RNA interference‐produced autoregulation of inducible nitric oxide synthase expression

Abstract

Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.

Contribution of microRNA-203 to the isoflurane preconditioning-induced neuroprotection

“…Quantitative PCR was carried out in triplicates using U6 snRNA as control as we described before [4]. Amplifying PCR and monitoring of the fluorescent emission in real time were performed in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems).…”

“…The sequence of miRNA-N is not similar to those of any known miRNAs. miRNA-N is recommended as a control miRNA or small interference RNA by Invitrogen and is used for this purpose in the literature [4]. Twenty fours later, cells were subjected to OGD or harvested for Western analysis.…”

Autoregulation of Inducible Nitric Oxide Synthase Expression by RNA Interference Provides Neuroprotection in Neonatal Rats

“…We have shown that autoregulation of iNOS expression is achievable in cell cultures 12 . We hypothesize that this autoregulation can provide neuroprotection against ischemia.…”

“…Three plasmids generated in our previous study 12 were used here. piNOS-N had an unrelated insert whose expression was controlled by the 3.2 Kb iNOS promoter.…”

Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor β2 Transcription in Human Neuroblastoma Cells

“…Total RNA Extraction and Real-time PCR-Real-time PCR was performed as described previously (12). The sequences of the primers were as follows: RFX1, forward, 5Ј-TCATCCGG-CTGCTCTACGA-3Ј, reverse, 5Ј-TGGCCAGATTGGCGA-ACT-3Ј; TGF␤2, forward, 5Ј-TCCAACCCAGCGCTAC-ATC-3Ј, reverse, 5Ј-GTGAAGCCATTCATGAACAGCAT-3Ј; TGF␤1, forward, 5Ј-TCGCCAGAGTGGTTATCTTTTG-3Ј, reverse, 5Ј-AGGAGCAGTGGGCGCTAAG-3Ј; TGF␤-R1, forward, 5Ј-GCAGAGCTGTGAAGCCTTGAG-3Ј, reverse, 5Ј-ATAATGTTTTCTTAATCCGCAATGC-3Ј; TGF␤-R2, forward, 5Ј-ACCACCAGGGCATCCAGAT-3Ј, reverse, 5Ј-CTGAAGCGTTCTGCCACACA-3Ј; and FGF1 (6), forward, 5Ј-ACAAGGGACAGGAGCGAC-3Ј, reverse, 5Ј-TCCAGCC-TTTCCAGGAACA-3Ј.…”