor cells. [J]. Nat Cell Biol, 2002, 2(2): 84-92.
9. Nibu Y, Zhang H, Levine M, et al. Interaction of short-range repressors with DrosopHila CtBP in the embryo. Science, 1998, 280: 101–104.
10. Zavadil J, Cermak L, Nieves N, et al. Integration of TGF-beta/Smad and Jagged1 /Notch signalling in epithelial to mesenchymal transition. [J]. EMBO, 2004, 23 (5): 1155-1165.
11. Yang YC, Piek E, Zavadil J, et al. Hierarchical model of gene regulation by transforming growth factor beta. [J]. Proc NatlAcad Sci, 2003, 100 (18): 10269-10274.
12. Zhou BP, Deng J, Xia W, et al. Dual regulation of Snail by GSK-3beta-mediatedpHospHorylation in control of epithelial-mesenchymal transition. [J]. Nat Cell Biol, 2004, 6 (10): 913-915.
13. Perez MA, Locascio A, Cano A, et al. A new role for E12/E47 in the repression of E-cadherin expression and epithelial mesenchymal transition. J Biol Chem, 2001, 276: 27424-27431.
14. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development, 2005, 132: 3151–3161.
15. Radisky DC. Epithelial - mesenchymal transition. [J]. Cell Sci, 2005, 118 (19): 4325 - 4326.
16. Li Y, Yang J, Dai C, et al. Role for integrin- linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis. J Clin Invest, 2003, 112 (4): 503-516.
17. Dai C, Yang J, Liu Y. Transforming growth factor-β1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J B iol Chem, 2003, 278 (14): 12537-12545.
18. Baritaki S, Bonavida B. Viral Infection and Cancer: The NF-kappa B/Snail/RK IP Loop Regulates Target Cell Sensitivity to Apoptosis by Cytotoxic LympHocytes. Immunology, 2010, 30:31-46.
19. Jayesh JK, Rosemarie MC, Mediha H, et al. Protein kinase B/Akt activity is involved in renal TGF-β1-driven epithelial-mesenchymal transition in vitro and in vivo. Am J PHysiol Renal PHysiol, 2008, 295: 215-225.
20. Daisuke S, Yohei M, Tatsuyo N, et al. Amelioration of renal alterations in obese type 2 diabetic mice by vasohibin-1, a negative feedback regulator of angiogenesis. AJP - Renal PHysiol, 2011, 4: 873-886.
21. Rukstalis JM, Mariano U, Megan V, et al. Endocrine Pancreatic Cell Lines Transcription Factor Snail Modulates Hormone Expression in Established. Endocrinology, 2006, 147: 2997-3006.
22. Messai Y, Noman MZ, Derouiche A, et al. Cytokeratin 18 expression pattern correlates with renal cell carcinoma progression: Relationship with Snail. Int J oncology, 2010, 36:1145-1154.
23. Schwensen KG, Burgess JS, Graf NS, et al. Early Cyst Growth Is Associated with the Increased Nuclear Expression of Cyclin D1/Rb Protein in an Autosomal- Recessive Polycystic Kidney Disease Rat Model. NepHrology, 2011, 117: 93-103.
24. Hiesberger T, Shao X, Gourley E, et al. Mutation of hepatocyte nuclear factor-1 inhibits Pkhd1 gene expression and produces renal cysts in mice. J Clin Invest, 2004, 113: 814–825.
25. Gresh L, Fischer E, Reimann A, et al. A transcriptional network in polycystic kidney disease. EMBO J, 2004, 23: 1657–1668.
26. Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 2002, 110: 341–350.
27. Melk A. Transcriptional analysis of the molecular basis of human kidney aging cDNA microarray profiling. Kidney Int, 2005, 68:2667–2679.
28.陶光利,张璟,卓文磊等. RNA干扰抑制Slug表达在TGF-β1诱导的肾小管细胞间质转分化中的抑制作用.免疫学杂志. 2010, 26(10):872-875.
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8. Elbashir SM, Harborth J, Weber K, et al. Analysis of gene function insomatic mammalian cells using small interfering RNAs. [J]. Methods, 2002, 26(2): 199-213.
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12. Myslinski E, Ame JC, Krol A, et al. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acid Res. 2001,29(12): 2502-2509.
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14. Hata E, Katsuda K, et al. Characteristics and epidemiologic genotyping of StapHylococcus aureus isolates from bovine mastitic milk in Hokkaido Japan. J Vet Med Sci, 2006, 68(2): 165-170.
15.钱亚芳,谷满.阳离子脂质体在基因转染载体中的研究进展.中国药师. 2008, 11(9): 1041-1043.
16. Pebernard S, Iggo RD. Determinants of interferon-stimulated gene induction by RNAi vectors. Differention, 2004, 72(2-3): 103-111.
17.刘娜,严海东,李雪竹.质粒表达型小分子干扰RNA对人肾小管上皮细胞单核细胞趋化蛋白-1抑制作用的研究.中国实用内科杂志. 2007, 27(6): 449-451.
