中药砒霜上调E-钙黏蛋白抑制宫颈癌Hela细胞生长、侵袭、迁移机制研究

Study on Mechanism of Arsenic Up-Regulating E-Cadherin to Inhibit Growth,Invasion and Migrationn of Cervical Cancer Hela Cells

目的:研究中药砒霜的主要成分三氧化二砷(As_(2)O_(3))抑制宫颈癌Hela细胞生长、侵袭、迁移的机制,观察As_(2)O_(3)对宫颈癌Hela细胞组蛋白脱乙酰基酶1(HDAC1)、E-钙黏蛋白表达的影响,以及联合HDAC1抑制剂伏立诺他(SAHA)的协同作用。方法:以宫颈癌Hela细胞作为载体,随机分为空白对照组、As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组4组,采用CCK-8法测定不同药物浓度对Hela细胞活性的抑制情况,并筛选后续实验药物浓度;采用Transwell法、划痕法检测各组药物对Hela细胞侵袭、迁移能力的影响;采用实时荧光定量聚合酶链式反应及蛋白免疫印迹法检测HDAC1和E-钙黏蛋白的mRNA表达情况与蛋白表达情况。结果:随着各组药物浓度增加,Hela细胞的活性逐渐降低。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组的Hela细胞活性均低于空白对照组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组穿越Transwell小室膜的细胞数量均少于空白对照组(P<0.05);As_(2)O_(3)+SAHA组的细胞数量少于As_(2)O_(3)组、SAHA组(P<0.05);As_(2)O_(3)组的细胞数量少于SAHA组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组的Hela细胞迁移百分比均低于空白对照组(P<0.05);As_(2)O_(3)+SAHA组的Hela细胞迁移百分比低于As_(2)O_(3)组、SAHA组(P<0.05);As_(2)O_(3)组的Hela细胞迁移百分比低于SAHA组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组的HDAC1 mRNA表达水平均低于空白对照组(P<0.05);As_(2)O_(3)组的HDAC1 mRNA表达水平高于SAHA组(P<0.05);As_(2)O_(3)+SAHA组的HDAC1 mRNA表达水平低于As_(2)O_(3)组、SAHA组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组的E-钙黏蛋白mRNA表达水平均高于空白对照组(P<0.05);As_(2)O_(3)+SAHA组的E-钙黏蛋白mRNA表达水平高于As_(2)O_(3)组、SAHA组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组的HDAC1蛋白表达水平均低于空白对照组(P<0.05);As_(2)O_(3)+SAHA组的HDAC1蛋白表达水平低于As_(2)O_(3)组、SAHA组(P<0.05)。As_(2)O_(3)组、SAHA组、As_(2)O_(3)+SAHA组E-钙黏蛋白的蛋白表达水平均高于空白对照组(P<0.05);As_(2)O_(3)+SAHA组E-钙黏蛋白的蛋白表达水平高于As_(2)O_(3)组、SAHA组(P<0.05)。结论:As_(2)O_(3)抑制Hela细胞的最佳浓度为8μmol/L,联合SAHA时,最佳抑制浓度为4μmol/L。As_(2)O_(3)可能通过抑制HDAC1的表达,同时促进升高E-钙黏蛋白的表达,与SAHA发挥协同作用抑制Hela细胞生长、侵袭、迁移。

Objective:To investigate the mechanism of arsenic trioxide(As_(2)O_(3)),the main component of Arsenic(Chinese medicine),inhibiting the growth,invasion and migration of Hela cells of cervical cancer,and to observe the effects of As_(2)O_(3)on the expression of histone deacetylase 1(HDAC1)and E-cadherin in Hela cells of cervical cancer,and the synergistic effect of HDAC1 inhibitor Vorinostat(SAHA).Methods:The oncomine database was used to investigate the expression differences of CDH1 gene in cervical cancer tissues with and without distant metastasis.Cervical cancer Hela cells were used as carriers,and were randomly divided into four groups:the blank control group,the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group.CCK-8 method was used to determine the inhibition of different drug concentrations on Hela cell growth,and the subsequent experimental drug concentrations were screened;Transwell method and scratch method were used to detect the effects of various medicine on invasion and migration ability of Hela cells;real-time fluorescence quantitative polymerase chain reaction and protein Western blotting methods were used to detect the protein and mRNA expression of HDAC1 and E-cadherin.Results:The Hela cell activity gradually decreased with the increasing drug concentration in each group.The Hela cell activity in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were all lower than that in the blank control group(P<0.05).The numbers of cells crossing the Transwell chamber membrane in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were all lower than that in the blank control group(P<0.05);the number of cells in the As_(2)O_(3)+SAHA group was lower than those in the As_(2)O_(3)group and the SAHA group(P<0.05);the number of cells in the As_(2)O_(3)group was lower than that in the SAHA group(P<0.05).The percentages of migration of Hela cells in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were lower than that in the blank control group(P<0.05);the percentage of migration of Hela cells in the As_(2)O_(3)+SAHA group was lower than those in the As_(2)O_(3)group and the SAHA group(P<0.05);the percentage of migration of Hela cells in the As_(2)O_(3)group was lower than that in the SAHA group(P<0.05).The HDAC1 mRNA expression levels in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were lower than that in the blank control group(P<0.05);the HDAC1 mRNA expression level in the As_(2)O_(3)group was higher than that in the SAHA group(P<0.05);the level in the As_(2)O_(3)+SAHA group was lower than those in the As_(2)O_(3)group and the SAHA group(P<0.05).The mRNA expression levels of E-cadherin in the As_(2)O_(3)group,the SAHA group,and the As_(2)O_(3)+SAHA group were all higher than that in the blank control group(P<0.05);the mRNA expression level of E-cadherin in the As_(2)O_(3)+SAHA group was higher than that in the As_(2)O_(3)group and the SAHA group(P<0.05).The HDAC1 protein expression levels in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were lower than that in the blank control group(P<0.05);the HDAC1 protein expression level in the As_(2)O_(3)+SAHA group was lower than those in the As_(2)O_(3)group and the SAHA group(P<0.05).The E-cadherin protein expression levels in the As_(2)O_(3)group,the SAHA group and the As_(2)O_(3)+SAHA group were higher than that in the blank control group(P<0.05);the E-cadherin protein expression level in the As_(2)O_(3)+SAHA group was higher than those in the As_(2)O_(3)group and the SAHA group(P<0.05).Conclusion:The optimal concentration of As_(2)O_(3)to inhibit Hela cells is 8μmol/L,and when combined with SAHA,the optimal inhibitory concentration is 4μmol/L.As_(2)O_(3)can inhibit the expression of HDAC1,promote the expression of E-cadherin,and play a synergistic role with SAHA to inhibit the growth,invasion and migration of Hela cells.