Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Summary

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Abstract

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 °C instead of the conventional 70-100 °C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other 'large' proteins like BRCA2 should be readily achievable.

Introduction

The BRCA2 (BReast CAncer susceptibility gene-2) gene encodes for a tumor suppressor protein that plays a crucial role in repairing DNA double-strand breaks by regulating the function of the recombinase enzyme Rad51 ¹. BRCA2 has also been implicated in the modulation of transcription and in cell cycle control ². Germline mutations in the BRCA2 gene induce an autosomal dominant susceptibility to breast and ovarian cancer in women and prostate cancer in men, as well as predisposition to other cancer types ^3,4. However, despite the increased risk in developing cancer, BRCA2 mutations that suppress or reduce BRCA2....

Protocol

1. Prepare siRNA solutions

1. Resuspend 5 nmol of scrambled (Ctrl) and 5 nmol of BRCA2 siRNA dried pellets in 100 µl of RNase-free water (final siRNA concentration: 50 µM). This is the siRNA stock and must be stored at -20 °C in 10 µl aliquots.
2. Take an aliquot of 10 µl from the siRNA stock and add 40 µl of RNase-free water to obtain the 10 µM siRNA working solution. This dilution must be stored at -20 °C until use.
   NOTE: The siRNA working solution should not undergo freezing/thawing more than 3 times.

2. Seeding of Human Epithelial Cell Lines

1. Remov....

Results

Before proceeding with a biological/biochemical assay to examine the effect of BRCA2 silencing on cell functions, the first step is to confirm the specificity of the silencing by using a scrambled siRNA (non-targeting siRNA) side by side with BRCA2 siRNA (Figure 1A). It is important to perform a second round of siRNA transfection with a high siRNA/transfection ratio to get a high efficiency of BRCA2 silencing. Indeed, performing only one round of siRNA transfection or using a lower siRN.......

Discussion

Because germline mutations of the BRCA2 gene lead to increased risk of several cancer types, including female and male breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, and melanoma ^3,4, a number of studies have been undertaken to understand the biological function of the BRCA2 protein. Most of these studies are genetic-based mainly due to technical difficulties in analyzing a giant protein like BRCA2. The method described here for silencing and analyzing BRCA2 protein expression prov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BRCA2 siRNA	Dharmacon	L-003462-00	SMARTpool: ON-TARGETplus BRCA2 siRNA
Ctrl siRNA	Dharmacon	D-001810-10-05	ON-TARGETplus Non-targeting Pool
Lipofectamine RNAiMAX Reagent	Life Technologies	13778075	Transfection Reagent
OPTI-MEM I Reduced Serum Medium	Life Technologies	31985-070	Medium for transfection procedure
NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast	Life Technologies	EA0378	Gel for protein electrophoresis
NuPAGE LDS Sample buffer	Life Technologies	NP0007	Reagent for protein electrophoresis
NuPAGE Sample Reducing agent (10x)	Life Technologies	NP0004	Reagent for protein electrophoresis
NuPAGE Antioxidant	Life Technologies	NP0005	Reagent for protein electrophoresis
HiMark Pre-stained protein standard	Life Technologies	LC5699	Prestained marker for gel electrophoresis
XCell SureLoc Mini-Cell  System	Life Technologies	EI0002	Equipment for protein electrophoresis/transfer
NuPAGE Tris-Acetate SDS Running Buffer	Life Technologies	LA0041	Reagent for Western blotting
PVDF membrane	Millipore	IPVH00010	Reagent for Western blotting
NuPAGE Transfer Buffer	Life Technologies	NP0006-1	Reagent for Western blotting
BRCA2 H-300 rabbit polyclonal antibody	Santa Cruz Biotechnology	sc-8326	1:300 dilution
Beta-tubulin monoclonal antibody	Sigma Aldrich	T4026-100UL	1:1,000 dilution
Skim Milk powder	Sigma Aldrich	70166	Reagent for Western blotting
Tween-20	Sigma Aldrich	P1379	Reagent for Western blotting
SuperSignal West Pico/Femto Chemiluminescent Substrate	Pierce Thermo Scientific	34080/34096	Chemiluminescence system
PNT1A cells	SIGMA Aldrich (for ECACC)	95012614	PNT1A human, normal prostate epithelium immortalized with SV40
Nthy cells	SIGMA Aldrich (for ECACC)	90011609	Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells
Cell Proliferation Kit I (MTT)	Roche	11465007001	Kit for cell proliferation assays