18.位红兰,曾锐,刘林等.高表达PTEN抑制TGF-β1诱导的肾小管上皮细胞转分化.华中科技大学学报. 2009, 38(6): 721-724.
19. Ioana ND, Mysore KP, Paul CN, et al. The TGFβ1-Induced Fibronectin in Human Renal Proximal Tubular Epithelial Cells Is p38 MAP Kinase Dependent and Smad Independent. NepHron Exp NepHrol, 2007, 105: 108-116.
20. Liu HK, Perrier S, Lipina C, et al. Functional characterisation of the regulation of CAAT enhancer binding protein alpHa by GSK-3 pHospHorylation of Threonines 222/226. BMC Mol Biol. 2006, 6: 14-26.
21. Bolos V, Peinado H, Perez-Moreno MA, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. [J]. Cell Sci, 2003, 116(3): 499-511.
22. Radisky DC. Epithelial - mesenchymal transition. [J]. Cell Sci, 2005, 118 (19): 4325-4326.
23. Kallui R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Ivest, 2003, 112: 1776-1784.
24. Carver EA, Jiang R, Lan Y, et al. The mouse Snail gene encodes a key regulator of the epithelial-mesenchymal transition. Mol Cell Biol, 2001, 21:8184-8188.
25. Nieto MA, Sargent MG, Wilkinson DG, et al. Control of cell behavior during vertebrate development by Slug, a zinc fnger gene. Science, 1994, 264: 835-839.
26. Masszi A, Fan L, Rosivall L, et al. Integrity of cell-cell contacts is a critical regulator of TGF-beta1 induced epithelial-to-myofibroblast transition: role for beta-catenin. Am J Pathol, 2004, 165: 1955–1967.
27. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, et al. Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK. J Cell Biol, 2003, 163: 847-857.
28. Barrallo-Gimeno, M.A. Nieto, A. The Snail genes as inducers of cell movement and survival: implications in development and cancer. [J]. Development, 2005, 132: 3151–3161.
29. Jun Y, Toshiaki M, Mihoko T, et al. Snail1 is involved in the renal epithelial- mesenchymal transition. [J]. BBRC, 2007, 362: 63–68.
30. Ohkubo T, Ozawa M. The transcription factor Snail down-regulates the tight junction components independently of E-cadherin downregulation. J Cell Sci, 2004, 117: 1675-1685.
31. Cho HJ, Baek KE, Saika S, et al. Snail is required for transforming growth factor-β-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway. BBRC, 2007; 353: 337-343.
32. Sato M, Muragaki Y, Saika S, et al. Targeted disruption of TGF-b1/Smad3 signaling protects against renal tubulointerstitial fbrosis induced by unilateral ureteral obstruction. J Clin Invest, 2003, 112: 1486-1494.
33. Morita T, Mayanagi T, Sobue K. Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via Slug induction and actin remodeling. J Cell Biol, 2007, 179: 1027-1042.
34. Vallin J, Thuret R, Giacomello E, et al. Cloning and characterization of three Xenopus Slug promoters reveal direct regulation by Lef/beta-catenin signaling. J Biol Chem, 2001, 276: 30350–30358.
35. Hoot KE, Lighthall J, Han G, et al. Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J Clin Invest, 2008, 118: 2722-2732.
36. Agnes B, Cristina A D,Patrick H M, et al. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J, 2006, 25:5603–5613.
37. Zhou BP, Deng J, Xia W, et al. Dual regulation of Snail by GSK-3beta-mediated pHospHorylation in control of epithelial-mesenchymal transition. [J]. Nat Cell Biol, 2004, 6 (10): 913-915.
38.郑颖,张璟,卓文磊等. TGF-β1对人肾小管上皮细胞表达糖原合成激酶-3β的影响.重庆医学. 2007, 36(5): 414-416.
39. Come C, Magnino F, Bibeau F, et al. Snail and slug play distinct roles during breast carcinoma progression. [J]. Clin Cancer Res, 2006, 12 (18): 5395-5402.
40. Cano, A, Perez-Moreno, M.A., Rodrigo, I., et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2000, 2: 76–83.
41. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial pHenotype? [J] Nat Rev Cancer, 2000, 7 (6): 415-428.
42. Batlle E, Sancho E, Franci C, et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000, 2: 84-89.
43. Hajra KM, Chen DY, Fearon ER. The SLUG zinc-fnger protein represses E-cadherin in breast cancer. Cancer Res, 2002, 62: 1613-1618.
44. Damian M, Eliszbeth D, Bjorn R. Snail and Slug Promote Epithelial-Mesenchymal Transition Throughβ-Catenin-T-Cell Factor-4-dependent Expression of Transforming Growth Factor-β3. MBoC, 2008, 19(11): 4875-4887.
45. Maralice CS, Inbal S, Tamar BY, et al. Autoregulation of E-cadherin expression by cadherin–cadherin interactions: the roles ofβ-catenin signaling, Slug, and MAPK. J Cell Biol, 2003, 4: 847-857.
