LRH1 silencing inhibits human colon cancer growth.

This study was undertaken to better determine the function of LRH-1 (NR5A2) in the colorectal cancers (CRC) through inducible shRNA-mediated knockdown within well-established CRC cell line Caco-2 Our work is the first to demonstrate that silencing of LRH-1 in established human CRC cell lines impairs proliferation though G0/G1 phase prolongation, and alteration in diverse cellular pathways consistent with the critical role of LRH-1 in CRC. Two separate DNA sequences targeting the LRH-1 LBD- were cloned into parent vector pTripZ RHS4696 (Open Biosystems), with incorporation of silencing vector confirmed by fluorescent assay for marker TurboRFP. Non-silencing control cell lines were produced similarly using manufacturer’s vector (Open Biosystems, RHS4743). Cells were plated at a concentration of 100,000 cells per ml in 12 well plates and allowed to attach overnight. shRNA expression was induced by the addition of 5 μg/ml of doxycycline when the cells approached 75% confluence. Cells were harvested at 96 hours post induction using Trizol reagent (Life Technologies), and Total RNA harvested using PureLink RNA Mini Kit (Ambion) according to the manufacturer’s protocol.