Fundamental study for therapy of liver cancers based on RNA interference

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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.