1. Vongwiwatana A, Tasanarong A, Rayner DC, et al. Epithelial to mesenchymal transition during late deterioration of human kidney transplants: the role of tubular cells in fibrogenesis. Am J Transplant 2005, 5: 1367–1374.
2. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005, 132: 3151–3161.
3. Manzanares M, Locascio A, Nieto M A. The increasing complexity of the Snail superfamily in metazoan evolution. [J]. Trends Genet, 2001, 17: 178-181.
4. Kataoka H, Murayama T, Kita T, et al. A novel Snail related transcription factor Smuc regulates basic helix–loop-helix transcription factor activities via specific E-box motifs. [J]. Nucleic Acids Res. 2000, 28: 626-633.
5. Nakayama H, Scott IC, Cross JC, et al. The transition to endore-duplication in tropHoblast giant cells is regulated by the mSNA zinc finger transcription factor. Dev. Biol. 1998, 199: 150–163.
6. Hemavathy K, Ashraf SI, Ip YT. Snail/Slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000, 257: 1-12.
7. Nieto MA. The Snail superfamily of zinc finger transcription factors. Nat Rev Mol Cell Biol. 2002, 3:155-166.
8. Battle E, Sancho E, Franci C, et al. The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumor cells. [J]. Nat Cell Biol, 2002, 2(2): 84-92.
9. Nibu Y, Zhang H, Levine M, et al. Interaction of short-range repressors with DrosopHila CtBP in the embryo. Science, 1998, 280: 101–104.
10. Zavadil J, Cermak L, Nieves N, et al. Integration of TGF-beta/Smad and Jagged1 /Notch signalling in epithelial to mesenchymal transition. [J]. EMBO, 2004, 23 (5): 1155-1165.
11. Yang YC, Piek E, Zavadil J, et al. Hierarchical model of gene regulation by transforming growth factor beta. [J]. Proc NatlAcad Sci, 2003, 100 (18): 10269-10274.
12. Zhou BP, Deng J, Xia W, et al. Dual regulation of Snail by GSK-3beta-mediatedpHospHorylation in control of epithelial-mesenchymal transition. [J]. Nat Cell Biol, 2004, 6 (10): 913-915.
13. Perez MA, Locascio A, Cano A, et al. A new role for E12/E47 in the repression of E-cadherin expression and epithelial mesenchymal transition. J Biol Chem, 2001, 276: 27424-27431.
14. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development, 2005, 132: 3151–3161.
15. Radisky DC. Epithelial - mesenchymal transition. [J]. Cell Sci, 2005, 118 (19): 4325 - 4326.
16. Li Y, Yang J, Dai C, et al. Role for integrin- linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis. J Clin Invest, 2003, 112 (4): 503-516.
17. Dai C, Yang J, Liu Y. Transforming growth factor-β1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J B iol Chem, 2003, 278 (14): 12537-12545.
18. Baritaki S, Bonavida B. Viral Infection and Cancer: The NF-kappa B/Snail/RK IP Loop Regulates Target Cell Sensitivity to Apoptosis by Cytotoxic LympHocytes. Immunology, 2010, 30:31-46.
19. Jayesh JK, Rosemarie MC, Mediha H, et al. Protein kinase B/Akt activity is involved in renal TGF-β1-driven epithelial-mesenchymal transition in vitro and in vivo. Am J PHysiol Renal PHysiol, 2008, 295: 215-225.
20. Daisuke S, Yohei M, Tatsuyo N, et al. Amelioration of renal alterations in obese type 2 diabetic mice by vasohibin-1, a negative feedback regulator of angiogenesis. AJP - Renal PHysiol, 2011, 4: 873-886.
21. Rukstalis JM, Mariano U, Megan V, et al. Endocrine Pancreatic Cell Lines Transcription Factor Snail Modulates Hormone Expression in Established. Endocrinology, 2006, 147: 2997-3006.
22. Messai Y, Noman MZ, Derouiche A, et al. Cytokeratin 18 expression pattern correlates with renal cell carcinoma progression: Relationship with Snail. Int J oncology, 2010, 36:1145-1154.
23. Schwensen KG, Burgess JS, Graf NS, et al. Early Cyst Growth Is Associated with the Increased Nuclear Expression of Cyclin D1/Rb Protein in an Autosomal- Recessive Polycystic Kidney Disease Rat Model. NepHrology, 2011, 117: 93-103.
24. Hiesberger T, Shao X, Gourley E, et al. Mutation of hepatocyte nuclear factor-1 inhibits Pkhd1 gene expression and produces renal cysts in mice. J Clin Invest, 2004, 113: 814–825.
25. Gresh L, Fischer E, Reimann A, et al. A transcriptional network in polycystic kidney disease. EMBO J, 2004, 23: 1657–1668.
26. Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 2002, 110: 341–350.
27. Melk A. Transcriptional analysis of the molecular basis of human kidney aging cDNA microarray profiling. Kidney Int, 2005, 68:2667–2679.
28.陶光利,张璟,卓文磊等. RNA干扰抑制Slug表达在TGF-β1诱导的肾小管细胞间质转分化中的抑制作用.免疫学杂志. 2010, 26(10):872-875.