Monocytic microRNA profile associated with coronary collateral artery function in chronic total occlusion patients

An expansive collateral artery network is correlated with improved survival in case of adverse cardiac episodes. We aimed to identify cellular microRNAs (miRNA; miR) important for collateral artery growth. Chronic total occlusion (CTO) patients (n = 26) were dichotomized using pressure-derived collateral flow index (CFIp) measurements; high collateral capacity (CFIp > 0.39; n = 14) and low collateral (CFIp < 0.39; n = 12) capacity. MiRNA profiling via next generation sequencing from various monocyte phenotypes (freshly isolated monocytes, monocytes cultured without stimulant, or stimulation with lipopolysaccharide, interleukin 4, transforming growth factor beta-1, or interferon gamma) revealed significantly different miRNA expression patterns between high versus low collateral capacity patients. Validation by real-time polymerase chain reaction demonstrated significantly decreased expression of miR339-5p in all stimulated monocyte phenotypes of low collateral capacity patients. MiR339-5p showed significant correlation with CFIp values in stimulated monocytes. Ingenuity pathway analysis of predicted gene targets of miR339-5p and differential gene expression data from high versus low CFIp patients (n = 20), revealed significant association with STAT3 pathway, and also suggested a possible regulatory role for this signaling pathway. These results identify a novel association between miR339-5p and coronary collateral function. Future work examining modulation of miR339-5p and downstream effects on the STAT3 pathway and subsequent collateral vessel growth are warranted.


RNA isolation from supernatant
Supernatants were thawed (on ice) and centrifuged (3000 g, 5 min, 4 ºC). An aliquot (200 μL) was transferred to a FluidX tube with 60 μL lysis solution BF containing carrier-RNA (1 μg/60 μL). RNA spike-in template mixture was also added to the sample, mixed and incubated at room temperature (7 min), after which 20 μL Protein precipitation solution BF was added. Samples were centrifuged and supernatants were subjected to Exiqon's internal proprietary bead based RNA isolation protocol.
Purified RNA was eluted in 50 μL final volume and stored at -80 ºC until further processing.

Next Generation Sequencing
Each RNA sample had adaptors ligated to its 3' and 5' ends and subsequently converted into complementary deoxyribonucleic acid (cDNA). The cDNA was pre-amplified with specific primers containing sample specific indexes. After 15 cycle pre-PCR the libraries were purified on QiaQuick columns and the insert efficiency evaluated by Bioanalyzer 2100 instrument on high sensitivity DNA chip (Agilent Inc.). MiRNA cDNA libraries were size fractionated on a LabChip XT (Caliper Inc.) and a band representing adaptors and 15-40 bp insert was excised according to the manufacturer's instructions.
Samples were then quantified using quantitative polymerase chain reaction (qPCR) and concentration standards. Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar concentrations. Library pools were quantified again with qPCR and optimal concentration of the library pool was used to generate the clusters on the surface of a flowcell. Sequencing was then conducted using NextSeq 500 sequencing methodology and reagents based on the manufacturer instructions (Illumina Inc.). On average 14.1 million 50-bp single-end reads were obtained per sample.
Adapters were trimmed and subsequently reads representing high quality sequencing data were mapped to the human reference genome (GRCh37/hg19) annotated with miRBase version 20 to count reads associated with known human microRNAs. The number of miRNAs identified in all samples with at least one tag per million (TPM) mapped reads was 262. Differential expression analysis was done using the EdgeR package in the statistical software environment R. Counts were normalized using the trimmed mean of M-values (TMM normalization) method 1 , to compensate for sample specific effects caused by variation in library size/sequencing depth between samples. TMM normalization was conducted in the EdgeR statistical software package (Bioconductor, http://www.bioconductor.org/). For each miRNA and each phenotype P-values for differential expression between high and low CFIp patients were determined by an exact test on the negative binomial distribution.

Validation of next generation sequencing results by real-time PCR
ExiLENT SYBR® Green master mix was utilized to assay each selected miRNA once by qPCR on the microRNA Ready-to-Use PCR Custom Pick and Mix Panel. Negative controls excluding template from the reverse transcription reaction were included and assayed the same as samples. Amplification was conducted in a LightCycler® 480 Real-Time PCR System (Roche) with 384 well plates. Roche LC software was employed to assess amplification curves for calculation of the quantification cycle (Cq) values by the 2nd derivative method as well as melting curve examination. Normalization of cellular and extracellular data was performed using stably expressed endogenous controls for each monocyte/macrophage phenotype (Supplemental Figure 3). These controls were preselected based on the NGS results. lipopolysaccharide; TGFβ1: transforming growth factor beta 1.   Table 5. MiR155 is an upstream regulator of differentially expressed genes in freshly isolated (CD14) and interleukin 4 (IL4) stimulated monocytes in patients with high versus low collateral capacity.

Supplemental
Gene set p-value Target Molecules