Construction of targeting human Na(+)-H(+) exchanger-1 gene adenovirus-mediated RNA interference expression vector and establishment of intracellular acidification model

Abstract

Objective To construct adenovirus-mediated RNA interference (RNAi) expression vector of targeting human Na(+)-H(+) exchanger-1 (NHE-1) gene to infect SH-SY5Y cells and establish intracellular acidification model in order to lay the foundation for researching the relation between intracellular pH value and sporadic Alzheimer's disease (AD). Methods Four kinds of recombined interfering plasmid pcDNATM 6.2 - GW/miR including specific pre-miRNA single stranded DNA oligonucleotide for NHE-1 and negative control plasmid pcDNATM 6.2-GW/neg-miR were constructed. The recombined plasmid with best interfere efficiency was detected by real⁃time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique. The best interfere fragment was recombined with pDONR221 vector and pAD/CMV/V5-DEST-GFP vector in turn by BP recombination system and LR recombination system. Recombinant adenovirus pAd-miR-NHE-1 was identified, purified, proliferated and titrated. The best multiplicity of infection (MOI) was determined by MOI gradient test. With optimum condition transfected SH-SY5Y, the intracellular pH was detected by fluorescent probe. Results PCR showed that the best RNAi expression vector of targeting human NHE-1 gene was constructed successfully. It could inhibit NHE-1 mRNA expression successfully. The best MOI was 160. According to the optimized MOI, SH-SY5Y cell line was infected by NHE-1 miRNA adenovirus. After 24, 48 and 72 h, the intracellular pH of adenovirus transfect group was 5.97 ± 0.03, 5.98 ± 0.02 and 5.98 ± 0.02, respectively; the intracellular pH of empty vector transfect group was 6.05 ± 0.04, 6.04 ± 0.07 and 6.03 ± 0.05, respectively; the intracellular pH of blank control group was 6.03 ± 0.06, 6.05 ± 0.04 and 6.03 ± 0.04, respectively. After 28, 48 and 72 h, the intracellular pH of adenovirus transfect group was decreased significantly compared to empty vector transfect group and blank control group (24 h: P = 0.002, 0.015; 48 h: P = 0.030, 0.012; 72 h: P = 0.018, 0.010). But at different period after transfection, the intracellular pH was of no obvious difference (P = 0.762). Conclusion The best RNAi expression vector of targeting human NHE-1 gene is successfully constructed and the intracellular acidification model is established which can provide an effective tool for further study of the relation between intracellular acid-base equilibrium and β-amyloid (Aβ) production.

DOI:10.3969/j.issn.1672-6731.2011.04.013