Ribonomic and short hairpin RNA gene silencing methods to explore functional gene programs associated with tumor growth arrest

Abstract

In 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.

Fig. 1

Overview of the strategy. Immunoprecipitation of hnRNPs from dormant/ non-tumorigenic cells allows for the identification of p38-target mRNAs (Subheading 3.1.). The upregulated messages may be knocked down via shRNA expressing vectors (Subheading 3.2.). The cells expressing shRNAs are introduced onto the chorioallantoinc membrame of a chick embryo (Subheading 3.3.). Growth of the shRNA cells above that of the parental cell line indicates that the knocked down gene is involved in tumor dormancy or survival (Subheading 3.4.). The other possible outcome (not shown, see Subheading 3.4.) is that the gene is required for survival and/or growth in vivo, so no tumor nodule will be detected.

Fig. 2

(A) Immunopurification of mRNA binding proteins HuR and hNRNP A1. Whole cell lysates (Subheading 3.1.1.) and cytoplasmic lysates (Subheading 3.1.2.) were immunopurified following the approprate procedure (Subheadings 3.1.4.1. and 3.1.4.2., respectively) using anti-HuR and anti-IgG and anti-hnRNP A1antibodies accordingly. A fraction of the immunopurified protein was analysed by Western blotting using anti-HuR antibody (left panel), or anti-hnRNP A1 antibody (right panel). Both HuR and hnRNP A1 are specifically immunopurified compared with their respective IgG controls. WCL, whole cell lysates; CL, cytoplasmic lysates; WB, Western blot. (B) RT-PCR analysis of mRNA associated with HuR and hnRNP A1 mRNP complex. RNA extracted from HuR and hnRNP A1 mRNP complexes (Subheadings 3.1.4.1. and 3.1.4.2., respectively) was subjected to RT-PCR analysis for the targets β-actin (left panel) or fibronectin-EDB (FN-EDB⁺, right panel). Fibronectin-EDB is the alternative spliced form of fibronectin mRNA. As hnRNP A1 is involved in alternative splicing, we tested for the association of EDB mRNA with hnRNP A1.

Fig. 3

Schematic representation of the retrovirus producing pSHAG-MAGIC vector. (A) Hairpin constructs are designed and ligated into pSHAG-MAGIC. The region flanked by two long terminal repeats contains the shRNA and the puromycin selection cassette. The vector contains the RK6γ origin which requires expression of PIR1 for replication. (B) Once the hairpin has been inserted into pSHAG-MAGIC, the vector is used to transfect packaging cells capable of producing retrovirus. Viral particles are then used to infect the target cell line. The barcode sequence can be used to follow hairpin construct in complex population via microarray hybridization as described in ref..

Fig. 4

A graph portraying three possible phenotypic outcomes of shRNA mediated downregulation of gene expression. (A) Growing nodule (open triangles, shRNA1): Genes are required for induction/maintenance of dormancy. (B) Dormant nodule (closed circles, shRNA2): genes are not required for induction/maintenance of dormancy. (C) No detectable growth (open diamonds, shRNA3): genes are required for survival in vivo.