MicroRNA expression in benign breast tissue and risk of subsequent invasive breast cancer

<div><p>MicroRNAs are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Abnormal expression of microRNAs is thought to contribute to the development and progression of cancer. A history of benign breast disease (BBD) is associated with increased risk of subsequent breast cancer. However, no large-scale study has examined the association between microRNA expression in BBD tissue and risk of subsequent invasive breast cancer (IBC). We conducted discovery and validation case-control studies nested in a cohort of 15,395 women diagnosed with BBD in a large health plan between 1971 and 2006 and followed to mid-2015. Cases were women with BBD who developed subsequent IBC; controls were matched 1:1 to cases on age, age at diagnosis of BBD, and duration of plan membership. The discovery stage (316 case-control pairs) entailed use of the Illumina MicroRNA Expression Profiling Assay (in duplicate) to identify breast cancer-associated microRNAs. MicroRNAs identified at this stage were ranked by the strength of the correlation between Illumina array and quantitative PCR results for 15 case-control pairs. The top ranked 14 microRNAs entered the validation stage (165 case-control pairs) which was conducted using quantitative PCR (in triplicate). In both stages, linear regression was used to evaluate the association between the mean expression level of each microRNA (response variable) and case-control status (independent variable); paired t-tests were also used in the validation stage. None of the 14 validation stage microRNAs was associated with breast cancer risk. The results of this study suggest that microRNA expression in benign breast tissue does not influence the risk of subsequent IBC.</p></div

Association between lifestyle factors and breast cancer risk based on subjects included in the discovery stage.

<p>Association between lifestyle factors and breast cancer risk based on subjects included in the discovery stage.</p

Association between benign breast disease histology and breast cancer risk.

<p>Association between benign breast disease histology and breast cancer risk.</p

Fold changes and p-values for the 14 miRNAs (plus 2 controls) evaluated in the validation stage.

<p>Fold changes and p-values for the 14 miRNAs (plus 2 controls) evaluated in the validation stage.</p

Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods.

<p>Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.</p

Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods.

<p>(A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.</p

DNA/RNA extractions using archived human specimens.

<p>Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.</p

Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods.

<p>The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.</p

Optimized TRIzol extraction of DNA from archived specimens.

<p>(A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig <i>et al.</i> 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034683#s2" target="_blank">materials and methods</a>. The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.</p

MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods.

<p>The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC_t (δC_t target miRNA - δC_t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.</p