Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing

Purpose: Prostate cancer (PCa) is the second leading cause of tumor mortality among males in western societies. In China, the diagnostic and fatality rate of PCa is increasing yearly. MicroRNAs (miRNAs) are small single stranded non-coding RNA molecules (~22 nucleotides) which impede protein production by directly interacting with 3’-untranslated regions of the target mRNAs. miRNAs are crucial regulators in malignant tumors. Recent profiling research suggests that miRNAs are aberrantly expressed in PCa, and these have been implicated in the regulation of apoptosis, cell cycle, epithelial to mesenchymal transition, PCa stem cells, and androgen receptor pathway.Methods: To find miRNAs differentially expressed in PCa and their relation to prognostic factors and therapeutic potentials, we studied 24 surgical specimens from men who underwent radical prostatectomy, through high-throughput Illumina sequencing and quantitative real-time PCR (qRT-PCR) methods. Moreover, a variety of biological information softwares and databases were applied to predict the target genes of miRNA, molecular functions, and signal pathways. We also discuss the functional significance of the differentially expressed miRNAs and the molecular pathways/targets regulated by these miRNAs.Results: Many miRNAs were differentially expressed (fold change 2, P&lt;0.05) by sequencing. This was confirmed by qRT-PCR in more clinical tissue samples. In the tumors, miRNAs (miR-125b-5p, miR-126-5p, miR-141, miR-151a-5p, miR-221-3p and miR-222-3p) were significantly upregulated with downregulation of miR-486-5p and miR-488. In addition, 13 novel miRNAs were identified from three prostate tissue libraries, with 12 of them assayed in 21 human normal tissues by qRT-PCR. Multiple databases indicated target genes for these differentially expressed miRNAs. Function annotation of target genes indicated that most of them tend to target genes involved in signal transduction and cell communication, especially cancer-related PI3K-Akt and p53 signaling pathway. Moreover, miR-141 and miR-488 post-transcriptionally regulated androgen receptor (AR) expression, and inhibited the growth and metastasis of prostate gland epithelial cells.Conclusion: The small RNA transcriptomes obtained in this study uncovers the differentially expressed miRNAs, and provides a better understanding of the expression and function of miRNAs in the development of PCa and reveals several miRNAs in PCa that may have biomarker and therapeutic potentials.-----------------------------------------Cite this article as:  Song C, Chen H, Ru G, Ding Q, Yang W. Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing. Int J Cancer Ther Oncol 2015; 3(4):3405.[This abstract was presented at the BIT’s 8th Annual World Cancer Congress, which was held from May 15-17, 2015 in Beijing, China.]</p

Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing

Purpose: Prostate cancer (PCa) is the second leading cause of tumor mortality among males in western societies. In China, the diagnostic and fatality rate of PCa is increasing yearly. MicroRNAs (miRNAs) are small single stranded non-coding RNA molecules (~22 nucleotides) which impede protein production by directly interacting with 3’-untranslated regions of the target mRNAs. miRNAs are crucial regulators in malignant tumors. Recent profiling research suggests that miRNAs are aberrantly expressed in PCa, and these have been implicated in the regulation of apoptosis, cell cycle, epithelial to mesenchymal transition, PCa stem cells, and androgen receptor pathway.Methods: To find miRNAs differentially expressed in PCa and their relation to prognostic factors and therapeutic potentials, we studied 24 surgical specimens from men who underwent radical prostatectomy, through high-throughput Illumina sequencing and quantitative real-time PCR (qRT-PCR) methods. Moreover, a variety of biological information softwares and databases were applied to predict the target genes of miRNA, molecular functions, and signal pathways. We also discuss the functional significance of the differentially expressed miRNAs and the molecular pathways/targets regulated by these miRNAs.Results: Many miRNAs were differentially expressed (fold change 2, P&lt;0.05) by sequencing. This was confirmed by qRT-PCR in more clinical tissue samples. In the tumors, miRNAs (miR-125b-5p, miR-126-5p, miR-141, miR-151a-5p, miR-221-3p and miR-222-3p) were significantly upregulated with downregulation of miR-486-5p and miR-488. In addition, 13 novel miRNAs were identified from three prostate tissue libraries, with 12 of them assayed in 21 human normal tissues by qRT-PCR. Multiple databases indicated target genes for these differentially expressed miRNAs. Function annotation of target genes indicated that most of them tend to target genes involved in signal transduction and cell communication, especially cancer-related PI3K-Akt and p53 signaling pathway. Moreover, miR-141 and miR-488 post-transcriptionally regulated androgen receptor (AR) expression, and inhibited the growth and metastasis of prostate gland epithelial cells.Conclusion: The small RNA transcriptomes obtained in this study uncovers the differentially expressed miRNAs, and provides a better understanding of the expression and function of miRNAs in the development of PCa and reveals several miRNAs in PCa that may have biomarker and therapeutic potentials.-----------------------------------------Cite this article as:  Song C, Chen H, Ru G, Ding Q, Yang W. Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing. Int J Cancer Ther Oncol 2015; 3(4):3405.[This abstract was presented at the BIT’s 8th Annual World Cancer Congress, which was held from May 15-17, 2015 in Beijing, China.

