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microRNA-145对TGF-β1诱导的人肾小管上皮细胞上皮间充质转化的影响
刘华, 何小解, 李国君, 丁庆雄, 梁晚霞, 樊娟
中国当代儿科杂志 ›› 2017, Vol. 19 ›› Issue (6) : 712-718.
PDF(2317 KB)
PDF(2317 KB)
microRNA-145对TGF-β1诱导的人肾小管上皮细胞上皮间充质转化的影响
Effects of microRNA-145 on epithelial-mesenchymal transition of TGF-β1-induced human renal proximal tubular epithelial cells
目的 研究microRNA-145（miR-145）对转化生长因子（TGF）-β1诱导人肾小管上皮细胞（HK-2）上皮间充质转化（EMT）的影响。方法 人工合成miR-145基因序列，构建真核重组质粒pCMV-miR-145。以未处理HK-2细胞为对照组；以TGF-β1处理HK-2细胞为TGF-β1组；以pCMV-myc空白质粒转染后经TGF-β1处理HK-2细胞为TGF-β1空白质粒组；以pCMV-miR-145质粒转染后经TGF-β1处理HK-2细胞为TGF-β1+miR-145组。采用实时荧光定量PCR检测miR-145表达。采用Western blot检测TGF-β/Smad信号传导蛋白TGF-β1、Smad3、Smad2/3、p-Smad2/3，以及EMT生物标记物蛋白α-平滑肌肌动蛋白（α-SMA）、纤维连接蛋白（FN）和I型胶原蛋白（ColⅠ）的表达水平。采用ELISA法检测培养细胞上清液中FN和Col I的含量。结果 miR-145表达质粒构建成功，重组质粒有效转染TGF-β1诱导HK-2细胞。与对照组比较，TGF-β1+miR-145组细胞中miR-145表达上调（P < 0.01）；与对照组和TGF-β1+miR-145组比较，TGF-β1组和TGF-β1空白质粒组细胞中miR-145表达均下降（P < 0.01）。与TGF-β1组和TGF-β1空白质粒组比较，TGF-β1+miR-145组细胞内TGF-β1、Smad3、Smad2/3和p-Smad2/3蛋白表达量减少（P < 0.05）；α-SMA、FN、ColⅠ蛋白表达亦明显减少（P < 0.05）；TGF-β1+miR-145组培养液上清中FN、ColⅠ含量减少（P < 0.05）。结论 miR-145参与TGF-β1处理HK-2细胞EMT的调控。miR-145可能通过抑制TGF-β依赖的Smad信号通路活性，从而抑制肾小管EMT。
Objective To investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells. Methods The gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups:control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA. Results pCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P < 0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P < 0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P < 0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P < 0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P < 0.05). Conclusions miR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.
microRNA-145 / TGF-β/Smad信号通路 / 上皮间充质转化 / 人肾小管上皮细胞
microRNA-145 / TGF-β/Smad signaling pathway / Epithelial-mesenchymal transition / Human proximal tubular epithelial cell
[1] Eddy AA, Neilson EG. Chronic kidney disease progression[J]. J Am Soc Nephrol, 2006, 17(11):2964-2966.
[2] Wang Y, Wang B, Du F, et al. Epigallocatechin-3-gallate attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in mice[J]. J Histochem Cytochem, 2015, 63(4):270-279.
[3] Sugiyama F, Kobayashi N, Ishikawa M, et al. Renoprotective mechanisms of telmisartan on renal injury and inflammation in SHRSP.Z-Leprfa/IzmDmcr rats[J]. Clin Exp Nephrol, 2013, 17(4):515-524.
[4] Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis[J]. J Clin Invest, 2002, 110(3):341-350.
[5] Huang Y, Tong J, He F, et al. miR-141 regulates TGF-β1-induced epithelial-mesenchymal transition through repression of HIPK2 expression in renal tubular epithelial cells[J]. Int J Mol Med, 2015, 35(2):311-318.
[6] Tang O, Chen XM, Shen S, et al. MiRNA-200b represses transforming growth factor-β1-induced EMT and fibronectin expression in kidney proximal tubular cells[J]. Am J Physiol Renal Physiol, 2013, 304(10):F1266-F1273.
[7] Wang JY, Gao YB, Zhang N, et al. miR-21 overexpression enhances TGF-β1-induced epithelial-to-mesenchymal transition by target smad7 and aggravates renal damage in diabetic nephropathy[J]. Mol Cell Endocrinol, 2014, 392(1-2):163-172.
[8] Kojima S, Enokida H, Yoshino H, et al. The tumor-suppressive microRNA-143/145 cluster inhibits cell migration and invasion by targeting GOLM1 in prostate cancer[J]. J Hum Genet, 2014, 59(2):78-87.
[9] Rangrez AY, Massy ZA, Metzinger-Le Meuth V, et al. miR-143 and miR-145:molecular keys to switch the phenotype of vascular smooth muscle cells[J]. Circ Cardiovasc Genet, 2011, 4(2):197-205.
[10] Zhang C. MicroRNA-145 in vascular smooth muscle cell biology:a new therapeutic target for vascular disease[J]. Cell Cycle, 2009, 8(21):3469-3473.
[11] Yang S, Cui H, Xie N, et al. miR-145 regulates myofibroblast differentiation and lung fibrosis[J]. FASEB J, 2013, 27(6):2382-2391.
[12] Megiorni F, Cialfi S, Cimino G, et al. Elevated levels of miR-145 correlate with SMAD3 down-regulation in cystic fibrosis patients[J]. J Cyst Fibros, 2013, 12(6):797-802.
[13] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method[J]. Nat Protoc, 2008, 3(6):1101-1108.
[14] Lan HY. Diverse roles of TGF β/Smads in renal fibrosis and inflammation[J]. Int J Biol Sci, 2011, 7(7):1056-1067.
[15] Meng XM, Chung AC, Lan HY. Role of the TGF β/BMP 7/Smad pathways in renal diseases[J]. Clin Sci (Lond), 2013, 124(4):243-254.
[16] Nawshad A, Hay ED. TGFβ3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development[J]. J Cell Biol, 2003, 163(6):1291-1301.
[17] Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[18] Yi R, O'Carroll D, Pasolli HA, et al. Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs[J]. Nat Genet, 2006, 38(3):356-362.
[19] Esquela Kerscher A, Slack FJ. Oncomirs-microRNAs with a role in cancer[J]. Nat Rev Cancer, 2006, 6(4):259-269.
[20] Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[J]. Nat Cell Biol, 2008, 10(5):593-601.
[21] Chen CH, Cheng CY, Chen YC, et al. MicroRNA-328 inhibits renal tubular cell epithelial-to-mesenchymal transition by targeting the CD44 in pressure-induced renal fibrosis[J]. PLoS One, 2014, 9(6):e99802.
[22] Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition[J]. J Clin Invest, 2009, 119(6):1420-1428.
[23] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J Am Soc Nephrol, 2010, 21(2):212-222.
