A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis - PubMed (original) (raw)

PTPN22 knockdown by RNAi increases antigen-receptor signaling in Jurkat T-cell line. Knockdown and NF-κB transcriptional response after T-cell receptor stimulation for cells transfected with two control siRNAs (Scramble) and two PTPN22 siRNAs. First, 4.5 μg siRNA (Dharmacon) and 1 μg pNF-κB-luc plasmid (Stratagene) were electroporated into

2×106

Jurkat cells (ATCC, clone E6-1, homozygous for 620R) in Cell Line Nucleofector Solution V, according to manufacturer’s protocol #S18 (amaxa). After 24 h,

5.5×104

cells were stimulated with anti-CD3 (2 μg/ml [BD Pharmingen, clone UCHT1]), anti-CD28 (2 μg/ml [BD Pharmingen, clone CD28.2]), and anti-mouse IgG1 (2 μg/ml [BD Pharmingen, clone A85-1]). Cells were lysed and assayed for luminescence 6 h after stimulation by use of Bright-Glo (Promega) and a 96-well MicroBeta plate reader (Wallac). PTPN22 siRNAs that were effective for knockdown were found by screening seven candidate siRNA sequences that were designed by use of Web-based tools from Ambion and Dharmacon. siRNA sequences used in this study were PTPN22.1 (5′-

GGCAGACAAAACCTATCCT), PTPN22.2 (5′-

GGATTCCAGCTACATCAAT), Scramble.1 (5′-

GAACGGCATCAAGGTGAAC), and Scramble.2 (5′-

TTCTCCGAACGTGTCACGT). RNA was harvested according to the manufacturer’s protocol (RNeasy 96 [Qiagen]), and TaqMan real-time quantitative RT-PCR (SDS-7900 [Applied Biosystems]) was used to measure PTPN22 expression levels. PTPN22 mRNA levels in each sample were normalized to total RNA amounts (RiboGreen [Molecular Probes]) and then expressed relative to PTPN22 levels in cells that were untransfected with siRNA. The PTPN22 TaqMan sequences were 5′-GGCCCAAAGCAAGAAAATTACTAAA (forward), 5′-TGTCTGCCTTGTACTTGGTAGATTG (reverse), and 5′-TTCAGCTTCAGAAATT (MGB probe).