Effects of vasoactive drugs on crystalloid fluid kinetics in septic sheep

<div><p>Purpose</p><p>Crystalloid fluid and vasoactive drugs are used in the early treatment of sepsis. The purpose of the present study was to examine how these drugs alter plasma volume expansion, peripheral edema, and urinary excretion.</p><p>Methods</p><p>Twenty-five anesthetized sheep were made septic by cecal puncture and a short infusion of lipopolysaccharide. After 50 min, a slow infusion of isotonic saline was initiated: the saline either contained no drug, norepinephrine (1 μg/kg/min), phenylephrine (3 μg/kg/min), dopamine (50 μg/kg/min), or esmolol (50 μg/kg/min). Ten min later, 20 mL/kg Ringer´s lactate solution was given over 30 min. Central hemodynamics, acid-base balance, and the urinary excretion were monitored. Frequent measurements of the blood hemoglobin concentration were used as input in a kinetic analysis, using a mixed effects modeling software.</p><p>Results</p><p>The fluid kinetic analysis showed slow distribution and elimination of Ringer´s lactate, although phenylephrine and dopamine accelerated the distribution. Once distributed, the fluid remained in the peripheral tissues and did not equilibrate adequately with the plasma. Overall, stimulation of adrenergic alpha₁-receptors accelerated, while beta₁-receptors retarded, the distribution and elimination of fluid. A pharmacodynamic E_max model showed that Ringer´s lactate increased stroke volume by 13 ml/beat. Alpha₁-receptors, but not beta₁-receptors, further increased stroke volume, while both raised the mean arterial pressure. Modulation of the beta₁-receptors limited the acidosis.</p><p>Conclusions</p><p>Stimulation of adrenergic alpha₁-receptors with vasoactive drugs accelerated, while beta₁-receptors retarded, the distribution and elimination of fluid. The tendency for peripheral accumulation of fluid was pronounced, in particular when phenylephrine was given.</p></div

Pharmacokinetic and pharmacodynamic parameters in the final model.

<p>Pharmacokinetic and pharmacodynamic parameters in the final model.</p

Hemodynamics.

<p>Early sepsis (-60 to -10 min) compared with treatment using vasoactive drugs and fluid (0 to 150 min). Data are mean (SD) based on patient mean values during the indicated period of time. Due to the small number in each group (n = 5), statistical comparions were made by using the Wilcoxon matched-pair test. * indicates statistical significance. <b><i>Abbreviations</i>:</b> CO = cardiac output, SV = stroke volume, MAP = mean arterial pressure; CVP = central venous pressure, BE = base excess; Nor = norepinephrine, Phe = phenylephrine, Dop = dopamine, Esm = esmolol.</p

The fluid kinetic model used to analyze the dilution of arterial plasma.

<p><b><i>Abbreviations</i>:</b><i>V</i>_c and <i>V</i>_t = central and peripheral fluid space expanded by infused fluid to <i>v</i>_c and <i>v</i>_t. <i>k</i>₁₂ and <i>k</i>₂₁ = rate constants governing the fluid transfer from <i>v</i>_c and <i>v</i>_t and vice versa. <i>k</i>₁₀ = elimination rate constant.</p

The curve-fitting procedure.

<p><b>A:</b> The measured plasma dilution over time in all sheep, regardless of vasoactive drug. <b>B:</b> The plasma dilution predicted from the base model (four parameters only) without consideration of covariates (drug effects), and <b>C:</b> The plasma dilution predicted from the final model with covariate effects.</p

Simulation of adrenergic effects on fluid distribution.

<p>Distribution of 20 ml/kg Ringer´s lactate infused over 30 min in sheep weighing 20 kg, depending on the degree of alpha₁-adrenergic <b>(A)</b> and beta₁-adrenergic <b>(B)</b> stimulation, graded between 0 and +3, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172361#pone.0172361.t001" target="_blank">Table 1</a>. The kinetic parameters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172361#pone.0172361.t003" target="_blank">Table 3</a> were used for the simulation.</p

Fluid distribution.

<p>Volume expansion of the central fluid space (<i>V</i>_c, the plasma), the peripheral fluid space (<i>V</i>_t) and the excreted urine (the elimination) during infusion experiments with 20 mL/kg of Ringer´s lactate over 30 min in septic sheep who were also given a vasoactive substance. Computer simulation using the parameter estimates shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172361#pone.0172361.t003" target="_blank">Table 3</a>.</p

The level of stimulation of receptors used in the volume kinetic analysis.

<p>Zero means no effect, +3 strong effect, and -1 means inhibition.</p