Values are means??SDs There was no significant change in the expression of NR2A\D mRNA after injection of NR1 shRNAmir1 (Figure?3D). of different doses of NR1 shRNAmir at different time points before injection of formalin. Pain behavior was assessed by monitoring the paw flinch response, paw withdrawal threshold, and thermal withdrawal latency. We then analyzed NR1 messenger RNA and protein expression in skin and the L5 dorsal root ganglion (DRG). Results We found that intradermal injection of 1 1, 5, and 10?g of shRNAmir significantly inhibited flinch responses (were designed based on GenBank accession number NM 017010. The following sequences were?the target sequences tested: sequence 1, GGGTAAACAACAGCAACAA (shRNAmir1); sequence 2, AGACTAAAGATAGTGACAA (shRNAmir2); sequence 3, nonsilencing shRNAmir (NS\shRNAmir) (Nonsilencing lentiviral shRNAmir, RHS 4346, Open Biosystems, Huntsville, AL, USA). No significant homology to known rat gene sequences was found in the GenBank database. Lentiviral vectors encoding shRNAmirs were made using the backbone of the pGIPZ vector (Open Biosystems), which was remodeled to express green fluorescent protein and shRNAmirs from a bicistronic transcript driven by a cytomegalovirus (CMV) promoter, followed by a PURO resistance gene (pGIPZ\shRNAmir). The template miRNA30\like DNA oligonucleotides targeting two positions of NR1 mRNAs (shRNAmir1 and shRNAmir2) were ligated into the assessments were used to compare the data in the time course study in the NR1 mRNA and western blot and in the analysis of NR2 subunits and interferon\ and pERK change in DRG. Except in the time course study, data from western blot of the NR1 subunit, rotarod test, CFA test, and antinociceptive effect of NR1 shRNAmir in each group were analyzed by one\way ANOVA with the Bonferroni post Compound E hoc test. Paired assessments were used to test the differences in NR1, NR2 subunits, and interferon\ between the left and right sides of the DRGs. Compound E A p\value of less than .05 was considered statistically significant. The analyses were performed with SPSS software (14.0; SPSS Inc., Chicago, IL, USA). 3.?Results 3.1. Effects of NR1 shRNAmir1 and Compound E shRNAmir2 on formalin\ and CFA\induced pain Injection of 1% formalin into the paw induced two phases of nociceptive response, with the first phase beginning immediately and persisting for 5?min. The second phase Compound E began approximately 15C20?min after injection of formalin and persisted for 20C40?min. Rats that received intradermal injections of 5?g shRNAmir1 or shRNAmir2 showed significantly fewer formalin\induced flinch responses during the post injection period of 20C50?min than rats that received 1?l PEI or 100?l saline (p?p?SLC3A2 received 5?g NR1 shRNAmir1 (Physique?1C). Open in a separate window Physique 1 Antinociceptive and gene silencing effects of two NR1 short hairpin (sh)RNAmirs. A significant decrease in flinch number between 20 and 50?min in formalin\induced nociceptive behavior (A) and a significant decrease in the expression of NR1 messenger RNA (mRNA) (B) were noted after injection of NR1 shRNAmir1 and shRNAmir2. *p?<?.05 NR1 shRNAmir1 and shRNAmir2 versus saline and polyethyleneimine (PEI) groups. (C) NR1 shRNAmir produced an antinociceptive effect on CFA\induced nociception. Compared with baseline values, a significant decrease in 50% paw withdrawal threshold was noted in groups of rats that received injection of saline or PEI, but not in group of rats that received 5?g NR1 shRNAmir1. *p?<?.05 compared with baseline values. Values are means??SDs 3.2. DoseCresponse antinociceptive and gene silencing effects of NR1 shRNAmir1 Intradermal injection of 5.