Interference of suppressor of cytokine signaling 3 promotes epithelial-mesenchymal transition in MHCC97H cells.
Interference of suppressor of cytokine signaling 3 promotes epithelial-mesenchymal transition in MHCC97H cells.
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DOI：
10.3748/wjg.v19.i6.866
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发表时间：
2013-02
影响因子：
4.3
通讯作者：
Yuan Ji;Zhi-Dong Wang;Zong-fang Li;Ke Li
中科院分区：
文献类型：
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作者：
Yuan Ji;Zhi-Dong Wang;Zong-fang Li;Ke Li
目的探讨细胞因子信号转导抑制因子3(SOCS3)沉默在人肝癌MHCC97H细胞上皮-间充质转化(EMT)中的作用。方法将SOCS3小干扰RNA(SiRNA)瞬时导入MHCC97H细胞。显微镜下观察转基因细胞的形态变化。免疫荧光法检测E-钙粘蛋白、波形蛋白和α-平滑肌肌动蛋白(α-SMA)的表达。采用Western blotting、实时定量聚合酶链式反应(RT-RT)等方法检测上皮细胞转化特征标志物(E-钙粘蛋白、波形蛋白、α-SMA和Snail)的蛋白表达和基因水平。酶联免疫吸附试验检测培养上清液中转化生长因子-β-1(TGFR-β-1)水平。结果转染SOCS3 siRNA的细胞形态由鹅卵石状向间充质梭形和梭形转变。SOCS3siRNA抑制MHCC97H细胞E-钙粘素的免疫荧光表达，而诱导Vimentin和α-SMA的免疫荧光表达。更重要的是，与阴性对照组相比，SOCS3的缺失导致MHCC97H细胞上皮标志物E-钙粘附素的表达降低(P<；0.05)，间质标志物Vimentin、α-SMA和转录因子Snail的表达增加(P<；0.05)。此外，与阴性对照组相比，SOCS3siRNA明显促进MHCC97H细胞分泌转化生长因子β1(200.20±29.02pg/mLvs 490.20±92.43pg/mLP<；0.05)。结论SOCS3沉默可通过改变MHCC97H细胞的表型特征和调节特征标志物来促进EMT的发生。
AIM To investigate the role of suppressor of cytokine signaling 3 (SOCS3) silencing in epithelial-mesenchymal transition (EMT) involved in a human hepatocellular carcinoma MHCC97H cell line. METHODS MHCC97H cells were transiently transfected with SOCS3 small-interfering RNA (siRNA). Morphological changes of the transfected cells were observed under microscope. Expressions of E-cadherin, Vimentin and α-smooth muscle actin (α-SMA) were identified with immunofluorescence. Furthermore, protein expressions and mRNA levels of characteristic markers of EMT (E-cadherin, Vimentin, α-SMA and Snail) were detected by Western blotting, quantitative real-time polymerase chain reaction. Transforming growth factor-β1 (TGF-β1) levels in the supernatant were measured with enzyme-linked immunosorbent assay. RESULTS The transfected cells with SOCS3 siRNA showed a morphological alteration from a typical cobblestone morphology to mesenchymal spindle-shaped and fusiform features. SOCS3 siRNA lessened immunofluorescent expression of E-cadherin, but elicited immunofluorescent expressions of Vimentin and α-SMA in MHCC97H cells. More importantly, compared with the negative control, depletion of SOCS3 resulted in the decrease of the epithelial marker E-cadherin (P < 0.05), and the increase of the mesenchymal markers Vimentin and α-SMA and the transcription factor Snail in MHCC97H cells (P < 0.05). Moreover, compared with the negative control, SOCS3 siRNA evidently enhanced TGF-β1 secretion in MHCC97H cells (200.20 ± 29.02 pg/mL vs 490.20 ± 92.43 pg/mL, P < 0.05). CONCLUSION SOCS3 silencing is able to promote EMT in MHCC97H cells via changing the phenotypic characteristics and modulating the characteristic markers.