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目的:探讨丹参酮ⅡA对肝癌细胞MHCC97-H Smad3、Smad7蛋白及上皮间质转移标志蛋白Ecadherin/N-cadherin表达水平及侵袭转移能力的影响。方法:10μM丹参酮ⅡA处理MHCC97-H细胞,实时定量PCR检测细检测Smad3、Smad7和snail的m RNA表达;用不同浓度丹参酮ⅡA及10%小牛血清对照组培养肝癌细胞MHCC97-H,48 h后,Western blot检测Smad3、p-Smad3、Smad7、Snail、E-cadherin和N-cadherin蛋白表达水平。划痕法和Transwell小室检测肝癌细胞侵袭/迁移能力。结果:10μM丹参酮ⅡA处理MHCC97-H细胞,不同时间点检测Smad3、Smad7和snail的m RNA表达:结果显示,与0 h相比,48 h及72 h Smad7的表达量明显升高,差异有统计学意义(P<0.05);Smad3及snail的差异无统计学意义。不同浓度丹参酮ⅡA处理MHCC97-H细胞48 h,Western blot检测Smad3、p-Smad3、Smad7、snail、E-cadherin、Ep CAm、N-cadherin和Vimentin蛋白表达:结果显示,与对照组相比,高浓度丹参酮ⅡA组(20μM)48 h的p-Smad3、Snail和N-cadherin、Vimentin明显降低,Smad7、E-cadherin和Ep CAM明显升高(P<0.05)。中浓度丹参酮ⅡA组(10μM)48 h的Smad7、E-cadherin和Ep CAM升高;p-Smad3、snail、Ncadherin和Vimentin降低(P<0.05)。低浓度丹参酮ⅡA组(5μM)48 h的E-cadherin升高,N-cadherin降低(P<0.05);而Smad3、p-Smad3及snail的结果与对照组无明显差异(P>0.05)。不同浓度的丹参酮ⅡA组和对照组(DMSO)Smad3的蛋白表达无影响。不同浓度的丹参酮ⅡA组均可以减少p-Smad3和增加E-cadherin的表达,且随着丹参酮ⅡA浓度增高p-Smad3的表达逐渐减少,E-cadherin的表达逐渐增加。划痕法结果显示,与对照组比较,24 h后中浓度和高浓度丹参酮ⅡA组肝癌细胞的划痕愈合较慢,差异均有统计学意义(P<0.05);48 h后中浓度和高浓度丹参酮ⅡA组与对照组相比,肝癌细胞的划痕愈合明显减慢,差异有统计学意义(P<0.01);且同等浓度丹参酮ⅡA组肝癌细胞的划痕愈合与24 h相比较,48 h肝癌细胞的划痕愈合减慢。transwell显示,与对照组相比,中浓度和高浓度丹参酮ⅡA处理细胞48 h可明显减少细胞的侵袭,差异有统计学意义(P<0.05)。而低浓度丹参酮ⅡA无明显影响,差异无统计学意义。结论:丹参酮ⅡA能抑制肝癌细胞侵袭转移,可能与与抑制Smad3蛋白磷酸化、上调Smad7蛋白,并抑制上皮间质转化有关。
Objective: To investigate the effect of Tanshinone ⅡA on the expression of MHCC97-H Smad3 and Smad7 and the expression of Ecadherin / N-cadherin and their invasion and metastasis in hepatocellular carcinoma cells. Methods: MHCC97-H cells were treated with 10μM tanshinone ⅡA, and the mRNA expression of Smad3, Smad7 and snail was detected by real-time quantitative PCR. The hepatoma cells MHCC97-H were cultured with different concentrations of tanshinone ⅡA and 10% fetal calf serum for 48 h The protein expression levels of Smad3, Smad3, Smad7, Snail, E-cadherin and N-cadherin were detected by Western blot. Scratch assay and Transwell chambers to detect hepatocellular carcinoma cell invasion / migration ability. Results: The expression of Smad3, Smad7 and snail mRNA was detected at different time points after treatment with 10μM tanshinone ⅡA: The expression of Smad7 in 48h and 72h was significantly higher than that in 0h Significance (P <0.05); there was no significant difference between Smad3 and snail. The expression of Smad3, p-Smad3, Smad7, snail, E-cadherin, Ep CAm, N-cadherin and Vimentin protein were detected by Western blot after treated with different concentrations of tanshinone ⅡA for 48 h: The results showed that compared with the control group, The concentrations of p-Smad3, Snail and N-cadherin, Vimentin in Tanshinone ⅡA group (20μM) for 48 h were significantly decreased, and the Smad7, E-cadherin and Ep CAM were significantly increased (P <0.05). The concentrations of Smad7, E-cadherin and Ep-CAM in middle-concentration tanshinone ⅡA group (10μM) increased at 48 h, while decreased in p-Smad3, snail, Ncadherin and Vimentin groups (P <0.05). E-cadherin and N-cadherin in low concentration tanshinoneⅡA group (5μM) for 48 h increased (P <0.05), but no significant difference between Smad3, p-Smad3 and snail in control group (P> 0.05). There was no effect on the protein expression of Smad3 in tanshinone ⅡA group and control group (DMSO) at different concentrations. The expressions of p-Smad3 and E-cadherin were decreased in different concentrations of tanshinoneⅡA group, and the expression of p-Smad3 decreased with the increase of tanshinoneⅡA concentration, and the expression of E-cadherin gradually increased. Scratch assay showed that the scratch healing of hepatocarcinoma cells in medium and high concentration of tanshinone ⅡA group was slower than that of control group after 24 h, the difference was statistically significant (P <0.05); medium concentration and high Compared with the control group, the wound healing of hepatoma cells in Tanshinone ⅡA group was significantly slower than that in the control group (P <0.01), and the wound healing of hepatoma cells with the same concentration of Tanshinone ⅡA group was significantly lower than that of the control group h Hepatoma cells scratch healing slows. Transwell showed that compared with the control group, Tanshinone ⅡA treated cells in medium and high concentrations for 48 h could significantly reduce the cell invasion, the difference was statistically significant (P <0.05). The low concentration of tanshinone Ⅱ A no significant effect, the difference was not statistically significant. Conclusion: Tanshinone ⅡA can inhibit the invasion and metastasis of hepatocellular carcinoma cells, which may be related to the inhibition of Smad3 phosphorylation, up-regulation of Smad7 protein and inhibition of epithelial-mesenchymal transition.