• BMB Reports
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• v.47 no.11
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• pp.619-624
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• 2014

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

• BMB Reports
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• v.41 no.8
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• pp.568-574
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• 2008

Antisense RNA molecules are powerful tools for controlling the expression of specific genes but their use in prokaryotes has been limited by their unpredictable antisense effectiveness. Moreover, appreciation of the molecular mechanisms associated with silencing in bacteria is still restricted. Here we report our attempts to define an effective antisense strategy in E. coli, and to dissect the observed silencing process. Antisense constructs complementary to different regions of lacZ were investigated, and silencing was observed exclusively upon expression of antisense RNA hybridising the 5'UTR of lac messenger. The level of lacZ mRNA was reduced upon expression of this antisense construct, and the silencing competence was found to be closely associated with its stability. These observations may help in the design of antisense molecules directed against prokaryotic genes.

• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.241-252
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• 2023

The era of innovative RNA therapies using antisense oligonucleotides (ASOs), siRNAs, and mRNAs is beginning. Since the emergence of the concept of ASOs in 1978, it took more than 20 years before they were developed into drugs for commercial use. Nine ASO drugs have been approved to date. However, they target only rare genetic diseases, and the number of chemistries and mechanisms of action of ASOs are limited. Nevertheless, ASOs are accepted as a powerful modality for next-generation medicines as they can theoretically target all disease-related RNAs, including (undruggable) protein-coding RNAs and non-coding RNAs. In addition, ASOs can not only downregulate but also upregulate gene expression through diverse mechanisms of action. This review summarizes the achievements in medicinal chemistry that enabled the translation of the ASO concept into real drugs, the molecular mechanisms of action of ASOs, the structure-activity relationship of ASO-protein binding, and the pharmacology, pharmacokinetics, and toxicology of ASOs. In addition, it discusses recent advances in medicinal chemistry in improving the therapeutic potential of ASOs by reducing their toxicity and enhancing their cellular uptake.

• Molecules and Cells
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• v.28 no.4
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• pp.341-345
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• 2009

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs. Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases. For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3203-3207
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• 2012

Backgrounds: To investigate the inhibiting effects of multi-target anti-microRNA antisense oligonucleotide (MTg-AMOs) on proliferation and migration of human gastric cancer cells. Methods: Single anti-microRNA antisense oligonucleotides (AMOs) and MTg-AMOs for miR-221, 21, and 106a were designed and transfected into SGC7901, a gastric cancer cell line, to target the activity of these miRNAs. Their expression was analyzed using stem-loop RT-PCR and effects of MTg-AMOs on human gastric cancer cells were determined using the following two assay methods: CCK8 for cell proliferation and transwells for migration. Results: In the CCK-8 cell proliferation assay, $0.6{\mu}mol/L$ was selected as the preferred concentration of MTg-AMOs and incubation time was 72 hours. Under these experimental conditions, MTg-AMOs demonstrated better suppression of the expression of miR-221, miR-106a, miR-21 in gastric cancer cells than that of single AMOs (P = 0.014, 0.024; 0.038, respectively). Migration activity was also clearly decreased as compared to those in randomized and blank control groups ($28{\pm}4$ Vs $54{\pm}3$, P <0.01; $28{\pm}4$ Vs $59{\pm}4$, P < 0.01). Conclusions: MTg-AMOs can specifically inhibit the expression of multiple miRNAs, and effectively antagonize proliferation and migration of gastric cancer cells promoted by oncomirs.

• BMB Reports
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• v.28 no.6
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• pp.538-545
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• 1995

When DNA replication of simian virus 40 (SV40) is initiated on the replication origin, the regions containing the initiation sites of DNA primase, which participates in the transient RNA primer synthesis for formation of Okazaki fragments in the lagging strand, were chosen as the target sites of antisense RNA for studies of the inhibition of SV40 DNA replication. Four recombinant transcription vectors, pUC-PrI, pUC-PrII, pGEM-PrBS, and pGEM-PrSN, coding antisense RNA, were constructed. Four antisense RNAs (named as I, II, BS, and SN) having the size of 18, 19,58, and 123 nts, respectively, were made from the transcription vectors by in vitro transcription. And then, antisense RNA in the concentration of 2${\mu}m$ were added to COS cells transfected with pATSV-W which is a recombinant plasmid containing the SV40 origin of replication. The inhibitory extent of DNA replication was measured by DpnI resistance and was confirmed by measurement of transient RNA primer synthesis. The result shows that six combinations of antisense RNA (I, II, BS, SN, I+SN, and BS+SN) lead to the inhibition of SV40 DNA replication by up to 85%.

• BMB Reports
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• v.50 no.4
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• pp.220-225
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• 2017

Antisense transcripts were initially identified as transcriptional noise, but have since been reported to play an important role in the quality control of miRNA functions. In this report, we tested the hypothesis that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates miRNA function via competitive endogenous RNAs, such as pseudogenes, long non-coding RNAs, and antisense transcripts. Based on analyses of RNA sequencing data, the knockdown of hnRNPK decreased the antisense PTOV1-AS1 transcript which harbors five binding sites for miR-1207-5p. We identified heme oxygenase-1 (HO-1) mRNA as a novel target of miR-1207-5p by western blotting and Ago2 immunoprecipitation. The knockdown of hnRNPK or PTOV1-AS1 suppressed HO-1 expression by increasing the enrichment of HO-1 mRNA in miR-1207-5p-mediated miRISC. Downregulation of HO-1 by a miR-1207-5p mimic or knockdown of hnRNPK and PTOV1-AS1 inhibited the proliferation and clonogenic ability of HeLa cells. Taken together, our results demonstrate that hnRNPK-regulated PTOV1-AS1 modulates HO-1 expression via miR-1207-5p.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7081-7084
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• 2013

MicroRNAs have been demonstrated to regulate proliferation and apoptosis in many types of cancers, but biological functions in osteosarcomas remain relatively unknown. Here, we found expression of miR-802 to be up-regulated in osteosarcoma tissues in comparison with adjacent normal tissues. Enforced expression of miR-802 was able to promote cell proliferation in U2OS and MG63 cells, while miR-802 antisense oligonucleotides (antisense miR-802) inhibited cell proliferation. At the molecular level, our results further revealed that expression of p27, a negative cell-cycle regulator, was negatively regulated by miR-802. Therefore, the data reported here indicate that miR-802 is an important regulator in osteosarcoma, our findings contributing to a better understanding of important mis-regulated miRNAs in this tumour type.

• Molecules and Cells
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• v.43 no.12
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• pp.975-988
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• 2020

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1A-AS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxia-induced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4127-4130
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• 2013

MicroRNA-16 (miR-16) has been demonstrated to regulate proliferation and apoptosis in many types of cancers, but its biological function in bladder cancer remains unknown. Here, we found expression of miR-16 to be downregulated in bladder cancer in comparison with the adjacent normal tissues. Enforced expression of miR-16 was able to inhibit cell proliferation in TCHu-1 cells, in line with results for miR-16 antisense oligonucleotides (antisense miR-16). At the molecular level, our results further revealed that cyclin D1 expression was negatively regulated by miR-16. Therefore, the data reported here demonstrate that miR-16 is an important regulator in bladder cancer, which will contribute to better understanding of important mis-regulated miRNAs.