Notch信号通路通过调控Snail/E-钙黏蛋白参与肝细胞肝癌侵袭迁移的机制

Mechanism of notch signaling pathway involved in invasion and migration of hepatocellular carcinoma by regulating Snail/E -cadherin

目的 探讨Notch信号通路在肝癌（HCC）细胞侵袭迁移中的作用机制.方法 分别用反转录-聚合酶链反应（RT-PCR）和Western blot检测肝癌细胞株HepG2和非肿瘤肝细胞株HL-7702中Snail和E-钙黏蛋白（E-cadherin）的mRNA和蛋白表达;Westem blot检测NICD蛋白的表达;200 pmol小干扰RNA（siRNA）-Snail和5μmol/L DAPT分别处理HepG2后,RT-PCR和Westem blot 检测Snail和E-cadherin的mRNA和蛋白表达;Transwell小室检测各处理组细胞的侵袭、迁移能力.结果 HepG2中Snail mRNA和蛋白的表达均高于非肿瘤肝细胞株HL-7702（0.11±0.06比0.08±0.02、0.46±0.06比0.21±0.04）,差异有统计学意义（P =0.021、0.032）,E-cadherin mRNA和蛋白的表达低于HL-7702（0.47±0.11比0.83±0.05、0.17 ±0.03比0.25±0.06）,差异有统计学意义（P =0.012、0.004）.与siRNA阴性对照组比较,siRNA-Snail组Snail mRNA（0.76±0.13比0.25±0.06,P=0.031）和蛋白（0.81±0.24比0.27 ±0.08,P=0.024）表达显著降低,E-cadherin mRNA（0.33±0.06比0.85±0.18,P=0.015）和蛋白（0.14 ±0.02比0.52 ±0.07,P=0.031）的表达显著上调,细胞侵袭（93.42±12.33比39.66±8.33,P=0.011）、迁移（375.66±65.21比98.52±12.76,P=0.008）能力均明显下降.HepG2中NICD蛋白的表达明显高于HL-7702（0.26±0.04比0.11±0.02）,差异有统计学意义（P=0.004）.与NT组比较,Notch信号通路特异性γ-分泌酶抑制剂（GSI）抑制剂DAPT能够降低NICD蛋白的表达（0.22±0.05,0.12 ±0.02,P=0.020）,说明DAPT能够有效抑制Notch信号通路的活化.DAPT能有效抑制Snail mRNA（0.56±0.08比0.23±0.05,P=0.004）和蛋白（0.72 ±0.06比0.27±0.04,P=0.030）的表达,上调E-cadherin mRNA（0.21±0.03比0.54±0.06,P=0.011）和蛋白（0.32±0.07比0.77 ±0.08,P =0.021）的表达,同时能够抑制肝癌细胞的侵袭（87.33±9.33比32.21 ±6.63,P=0.008）、迁移（384.23±43.33比78.31±9.33,P=0.012）.结论 Notch信号通路在肝癌侵袭迁移过程中起重要作用,其作用机制是通过调控Snail/E-cadherin来实现的.

Objective To investigate the mechanism of Notch signaling pathway in invasion and migration of hepatocellular carcinoma （HCC） cells.Methods The mRNA and protein expression levels of Snail and E-cadherin in hepatoma cells （HepG2） and non-tumor liver cell line （HL-7702） were detected by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting respectively.Western blotting was used to detect the expression of NICD protein.The expression of Snail and E-cadherin mRNA and protein was detected by RT-PCR and Western blotting respectively after HepG2 cells were treated with 200 pmol small interfering RNA （siRNA）-Snail siRNA-Snail and 5 μmol/L DAPT.The invasive ability of HepG2 cells was examined by Transwell chamber.Results The expression levels of Snail mRNA and protein in HepG2 cells were significantly higher than those in HL-7702 cells （0.11 ±0.06 vs.0.08±0.02,P=0.021;0.46±0.06 vs.0.21 ±0.04,P=0.032）.The expression levels of E-cadherin mRNA and protein in HepG2 cells were significantly lower than those in HL-7702 cells （0.47±0.11 vs.0.83±0.05,P=0.012;0.17±0.03 vs.0.25 ±0.06,P=0.004）.As compared with the negative control group,the expression levels of Snail mRNA （0.76 ± 0.13 vs.0.25 ± 0.06,P =0.031） and protein （0.81 ± 0.24 vs.0.27 ± 0.08,P =0.024） in siRNA-Snail-treated group （Ss） decreased significantly,and the expression levels of E-cadherin mRNA （0.33 ± 0.06 vs.0.85 ± 0.18,P =0.015） and protein （0.14 ± 0.02 vs.0.52 ± 0.07,P =0.031） increased significantly,and the invasive ability （93.42 ± 12.33 vs.39.66 ± 8.33,P =0.011） and migration （375.66 ± 65.21 vs.98.52 ± 12.76,P =0.008） decreased significantly.The expression levels of NICD protein in HepG2 cells were significantly higher than those in HL-7702 cells （0.26 ± 0.04 vs.0.11 ± 0.02,P =0.004）.As compared with NT group,the expression of NICD protein was significantly inhibited by Notch pathway selective inhibitor DAPT （0.22 ±0.05 vs.0.12 ±0.02,P =0.020）,indicating that DAPT can effectively inhibit the activation of notch signaling pathway.The expression levels of Snail mRNA （0.56 ± 0.08 vs.0.23 ± 0.05,P =0.004） and protein （0.72 ± 0.06 vs.0.27 ± 0.04,P =0.030） could be down-regulated,and the expression levels of E-cadherin mRNA （0.21 ± 0.03 vs.0.54 ± 0.06,P =0.011） and protein （0.32 ±0.07 vs.0.77 ±0.08,P =0.021） could be upregulated by DAPT.Furthermore,DAPT could inhibit cell invasion （87.33 ±9.33 vs.32.21 ±6.63,P =0.008） and migration （384.23 ±43.33 vs.78.31 ± 9.33,P =0.012） in HepG2 cells.Conclusion Notch signaling pathway plays an important role in invasion and migration of HCC cells probably through the regulation of Snail/E-cadherin.