NF-kappaBp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1beta-induced and TNF-alpha-induced chondrocytes.
OBJECTIVE
Small interfering RNA (siRNA) triggers RNA interference in mammalian somatic cells. Nuclear factor kappaB (NF-kappaB) is a transcription factor that is implicated in inflammation and immune activation. This study was to use NF-kappaBp65-specific siRNA to inhibit the expression of genes of cyclooxygenase-2 (COX-2), nitric oxide synthase-2 (NOS-2) and matrix metalloproteinase-9 (MMP-9), which is paralleled with the initiation and progression of cartilage lesions in osteoarthritis (OA) model, in induced chondrocytes, and therefore to explore a new gene therapy for OA.


METHODS
Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to optimize the silencing effects of NF-kappaBp65-specific siRNA in cultured rat chondrocytes, and then to determine the expression of COX-2, NOS-2 and MMP-9 in induced chondrocytes. The activation of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). Western blot and RT-PCR were subjected to densitometric analysis and then band intensities were also determined.


RESULTS
The NF-kappaBp65-specific siRNA inhibited the expression of NF-kappaBp65 and activation of NF-kappaB, reducing significantly the expression of COX-2, NOS-2 and MMP-9 induced by interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in cultured chondrocytes.


CONCLUSIONS
NF-kappaBp65-specific siRNA can inhibit the expression of COX-2, NOS-2 and MMP-9 in IL-1beta-induced and TNF-alpha-induced chondrocytes. This suggests that NF-kappaBp65-specific siRNA has potential to be a useful, preventive and therapeutic agent for OA at early stages.
Lianxu
C
C
Institute of Sports Medicine, Peking University Third Hospital, No. 49, North Garden Road, Haidian District, Beijing 100083, China.
Hongti
J
J
Changlong
Y
Y
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism; D019902:Chondrocytes / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics; D007375:Interleukin-1 / Q000037:antagonists & inhibitors*; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D020370:Osteoarthritis, Knee / Q000378:metabolism / Q000473:pathology; D034741:RNA, Small Interfering; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D007375:Interleukin-1; D016328:NF-kappa B; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D020780:Matrix Metalloproteinase 9