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目的探讨miR-205对人食管鳞癌干细胞转移、分化能力的影响。方法采用RT-PCR检测20例食管鳞癌细胞、20例正常食管上皮细胞、20例食管非典型增生上皮细胞和20例食管鳞癌干细胞中miR-205、锌指E-盒结合同源异形盒1(ZEB1)、锌指E-盒结合同源异形盒2(ZEB2)以及E-钙黏蛋白(E-cadherin)的mRNA表达情况。分别利用脂质体Lipofectamine2000将人工合成的miR-205、抗miR-205的抑制剂瞬时转染食管鳞癌干细胞,采用RT-PCR测定食管鳞癌干细胞中miR-205、ZEB1、ZEB2以及E-cadherin的mRNA转录水平,并采用Western blot方法检测食管鳞癌干细胞中miR-205、ZEB1、ZEB2以及E-cadherin蛋白的表达。Transwell小室法和划痕实验检测转染的食管鳞癌干细胞的侵袭及迁移能力。结果食管鳞癌细胞和食管鳞癌干细胞中miR-205、ZEB1和ZEB2的mRNA表达相对强度均高于正常食管上皮细胞和食管非典型增生上皮细胞;食管鳞癌细胞和食管鳞癌干细胞中E-cadherin的mRNA表达相对强度则低于正常食管上皮细胞和食管非典型增生上皮细胞(P<0.05)。人工合成的miR-205瞬时转染后,食管鳞癌干细胞中ZEB1和ZEB2的mRNA表达相对强度和蛋白表达水平均较转染前下降,miR-205和E-cadherin的mRNA表达相对强度、蛋白表达水平则均较转染前升高,细胞侵袭及迁移能力降低(P<0.05)。抗miR-205的抑制剂瞬时转染后,食管鳞癌干细胞中ZEB1和ZEB2的mRNA表达相对强度和蛋白表达水平均较转染前升高,miR-205和E-cadherin的mRNA表达相对强度和蛋白表达水平则均较转染前降低,细胞侵袭及迁移能力升高(P<0.05)。结论 miR-205在人食管鳞癌细胞中的表达上调,且可能通过下调ZEB1和ZEB2的mRNA表达相对强度和蛋白表达水平和上调E-cadherin的mRNA表达相对强度和蛋白表达水平起到抑癌基因的作用,上调miR-205表达可能是治疗食管鳞癌的有效方法。
Objective To investigate the effect of miR-205 on the metastasis and differentiation of human esophageal squamous carcinoma stem cells. Methods RT-PCR was used to detect the expression of miR-205 and zinc finger E-box in 20 esophageal squamous cell carcinoma cells, 20 normal esophageal epithelial cells, 20 esophageal atypical hyperplastic epithelial cells and 20 esophageal squamous cell carcinoma stem cells. 1, ZEB1, ZEB2 and E-cadherin. The synthetic miR-205 and anti-miR-205 inhibitors were transiently transfected into esophageal squamous carcinoma stem cells using lipofectamine 2000. The expressions of miR-205, ZEB1, ZEB2 and E-cadherin in esophageal squamous carcinoma stem cells were detected by RT- MRNA expression levels of miR-205, ZEB1, ZEB2 and E-cadherin in esophageal squamous carcinoma stem cells were detected by Western blot. Transwell chamber assay and scratch assay were used to detect the invasion and migration of transfected esophageal squamous carcinoma stem cells. Results The relative intensity of mRNA expression of miR-205, ZEB1 and ZEB2 in esophageal squamous cell carcinoma and esophageal squamous cell carcinoma stem cells was higher than that in normal esophageal epithelial cells and esophageal atypical hyperplasia epithelial cells. In esophageal squamous cell carcinoma and esophageal squamous cell carcinoma stem cells, The relative mRNA expression of cadherin was lower than that of normal esophageal epithelial cells and esophageal atypical hyperplasia epithelial cells (P <0.05). The relative intensity and protein expression of ZEB1 and ZEB2 mRNA in esophageal squamous carcinoma stem cells were lower than those before transfection after transiently transfection of synthetic miR-205. The relative intensity of miR-205 and E-cadherin mRNA expression, protein expression The levels were higher than before transfection, cell invasion and migration decreased (P <0.05). After transient transfection with anti-miR-205 inhibitor, the relative intensity and protein expression of ZEB1 and ZEB2 in esophageal squamous carcinoma stem cells were higher than those before transfection, and the relative intensity of mRNA expression of miR-205 and E-cadherin The protein expression levels were lower than before transfection, cell invasion and migration ability increased (P <0.05). Conclusion The expression of miR-205 in human esophageal squamous cell carcinoma cells is up-regulated, and may play a role in tumor suppressor gene by down-regulating the relative intensity and protein expression level of ZEB1 and ZEB2 mRNA and up-regulating the relative expression of E-cadherin mRNA and protein level The up-regulation of miR-205 expression may be an effective method for the treatment of esophageal squamous cell carcinoma.