Mamld1 knockdown with siRNA in mouse Leydig tumor cells. Mus musculus

MAMLD1 (mastermind-like domain containing 1, alias CXorf6) on human chromosome Xq28 is a causative gene for hypospadias, a mild form of 46,XY disorders of sex development. To date, multiple mutations have been identified in patients with various types of hypospadias. In this regard, the mouse homologous gene Mamld1 is transiently expressed in fetal Sertoli and Leydig cells around the critical period for sex development, and transient Mamld1 knockdown using small interfering RNAs (siRNAs) reduces testosterone production in cultured mouse Leydig tumor cells (MLTCs). We performed detailed analyses in Mamld1 knockdown experiments using MLTCs. We used microarrays to display the global gene expression profile change, after knockdown of Mamld1 expression in MLTCs, to find the gene set regulated by Mamld1. To compare changes in gene expression profile in MLTCs, MLTCs were transfected with control siRNA or Mamld1 siRNA, 48h later, total RNA was subjected to Q-PCR to confirm a significantly decreased Mamld1 mRNA, and then these total RNA samples were for microarray analysis. The microarray data was analyzed to explore gene sets/pathways, which were significantly impacted by knockdown of Mamld1. Real-time RT-PCR and microarray analyses showed significantly decreased Cyp17a1 expression (~70%) in both siRNA1- and siRNA2-transfected MLTCs. The siRNAs knockdown did not affect the expressions of Nr5a1 (Sf1), Star, Por, and Insl3. 47 genes including a Notch-related gene Hey1 were significantly up-regulated (fold change >2.0) and 38 genes were significantly down-regulated (fold change =2.0