Genes regulated by Nkx2-3 in siRNA-mediated knockdown B cells: Implication of endothelin-1 in inflammatory bowel disease

Nkx2-3 gene variants are strongly associated with inflammatory bowel disease (IBD) and its expression is up-regulated in Crohn's disease (CD). However, the nature of its role underlying IBD pathogenesis is unknown. We investigated the genes regulated by Nkx2-3 using cDNA microarray. A small interfering RNA (siRNA)-mediated knockdown of Nkx2-3 in a B cell line from a CD patient was generated. Gene expression was profiled on high-density cDNA microarrays representing over 25,000 genes. Ingenuity pathway analysis (IPA) was used to identify gene networks according to biological functions and associated pathways. Expression profiling analysis by cDNA microarray showed that 125 genes were regulated by Nkx2-3 knockdown (fold change ⩾3.0, p < 0.01), among which 51 genes were immune and inflammatory response genes. Microarray results were validated by RT-PCR and further confirmed in a B cell line expressing siRNA of Nkx2-3 from an additional CD patient. The results showed that Nkx2-3 was up-regulated (p < 0.05) and EDN1 was down-regulated (p < 0.05) in B cell lines from CD patients. mRNA expression levels of Nkx2-3 were negatively correlated with those of EDN1 (r = −0.6044, p < 0.05). EDN1 was also down-regulated in intestinal tissues from UC patients (p < 0.05). Our present results demonstrate that a decrease in Nkx2-3 gene expression level can profoundly alter the expression of genes and cellular functions relevant to the pathogenesis and progression of IBD, such as EDN1.