Apoptosis of Leukemic Cells by Using tRNase ZL Under the Direction of Oncogene-Targeting Small Guide RNA.
Authors: Watanabe Norihiro , Narita Miwako , Yamahira Akie , Taniguchi Tomoyo , Furukawa Tatsuo , Yoshida Tetsuo , Miyazawa Tatsuya , Nashimoto Masayuki , Takahashi Masuhiro 
Published: October 2019

Abstract
Abstract 3763

Introduction:
tRNase ZL is one of the enzymes for tRNA maturation and removes a 3′ trailer of pre-tRNA by recognizing four leaves-clover-like structure of pre-tRNA. In addition, we found that tRNase ZL can recognize 12-bases hairpin structures and cleaves 3′ portion of them. Furthermore, we have demonstrated that tRNase ZL could cleave 3′ portion of target mRNA by recognizing a complex consisting of both the target mRNA and artificially designed small guide RNA (sgRNA) which possesses the complementary sequences with target RNA and form a four leaves-clover-like structure or 12-bases hairpin structures with target mRNA. Currently, sgRNA is divided into five categories; 3′-truncated tRNA (form a four leaves-clover-like structure with target mRNA), 5′-half tRNA (form a half of four leaves-clover-like structure with target mRNA), 14-nucleotides linear type sgRNA (form a 14-bases stem with target mRNA), hook type sgRNA (containing a single loop structure and form 12-bases hairpin structure with target mRNA) and heptamer sgRNA (7-bases RNA structure complementary to target mRNA and form a 12-bases hairpin structure with target mRNA). The use of sgRNA, especially heptamer sgRNA, to knockdown specific mRNAs has several advantages over long form RNAs (antisense, shRNA or siRNA); 1) heptamer sgRNA can be introduced into cells without any transfection operation, 2) less off-target effect, 3 less interferon responses, 4) the synthesis of heptamer sgRNA is inexpensive than that of long form RNAs. Thus, gene silencing methods using tRNase ZL and sgRNA are presumed to induce effective and specific target RNA inhibition.
In the present study, in order to explore the application of sgRNA (especially heptamer sgRNA) as novel nucleic acid medicine, we investigated the effects of heptamer sgRNA targeting WT1 gene (WT1sgRNA) and PRMAE gene (PRAMEsgRNA) on apoptosis, proliferation and mRNA suppression in leukemic cell lines.

Materials and Methods:
The 5′-phosphorylated 2′-O-methyl heptamer sgRNAs, which were designed to form 12-bases hairpin structure with target mRNA of leukemia associated antigens (LAA) were synthesized and purified by high-performance liquid chromatography. The leukemic cell lines (HL60, C2F8 and U937) were cultured with heptamer sgRNA at the concentration of 1 mM. Introduction of heptamer sgRNA into cells was investigated by using FITC-conjugated heptamer sgRNA and evaluated by flow cytometry and fluorescence microscopy. Quantification of target mRNA was determined by real-time quantitative PCR. Effects of heptamer sgRNA on apoptosis of leukemic cells were evaluated by flow cytometry. Inhibition of cell proliferation was evaluated by TetraColor ONE reagent.

Results:
Heptamer sgRNA could be introduced into cells by just adding them to culture medium by both flow cytometry and fluorescent microscopy. Expression level of WT1 and PRAME mRNA in HL60 cells was reduced by culturing HL60 cells with WT1sgRNA and PRAMEsgRNA (up to 75% and 30% reduction, respectively) compared to control EGFPsgRNA (estimated as 0% reduction). Likewise, WT1sgRNA suppressed WT1 mRNA expression in C2F8 cells by just adding WT1sgRNA to culture medium (30% reduction). In addition, WT1sgRNA and PRAMEsgRNA could induce apoptosis in HL60 cells but not in WT1(–)PRAME(–) U937 cells. Control EGFPsgRNA did not induce apoptosis. WT1sgRNA and PRAMEsgRNA could inhibit cell proliferation in HL60 cells (70% and 40% inhibition) respectively, compared to control EGFPsgRNA (estimated as 0% inhibition). WT1sgRNA could also inhibit cell proliferation in C2F8 cells (70% inhibition).

Conclusion:
These data indicate that WT1sgRNA and PRAMEsgRNA specifically bind to target mRNA and forms 12-bases hairpin structure with target mRNA, which complex is recognized by tRNase ZL. Then 3′ portion of target mRNA is cleaved by tRNase ZL by target-specific manner. Taken together, these findings suggested a possibility that oncogene-targeting sgRNA (especially heptamer sgRNA) could be applied as a novel nucleic acid medicine against leukemia and other tumors.

Disclosures:
No relevant conflicts of interest to declare.