Ribonomic and Short Hairpin RNA Gene Silencing Methods to Explore Functional Gene Programs Associated With Tumor Growth Arrest

Abstract

SummaryIn this chapter, we present an approach using genomic and ribonomic profiling to investigate functional gene programs in a tumor growth model. To reach this goal, ribonomic profiling was combined with RNA interference in a tumor dormancy model. Strategies merging functional genomic technologies are outlined for the identification of novel posttranscriptionally regulated targets of p38 to show that they are functionally linked to the induction or interruption of cellular growth in cancer. In the first section of this chapter, we describe a method for the detection of mRNA subsets associated with RNA-binding proteins such as hnRNP A1 using (1) immunopurification of mRNA-protein complexes, from either whole cell lysates or subcellular fractions and (2) gene expression arrays to find those mRNAs bound to hnRNP A1. In the second section, short hairpin RNA technology was used to create a library of shRNAs that target p38 induced mRNAs expression libraries are utilized to "knockdown" the genes identified in the first section. Finally, this library of gene candidates is evaluated in vivo to address their functional role in the induction or maintenance of dormancy.

HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes

“…[59]. All steps took place at 0 to 4 °C, starting with the preparation of antibody coated protein A-sepharose (PAS) bead matrix (Sigma-Aldrich, St. Louis, MO, USA).…”

“…Fractionated nuclear extracts from the triplicate experiments (each containing about 3 × 10 7 cells) were subjected to immunoprecipitation (IP) assays applying the basic procedure used before [ 31 ] slightly modified according to the protocol of Baroni et al . [ 59 ]. All steps took place at 0 to 4 °C, starting with the preparation of antibody coated protein A-sepharose (PAS) bead matrix (Sigma-Aldrich, St. Louis, MO, USA).…”

“…Thereafter, the quickly thawed nuclear extracts were first clarified at 13,000 rpm, 10 min to remove any insoluble material and, then, brought to a final volume of 1 mL in buffer A with the addition of PMSF, EDTA, DTT, RNase out and placental RNase inhibitor at the amounts indicated in Baroni et al . [ 59 ]. Before IP, pre-clearing of the nuclear extracts was performed to reduce unspecific binding by 30 min incubation with PAS beads been pre-coated with 6 μg IgG2.…”

The <em>In Ovo </em>Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

“…The CAM assay has been used extensively to study angiogenesis 4 . It has also been successfully developed into a tumor xenograft model of cancers, including glioblastoma 5 , pancreatic cancer 6 , melanoma [7][8][9] , and osteosarcoma [10][11] . Both in ovo 12 and ex ovo 13 techniques have been utilized in the literature, with details varying from protocol to protocol.…”