miR133a通过下调Gdnf基因表达抑制输尿管芽的发育
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摘要
该研究预测并验证了在后肾间充质细胞中mi R133a对输尿管芽发育关键因子Gdnf(glial cell line derived neurotrophic factor)基因表达的调控作用,探讨了其抑制输尿管芽发育的作用。通过生物信息学分析发现,Gdnf m RNA 3′非翻译区(3′untranslated regions,3′UTR)在不同物种间高度保守,并且存在mi R133a等的结合位点。双荧光素酶报告基因实验显示,mi R133a可以结合在Gdnf m RNA 3′UTR上。Real-time PCR和Western blot结果证实,mi R133a显著地抑制Gdnf的m RNA水平和蛋白质水平。解离重聚胚肾离体培养实验结果显示,mi R133a抑制输尿管芽的发育。该研究结果表明,mi R133a通过靶向结合于Gdnf m RNA的3′UTR,下调Gdnf基因的表达,从而抑制输尿管芽的发育。
The present study predicted and testified that miR133 a could regulate glial cell line derived neurotrophic factor(Gdnf), a protein generated by metanephrogenic mesenchyme(MM), which is of great importance in the development of ureteric bud(UB) and investigated inhibitory effect of miR133 a towards the development of UB. Bioinformatics analysis displayed the high conservation of the Gdnf m RNA 3′ untranslated regions(3′UTR) between different species and predicted that miR133 a could probably bind to it. The results of luciferase assay confirmed that miR133 a could directly target Gdnf m RNA 3′UTR. The results of Real-time PCR and Western blot revealed that miR133 a down-regulated the expression of Gdnf both at m RNA and protein levels. Furthermore, we found significant reduction in the ability of miR133 a treated cells to contribute to ureteric bud formation in embryonic kidney rudiments culture system. Taken together, our results suggest that miR133 a might inhibit the formation of ureteric bud through its binding to complementary sequences in the Gdnf m RNA 3′UTR.
The present study predicted and testified that miR133 a could regulate glial cell line derived neurotrophic factor(Gdnf), a protein generated by metanephrogenic mesenchyme(MM), which is of great importance in the development of ureteric bud(UB) and investigated inhibitory effect of miR133 a towards the development of UB. Bioinformatics analysis displayed the high conservation of the Gdnf m RNA 3′ untranslated regions(3′UTR) between different species and predicted that miR133 a could probably bind to it. The results of luciferase assay confirmed that miR133 a could directly target Gdnf m RNA 3′UTR. The results of Real-time PCR and Western blot revealed that miR133 a down-regulated the expression of Gdnf both at m RNA and protein levels. Furthermore, we found significant reduction in the ability of miR133 a treated cells to contribute to ureteric bud formation in embryonic kidney rudiments culture system. Taken together, our results suggest that miR133 a might inhibit the formation of ureteric bud through its binding to complementary sequences in the Gdnf m RNA 3′UTR.
引文
1 Pillai RS,Bhattacharyya SN,Filipowicz W.Repression of protein synthesis by mi RNAs:How many mechanisms?Trends Cell Biol2007;17(3):118-26.
2 GarofaloM,CondorelliGL,CroceCM,CondorelliG.Micro RNAs as regulators of death receptors signaling.Cell Death Differ 2010;17(2):200-8.
3 Yosypiv IV.Renin-angiotensin system-growth factor cross-talk:A novel mechanism for ureteric bud morphogenesis.Pediatr Nephrol 2009;24(6):1113-20.
4 Basson MA,Watson-Johnson J,Shakya R,Akbulut S,Hyink D,Costantini FD,et al.Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1.Dev Biol 2006;299(2):466-77.
5 Pichel JG,Shen L,Sheng HZ,Granholm AC,Drago J,Grinberg A,et al.Defects in enteric innervation and kidney development in mice lacking GDNF.Nature 1996;382(6586):73-6.
6 Moore MW,Klein RD,Farinas I,Sauer H,Armanini M,Phillips H,et al.Renal and neuronal abnormalities in mice lacking GDNF.Nature 1996;382(6586):76-9.
7 Costantini F.Renal branching morphogenesis:Concepts,questions,and recent advances.Differentiation 2006;74(7):402-21.
8 Costantini F,Shakya R.GDNF/Ret signaling and the development of the kidney.Bioessays 2006;28(2):117-27.
9 Bryksin AV,Matsumura I.Overlap extension PCR cloning:A simple and reliable way to create recombinant plasmids.Biotechniques 2010;48(6):463-5.
