Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing

Tamura,Masato; Kawano,Mitsuoki; Sato,Mari; Nashimoto,Masayuki

doi:10.3892/mmr.2015.4160

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing

Authors:
- Masato Tamura
- Mitsuoki Kawano
- Mari Sato
- Masayuki Nashimoto
View Affiliations

Affiliations: Department of Biochemistry and Molecular Biology, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido 060‑8586, Japan, Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Niigata 956‑8603, Japan
Published online on: July 31, 2015 https://doi.org/10.3892/mmr.2015.4160
Pages: 6365-6369

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl‑β‑cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis‑inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin‑, caveolae‑ and raft‑independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis‑inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA‑mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically‑targeted and potent sgRNA drugs.

View Figures

View References

1	Nashimoto M: Specific cleavage of target RNAs from HIV-1 with 5′ half tRNA by mammalian tRNA 3′ processing endoribo-nuclease. RNA. 2:523–524. 1996.PubMed/NCBI
2	Tamura M, Nashimoto C, Miyake N, Daikuhara Y, Ochi K and Nashimoto M: Intracellular mRNA cleavage by 3′ tRNase under the direction of 2′-O-methyl RNA heptamers. Nucleic Acids Res. 31:4354–4360. 2003. View Article : Google Scholar : PubMed/NCBI
3	Nashimoto M: Anomalous RNA substrates for mammalian tRNA 3′ processing endoribonuclease. FEBS Lett. 472:179–186. 2000. View Article : Google Scholar : PubMed/NCBI
4	Takaku H, Minagawa A, Takagi M and Nashimoto M: A novel 4-base-recognizing RNA cutter that can remove the single 3′ terminal nucleotides from RNA molecules. Nucleic Acids Res. 32:e912004. View Article : Google Scholar
5	Shibata HS, Takaku H, Takagi M and Nashimoto M: The T loop structure is dispensable for substrate recognition by tRNase ZL. J Biol Chem. 280:22326–22334. 2005. View Article : Google Scholar : PubMed/NCBI
6	Elbarbary R, Takaku H, Tamura M and Nashimoto M: Inhibition of vascular endothelial growth factor expression by TRUE gene silencing. Biochem Biophys Res Commun. 379:924–927. 2009. View Article : Google Scholar : PubMed/NCBI
7	Nakashima A, Takaku H, Shibata HS, Negishi Y, Takagi M, Tamura M and Nashimoto M: Gene silencing by the tRNA maturase tRNase Z(L) under the direction of small-guide RNA. Gene Ther. 14:78–85. 2007. View Article : Google Scholar
8	Sano T, Takahashi M, Nozaki T, Takahashi Y, Tamura M and Nashimoto M: Expanding the utility of heptamer-type sgRNA for TRUE gene silencing. Biochem Biophys Res Commun. 416:427–432. 2011. View Article : Google Scholar : PubMed/NCBI
9	Takahashi M, Elbarbary RA, Nakashima A, Abe M, Watanabe N, Narita M, Takahashi M, Tamura M, Yoshida T and Nashimoto M: A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells. Cancer Letters. 328:362–368. 2013. View Article : Google Scholar
10	Watanabe N, Narita M, Yamahira A, Taniguchi T, Furukawa T, Yoshida T, Miyazawa T, Nashimoto M and Takahashi M: Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA. Leuk Res. 37:580–585. 2013. View Article : Google Scholar : PubMed/NCBI
11	Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, Høg A, Worm J, Hedtjärn M, Souleimanian N, Miller P, et al: Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 38:e32010. View Article : Google Scholar :
12	Soifer HS, Souleimanian N, Wu S, Voskresenskiy AM, Collak FK, Cinar B and Stein CA: Direct regulation of androgen receptor activity by potent CYP17 inhibitors in prostate cancer cells. J Biol Chem. 287:3777–3787. 2012. View Article : Google Scholar :
13	Souleimanian N, Deleavey GF, Soifer H, Wang S, Tiemann K, Damha MJ and Stein CA: Antisense 2′-deoxy, 2′-fluroarabino nucleic acids (2′F-ANAs) oligonucleotides: In vitro gymnotic silencers of gene expression whose potency is enhanced by fatty acids. Mol Ther Nucleic Acids. 1:e432012. View Article : Google Scholar
14	Koller E, Vincent TM, Chappell A, De S, Manoharan M and Bennett CF: Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res. 39:4795–4807. 2011. View Article : Google Scholar : PubMed/NCBI
15	Sato MM, Nakashima A, Nashimoto M, Yawaka Y and Tamura M: Bone morphogenetic protein-2 enhances Wnt/beta-catenin signaling-induced osteoprotegerin expression. Genes Cells. 14:141–153. 2009. View Article : Google Scholar : PubMed/NCBI
16	Uyama M, Sato MM, Kawanami M and Tamura M: Regulation of osteoblastic differentiation by the proteasome inhibitor bortezomib. Genes Cells. 17:548–558. 2012. View Article : Google Scholar : PubMed/NCBI
17	Dutta D and Donaldson JG: Search for inhibitors of endocytosis: Intended specificity and unintended consequences. Cell Logist. 2:203–208. 2012. View Article : Google Scholar
18	Donaldson JG, Finazzi D and Klausner RD: Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature. 360:350–352. 1992. View Article : Google Scholar : PubMed/NCBI
19	Kastelein JJ, Wedel MK, Baker BF, Su J, Bradley JD, Yu RZ, Chuang E, Graham MJ and Crooke RM: Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipo-protein B. Circulation. 114:1729–1735. 2006. View Article : Google Scholar : PubMed/NCBI
20	Sazani P, Gemignani F, Kang SH, Maier MA, Manoharan M, Persmark M, Bortner D and Kole R: Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol. 20:1228–1233. 2002. View Article : Google Scholar : PubMed/NCBI
21	Vickers TA, Zhang H, Graham MJ, Lemonidis KM, Zhao C and Dean NM: Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2′-O-methoxyethyl-modified oligonucleotide. J Immunol. 176:3652–3661. 2006. View Article : Google Scholar : PubMed/NCBI
22	Doherty GJ and McMahon HT: Mechanisms of endocytosis. Annu Rev Biochem. 78:857–902. 2009. View Article : Google Scholar : PubMed/NCBI
23	Tonkinson JL and Stein CA: Patterns of intracellular compartmentalization, trafficking and acidification of 5′-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells. Nucleic Acids Res. 22:4268–4275. 1994. View Article : Google Scholar : PubMed/NCBI
24	Bennett CF and Swayze EE: RNA targeting therapeutics: Molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol. 50:259–293. 2010. View Article : Google Scholar : PubMed/NCBI