Effects of astragalus injection on the expression of β-Catenin and E-cadherin in human endometrial carcinoma cells
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摘要:
目的研究黄芪注射液对子人宫内膜癌HEC-1-B 细胞β-catenin 和E-cadherin 基因和蛋白表达的影响。 方法分别采用RT-PCR和免疫组化的方法检测β-catenin和E-cadherin基因和蛋白的表达。用含不同浓度的黄芪注射液的条件培养基分别培养人子宫内膜癌细胞株HEC-1-B 24、48 、72 h后，应用RT-PCR检测各组细胞中β-catenin、E-cadherin基因表达，应用免疫细胞化学法检测HEC-1-B中β-catenin、E-cadherin蛋白表达。 结果RT-PCR显示，随着子宫内膜癌细胞在含有黄芪注射液的条件培养基中培养时间的延长，细胞中E-cadherin基因表达逐步增强（P＜0.05），而β-catenin基因表达逐步减弱（P＜0.05）。免疫组化染色结果显示，与对照组比较，黄芪注射液可明显降低子宫内膜癌HEC-1-B细胞中β-catenin蛋白的表达、增加E-cadherin蛋白的表达（P＜0.05），而且黄芪注射液浓度越高处理时间越长β-catenin表达更低（P＜0.05），黄芪注射液浓度越高E-cadherin表达更高（P＜0.05）。结论黄芪注射液可呈剂量和时间依赖性促进E-cadherin基因和蛋白的表达、抑制β-catenin基因和蛋白的表达，从而使WNT信号通路受到抑制。这可能是其发挥抗肿瘤作用的机制之一。 -
关键词:
- 黄芪注射液 /
- 人子宫内膜癌细胞 /
- β-catenin /
- E-cadherin
Abstract:Objective To investigate the effects of astragalus injection on the mRNA and protein expression of β-catenin and E-cadherin in HEC-1-Bendometrial carcinoma cells. Methods Cell climbing slices of HEC-1-B cells were prepared for the intervening experiment of astragalus injection on endometrial carcinoma cells, then their mRNA and protein expression of β-catenin and E-cadherin detected by RT-PCR and immunohistochemistry. Results RT-PCR results showed that with the incubation time of endometrial cancer cells in conditioned medium containing astragalus injection increased, E-cadherin gene expression of the cells gradually increased (P＜0.05), while the β-catenin gene expression gradually decreased (P＜0.05). The immunocytochemistry result indicated thatastragalus could significantly decrease β-catenin expression and increase E-cadherin expression in HEC-1-B cells（P＜0.05）. Moreover, accompanied by elevated concentration and treatment time of astragalus injection, β-catenin expression dropped progressively (P＜0.05), meanwhile with the concentration of astragalus injection raised, E-cadherin expression gradually increased (P＜0.05). Conclusion Astragalus injection could significantly inhibit WNT signaling pathway by promoting mRNA andprotein expression of E-cadherin,decreasing mRNA and protein expression ofβ-cateninin a dose- and time-dependent manner, which might be one of the mechanisms for its anti-tumor effect. -
Key words:
- astragalus /
- endometrial carcinoma cells /
- β-catenin /
- E-cadherin
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图 1 黄芪注射液培养后细胞内转录因子表达水平测定结果
A: 图为RT-PCR电泳图; B: 各组统计结果：分为24 h处理组、48 h处理组、72 h处理组，*P ＜0.05; **P ＜0.05.
图 2 各组细胞β-catenin蛋白表达（IHC, ×200）
A: 100 mg/mL黄芪注射液处理24 h; B: 100 mg/mL黄芪注射液处理48 h; C: 100 mg/mL黄芪注射液处理72 h; D: 200 mg/mL 黄芪注射液处理24 h; E: 200 mg/mL 黄芪注射液处理48 h; F: 200 mg/mL 黄芪注射液处理72 h; G: 300 mg/mL黄芪注射液处理24 h; H: 300 mg/mL黄芪注射液处理48 h; I: 300 mg/mL黄芪注射液处理72 h.
图 3 各组细胞E-cadherin蛋白表达（IHC, ×200）
A: 100 mg/mL黄芪注射液处理72 h; B: 200 mg/mL黄芪注射液处理72 h; C: 300 mg/mL黄芪注射液处理72 h.
表 1 设计合成的引物及碱基序列
目的基因 引物序列 退火温度和循环数 产物大小（bp） β-Catenin 上游5´-GGGATAAAATTGCGAAGGA-3´ 57 ℃ 583 下游5´-TGCTGTGAGGCATCGTCTG-3´ 30 个循环 E-cadherin 上游5´-CGACAAAGGACAGCCTATTT-3´ 56 ℃ 582 下游5´-ATTCTGATCGGTTACCGTGA-3´ 27 个循环 GAPDH 上游5´-CGCTGAGTACGTCGTGGAGT-3´ 60 ℃ 172 下游5´-GCTGATGATCTTGAGGCTGTTG-3´ 25 个循环 
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表 2 黄芪注射液对子宫内膜癌细胞β-catenin和E-cadherin蛋白表达的影响（n=6, x±s）
组别 24 h 48 h 72 h β-catenin E-cadherin β-catenin E-cadherin β-catenin E-cadherin 对照组 92.86±0.21 28.79±0.18 93.10±0.16 29.10±0.22 93.24±0.14 29.23±0.15 100 mg/mL黄芪注射液组 91.17±0.31* 31.43±0.24* 89.21±0.25* 32.27±0.30* 85.42±0.32*Δ 36.18±0.34*Δ 200 mg/mL黄芪注射液组 88.72±0.33*# 32.05±0.42*# 86.56±0.20*# 35.94±0.40*# 82.96±0.37*#Δ 38.25±0.29*#Δ 300 mg/mL黄芪注射液组 85.04±0.28*# 37.56±0.37*# 82.84±0.36*# 40.13±0.24*# 77.52±0.16*#Δ 46.37±0.33*#Δ 与对照组比较, *P ＜0.05; 与100 mg/mL黄芪注射液组比较, #P＜0.05; 与24 h黄芪注射液组比较, ΔP＜0.05.
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