Fundamental study for therapy of liver cancers based on RNA interference

AFP（甲胎蛋白）被认为是肝癌的肿瘤标志物，最近有报道称它与细胞生长有关。我们测试了AFP的抑制是否会影响肝癌细胞的生长。Huh7细胞是一种肝癌来源的细胞系，我们通过脂肪转染了几种用于AFP的si RNA。培养基酶联免疫吸附试验和细胞RT-PCR检测结果显示sirna最有效。然后，我们用该sirna检查细胞计数和BrdU标记，并注意到抑制作用。我们现在正试图利用稳定的细胞系组成性地表达短发夹RNA来阐明其分子机制。接下来，我们专注于JNKs，应激诱导的MAP激酶也参与肝脏再生和致癌。JNK的两种亚型JNK1和JNK2的si rna都能抑制肝癌细胞的生长。然而，它们与底物的反应是不同的。c-Jun和ATF2的磷酸化仅被JNK1的si RNA抑制。此外，敲低Elk1对细胞生长影响不大。因此，JNK2似乎通过包括其他底物在内的途径促进细胞生长。然后，我们研究了si RNA给药大鼠肝脏的有效方法。经门静脉或尾静脉注射时，将胶原蛋白与si RNA混合后均不能很好地发挥作用。接下来，我们采用流体动力学方法通过尾静脉注射sirna和shrna产生载体。我们成功地抑制了c-Jun在大鼠肝脏中的表达。我们还建立了den诱导的大鼠肝癌模型，并通过超声观察其癌变过程。现在，我们已经开始研究sirna或shrna产生载体在治疗或预防肝癌方面的作用。

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.