全反式维甲酸抑制肝癌细胞增殖的作用机制研究

Human hepatocellular carcinoma cell growth is suppressed by all-trans-retinoicacid through down-regula-ting miR-18a expression

目的研究全反式维甲酸（all—trans—retinoicacid，ATRA）对肝癌细胞增殖的影响并探讨可能的作用机制。方法利用ATRA处理肝癌细胞系HepG2与SMMC-7721，选择MTT法分析细胞增殖。利用实时PCR方法研究ATRA对miR-18a表达的影响，并选择特异抑制剂Anti-miR-18a处理肝癌细胞，利用MTT法分析细胞增殖。设计拯救实验，利用ATRA处理转染miR-18a模拟物的肝癌细胞系，利用MTT法分析细胞增殖情况。结果ATRA可以有效抑制肝癌细胞系HepG2和SMMC-7721的增殖，A490吸光度值分析显示，HepG2经ATRA处理后，细胞增殖分别被抑制74％（P〈0．05，36h）、72％（P〈0．01，48h）、67％（P〈0．05，72h）；SMMC-7721经ATRA处理后，细胞增殖分别被抑制68％（P〈0．05，48h）、64％（P〈0．01，72h）。miR-18a在肝癌细胞HepG2与SMMC-7721中的表达水平分别上调4．7倍（P〈0．05）、3．8倍（P〈0．05）；ATRA处理后，HepG2与SMMC-7721内源性miR-18a的表达水平分别下调67％（P〈0．05）与56％（P〈0．05）。过表达miR-18a的肝癌细胞HepG2与SMMC-7721对ATRA的抑制作用出现耐受，其中HepG2细胞增殖上调1．2倍（P〈0．05），SMMC-7721细胞增殖上调1．25倍（P〈0．05，24h）与1．2倍（P〈0．05，48h）。结论ATRA可通过下调肝癌细胞内源性miR-18a的表达，抑制细胞增殖。

Objective To investigate the inhibitory effect of all-trans-retinoicacid （ATRA） on HCC cell growth and probe the potential molecular mechanism. Methods HCC cell lines, HepG2 and SMMC-7721 were treated by ATRA and cell growth was analyzed by using MTT assay. The expression levels of miR-18a were evaluated in HepG2 and SMMC-7721, compared with the normal livers pool, by using Real- Time PCR analysis. Cell growth analysis by using MTT assay was performed on HepG2 and SMMC-7721 after transfection with anti-miR-18a. Rescued assay was designed to probe the mechanism of ATRA on cell growth by using ATRA with or without miR-18a mimic. Results HepG2 cell growth was suppressed about 74% （P〈0.05, 36 h）, 72% （P〈0. 01, 48 h）, and 670〈 （P〈0. 05, 72 h） and SMMC-7721 cell growth was inhibited about 68% （P〈0.05, 48 h）, and 64% （P〈0.05, 72 h） after treatment with ATRA, com- pared with the cells treated with Ethanol. MiR-18a expression was up-regulated in HepG2 and SMMC-7721 cell lines about 4.7- and 3.8-fold （P〈0. 05）, respectively. Endogenous miR-18a levels were down-regulated by ATRA about 67% and 56% （P〈0. 05）. The inhibitory effect of ATRA on HCC cell growth was re- versed about 1.2-fold （P%0. 05, 48 h） by overexpression of miR-18a in HepG2 cells and cell growth of SMMC-7721 was enhanced about 1.25-and 1.2-fold （P〈0.05, 24 and 48 h） with ectopic expression of miR-18a. Conclusion HCC cells growth is suppressed by ATRA through miR-18a mediated network.