Identification of genes that are linked with optineurin expression using a combined RNAi–microarray approach

Abstract

Mutations in the optineurin gene are associated with open-angle glaucoma. Its gene product is a 74 kDa protein implicated in several cellular pathways. Although a range of interacting partners of optineurin have been identified, its physiological and pathophysiological role remains unclear. To understand comprehensive molecular mechanisms by which optineurin mediates, we identified genome-wide molecular changes upon silencing optineurin in HeLa cells by using microarray technology. A series of differentially expressed genes due to reduced expression of optineurin was identified. Network analyses showed that most of the functional categories of identified genes are associated with cellular function and maintenance as well as cellular assembly and organization. From these networks 22 genes were selected for confirmation by quantitative real-time PCR (Q-RT–PCR). To eliminate false-positive results due to off-target effects, a second siRNA was used to transfect HeLa cells and candidate genes were re-analyzed in these samples applying Q-RT–PCR. Several genes turned out to be differentially expressed in both siRNA experiments and changes in expression were confirmed on protein level. Coupling RNAi knockdown with microarray and Q-RT–PCR analyses provided several candidate genes that are linked with optineurin expression and confirms the assumption that optineurin is involved in trafficking processes and cellular morphology.