摘要
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 Ⅱ A on Smad3/Smad7 protein and epithelial mesenehymal transition （EMT） essential protein （E-cadherin/N-cadherin） of hepatocellular carcinoma cell line MHCC97-H. Methods： Hepatocellular carcinoma cell line MHCC97-Ⅱ was cultured in Tanshinone Ⅱ A （10 μM） and 10% blank serum, respectively. Real-time PCR（qPCR） was used to detect the mRNA level of Smad3, Smad7, and Snail; Hepatocellular carcinoma cell line MHCC97-H was cultured in different concentrations （5 μM, 10 μM and 20 μM） of Tanshinone Ⅱ A and 10% blank serum for 48 hours, respectively. Western blot was used to detect the protein level of Smad3, p-Smad3, Smad7, Snail, E-cadherin, and N-cadherin, respectively. Wound healing assays and Transwell chamber was respectively used to detect the invasion and migration ability of MI-ICC97-Ⅱ cell. Results： qPCR results revealed that mRNA level of Smad7 of Hepatocellular carcinoma cell line MHCC97-Ⅱ that was cultured in Tanshinone Ⅱ A（10μM） and 10% blank serum for 48 hours or for 72 hours was obviously higher than that for 0 hours, respectively. While the difference of Smad3 and Snail was not significant, respectively. The protein level of p-Smad3, Snail, N-cadherin, Vimentin of high-concentration group was lower than that of control group, as well as the protein level of the middle-concentration group, respectively （P〈0.05）; protein level of Smad7, E-cadherin, EpCAM of high-concentration group was higher than that of control group, as well as the trend of the protein level of the middle-concentration group, respectively （P〈0.05）. Difference of protein level of N-cadherin and E-cadherin between low-concentration group and control group was significant （P〈0.05）; while the difference of Smad3, p-Smad3 and Snail was not significant （P〉0.05）. The protein level of-Smad3 of the different concentrations（5 μM,10 μM and 20 μM） of Tanshinone Ⅱ A were lower than that of control group, while protein level of E-cadherin of the different concentrations（5 μM,10 μM and 20μM） of Tanshinone Ⅱ were higher than that of control group. Also the protein level of Smad3 decreased as the increase of the concentration of Tanshinone Ⅱ A, which was contrary to the protein level of E-cadherin. Would healing assays revealed that, after 24 hours, the speed of would healing of high- concentration group and middle-concentration group was slower than that of control group, respectively （P〈0.05）. After 48 hours, the speed of would healing of high-concentration group and middle-concentration group was slower than that of control group, respectively （P〈0.05）. Also the speed of would healing of same-concentration group which was cultured for 48 hours was lower than that of control group, which was cultured for 24 hours. Consistent with the results of would healing assays, Transwell assay showed that the migration of MHCC97-Ⅱ cell of high-concentration group and middle-concentration group was inhibited than that of control group, respectively （P〈0.05）. While the difference of the low-concentration group was not significant, respectively. Conclusion： Tanshinone Ⅱ A inhibited the invasion and migration of HCC, potential mechanism was inhibiting the phosphorylation of Smad3, the increase of Smad7 and epithelial mesenchymal transition of HCC.
出处
《中医药导报》
2017年第10期30-34,共5页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
2014年国家自然科学青年基金项目(81403397)
2014年广东省自然科学基金项目(2014A030313408)
2016年广东省科技计划项目(2016A020226052)
