Fundamental study for therapy of liver cancers based on RNA interference

AFP（甲胎蛋白），被称为肝癌的肿瘤标志物，最近有报道称参与细胞生长。为探讨抑制AFP对肝癌细胞生长的影响，采用脂质体转染法将不同类型的AFP siRNA转染肝癌细胞株Huh 7。培养基的ELISA和细胞的RT-PCR显示最有效的siRNA。然后，我们检查了细胞计数和BrdU标记的siRNA，并注意到抑制效果。我们现在正试图使用稳定表达sh（短发夹）RNA的细胞系来阐明分子机制。接下来，我们专注于JNK，应激诱导的MAP激酶，也已知参与肝再生和癌变。JNK的两种亚型JNK 1和JNK 2的siRNA均抑制肝癌细胞的生长。但是，它们与底物的反应存在差异。c-Jun和ATF 2的磷酸化仅被JNK 1的siRNA抑制。此外，Elk 1的敲低对细胞生长没有太大影响。因此，JNK 2可能通过包括其他底物的途径促进细胞生长。然后，我们研究了siRNA在大鼠肝脏中的有效给药方法。当通过门静脉或尾静脉注射时，与siRNA混合的去端胶原不起作用。我们接下来使用基于流体动力学的siRNA和shRNA产生载体通过尾静脉注射。我们成功地抑制了c-Jun在大鼠肝脏中的表达，并建立了DEN诱导的大鼠肝癌模型，通过超声观察了其癌变过程。现在，我们已经开始研究siRNA或shRNA生产载体对肝癌治疗或预防的作用。

AFP(alphafeto protein), known as a tumor marker for liver cancers, has recently been reported to be involved in cell growth. We tested whether inhibition of AFP could influence the growth of liver cancer cells.Huh7 cells, a liver cancer-derived cell line, were transfected with several types of si RNA for AFP by lipofection. ELISA of culture media and RT-PCR of cells showed the most effective si RNA. Then, we checked cell count and BrdU labeling with this si RNA and noticed the suppressive effect. We are now trying to clarify the molecular mechanism using stable cell lines expressing sh(short hairpin) RNA constitutively.Next, we focused on JNKs, stress-induced MAP kinases also known to be involved in liver regeneration and carcinogenesis. Both of si RNAs for two isoforms of JNK, JNK1 and JNK2, inhibited the growth of liver cancer cells. However, there is a difference of their reaction with substrates. Phosphorylation of c-Jun and ATF2 was inhibited only by si RNA for JNK1. In addition, knockdown of Elk1 did not influence cell growth much. Therefore, JNK2 seems to contribute to cell growth through the pathway including other substrates.Then, we studied effective methods for administration of si RNA to rat livers in vivo. Atelocollagen, mixed with si RNA, did not work well when injected through either portal vein or tail vein. We next used hydrodynamics-based injection of si RNA and shRNA-producing vector via tail veins. We successfully inhibited the expression of c-Jun in rat livers.We also established a rat model of DEN-induced liver cancers and observed the carcinogenetic process by ultrasound. Now, we have started to examine the effect of si RNA or shRNA-producing vectors on therapy or prevention of liver cancers.