10 Unbekandt M,Davies JA.Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues.Kidney Int 2010;77(5):407-16.
11 Sakr M,Takino T,Sabit H,Nakada M,Li Z,Sato H.mi R-150-5p and mi R-133a suppress glioma cell proliferation and migration through targeting membrane-type-1 matrix metalloproteinase.Gene 2016;587(2):155-62.
12 Tao J,Wu D,Xu B,Qian W,Li P,Lu Q,et al.micro RNA-133inhibits cell proliferation,migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor.Oncol Rep 2012;27(6):1967-75.
13 Suzuki S,Yokobori T,Tanaka N,Sakai M,Sano A,Inose T,et al.CD47 expression regulated by the mi R-133a tumor suppressor is a novel prognostic marker in esophageal squamous cell carcinoma.Oncol Rep 2012;28(2):465-72.
14 Wang J,Li J,Guo F,Yan Y.Micro RNA-133a inhibits the malignant behavior of glioma via downregulation of matrix metallopeptidase 9.Mol Med Rep 2016;13(4):3220-6.
15 Ma J,Wang T,Guo R,Yang X,Yin J,Yu J,et al.Micro RNA133a and micro RNA326 cocontribute to hepatocellular carcinoma5 fluorouracil and cisplatin sensitivity by directly targeting Bcell lymphomaextra large.Mol Med Rep 2015;12(4):6235-40.
16 Yamamoto N,Nishikawa R,Chiyomaru T,Goto Y,Fukumoto I,Usui H,et al.The tumor-suppressive micro RNA-1/133a cluster targets PDE7A and inhibits cancer cell migration and invasion in endometrial cancer.Int J Oncol 2015;47(1):325-34.
2 GarofaloM,CondorelliGL,CroceCM,CondorelliG.Micro RNAs as regulators of death receptors signaling.Cell Death Differ 2010;17(2):200-8.
3 Yosypiv IV.Renin-angiotensin system-growth factor cross-talk:A novel mechanism for ureteric bud morphogenesis.Pediatr Nephrol 2009;24(6):1113-20.
4 Basson MA,Watson-Johnson J,Shakya R,Akbulut S,Hyink D,Costantini FD,et al.Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1.Dev Biol 2006;299(2):466-77.
5 Pichel JG,Shen L,Sheng HZ,Granholm AC,Drago J,Grinberg A,et al.Defects in enteric innervation and kidney development in mice lacking GDNF.Nature 1996;382(6586):73-6.
6 Moore MW,Klein RD,Farinas I,Sauer H,Armanini M,Phillips H,et al.Renal and neuronal abnormalities in mice lacking GDNF.Nature 1996;382(6586):76-9.
7 Costantini F.Renal branching morphogenesis:Concepts,questions,and recent advances.Differentiation 2006;74(7):402-21.
8 Costantini F,Shakya R.GDNF/Ret signaling and the development of the kidney.Bioessays 2006;28(2):117-27.
9 Bryksin AV,Matsumura I.Overlap extension PCR cloning:A simple and reliable way to create recombinant plasmids.Biotechniques 2010;48(6):463-5.
10 Unbekandt M,Davies JA.Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues.Kidney Int 2010;77(5):407-16.
11 Sakr M,Takino T,Sabit H,Nakada M,Li Z,Sato H.mi R-150-5p and mi R-133a suppress glioma cell proliferation and migration through targeting membrane-type-1 matrix metalloproteinase.Gene 2016;587(2):155-62.
12 Tao J,Wu D,Xu B,Qian W,Li P,Lu Q,et al.micro RNA-133inhibits cell proliferation,migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor.Oncol Rep 2012;27(6):1967-75.
13 Suzuki S,Yokobori T,Tanaka N,Sakai M,Sano A,Inose T,et al.CD47 expression regulated by the mi R-133a tumor suppressor is a novel prognostic marker in esophageal squamous cell carcinoma.Oncol Rep 2012;28(2):465-72.
14 Wang J,Li J,Guo F,Yan Y.Micro RNA-133a inhibits the malignant behavior of glioma via downregulation of matrix metallopeptidase 9.Mol Med Rep 2016;13(4):3220-6.
15 Ma J,Wang T,Guo R,Yang X,Yin J,Yu J,et al.Micro RNA133a and micro RNA326 cocontribute to hepatocellular carcinoma5 fluorouracil and cisplatin sensitivity by directly targeting Bcell lymphomaextra large.Mol Med Rep 2015;12(4):6235-40.
16 Yamamoto N,Nishikawa R,Chiyomaru T,Goto Y,Fukumoto I,Usui H,et al.The tumor-suppressive micro RNA-1/133a cluster targets PDE7A and inhibits cancer cell migration and invasion in endometrial cancer.Int J Oncol 2015;47(1):325-34.