Long non-coding RNAs modulate tumor microenvironment to promote metastasis: novel avenue for therapeutic intervention

Cancer is a devastating disease and the primary cause of morbidity and mortality worldwide, with cancer metastasis responsible for 90% of cancer-related deaths. Cancer metastasis is a multistep process characterized by spreading of cancer cells from the primary tumor and acquiring molecular and phenotypic changes that enable them to expand and colonize in distant organs. Despite recent advancements, the underlying molecular mechanism(s) of cancer metastasis is limited and requires further exploration. In addition to genetic alterations, epigenetic changes have been demonstrated to play an important role in the development of cancer metastasis. Long non-coding RNAs (lncRNAs) are considered one of the most critical epigenetic regulators. By regulating signaling pathways and acting as decoys, guides, and scaffolds, they modulate key molecules in every step of cancer metastasis such as dissemination of carcinoma cells, intravascular transit, and metastatic colonization. Gaining a good knowledge of the detailed molecular basis underlying lncRNAs regulating cancer metastasis may provide previously unknown therapeutic and diagnostic lncRNAs for patients with metastatic disease. In this review, we concentrate on the molecular mechanisms underlying lncRNAs in the regulation of cancer metastasis, the cross-talk with metabolic reprogramming, modulating cancer cell anoikis resistance, influencing metastatic microenvironment, and the interaction with pre-metastatic niche formation. In addition, we also discuss the clinical utility and therapeutic potential of lncRNAs for cancer treatment. Finally, we also represent areas for future research in this rapidly developing field

Genetic and epigenetic characterization of the sphingosine-1-phosphate signalling system in macrophages in chronic obstructive pulmonary disease

Alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic bronchial epithelial cells (a process termed ‘efferocytosis’) and bacteria. These defects may contribute to COPD pathogenesis in several ways. Secondary necrosis of uncleared apoptotic material may result in chronic airways inflammation and perpetuation of COPD disease. A reduced alveolar macrophage phagocytic host response to bacteria, especially non-typeable H influenzae (NTHi), may contribute to neutrophilic inflammation and NTHi colonization of the lower airway. However, the exact mechanism that leads to the phagocytic dysfunction is still unknown. The sphingosine 1-phosphate (S1P) signalling system is known to regulate macrophage function. Experiments described in Chapter 2 of the thesis therefore applied a novel approach of measuring all S1P signalling system components in alveolar macrophages from COPD patients and healthy controls. Several components of the S1P system, in particular relative mRNA levels for sphingosine kinases SPHK1 and S1P receptor S1PR5, were dysregulated in COPD and were strongly correlated with efferocytosis, suggesting a potential link to the defective alveolar macrophage phagocytic ability in COPD. Oxidative stress and inflammation have been shown to contribute to many COPD characteristics, such as uncontrolled activation of cell signalling pathways, increased airway epithelial cell apoptosis, and defective alveolar macrophage phagocytic ability. Chapter 3 describes the effect of two models of oxidative stress and inflammation, cigarette smoke (potential oxidative conditions) and lipopolysaccharide (LPS) (potential inflammatory conditions) on components of S1P signalling and on efferocytosis and phagocytosis of NTHi, using a human macrophage cell line in vitro. Cigarette smoke and LPS increased the mRNA expression of SPHK1 and S1PR5 in macrophages, extending the results in Chapter 2 and further supporting the potential link between the S1P signalling system and macrophage phagocytic ability. Cigarette smoke decreased the capacity of macrophages to phagocytose apoptotic cells and bacteria. However, LPS reduced phagocytosis of bacteria only. Treatment option for oxidative stress is anti-oxidants and thymoquinone (TQ) is anti-oxidant/anti-inflammatory agent that has been shown to modulate macrophage inflammatory responses and has successfully been trialled in human clinical studies. Chapter 3 further reports that TQ per se had a pro-phagocytic effect on macrophage phagocytic ability. TQ also rescued macrophages from the negative effects of cigarette smoke, and to lesser extent LPS, on macrophage efferocytosis and the mRNA expression respectively. In addition, TQ demonstrated a pro-survival effect on bronchial epithelial cells treated with cigarette smoke. The effects on relative mRNA expression of SPHK1 and S1PR5 in the cell line were mirrored using acutely isolated alveolar macrophages from COPD patients. COPD patients are at increased risk for developing lung cancer and there is strong evidence that pulmonary macrophage dysfunction plays an important role in the pathogenesis of both diseases. DNA methylation has been shown to be modified in COPD and lung cancer. However, it unknown whether the change in mRNA expression of the S1P system (Chapter 2) are controlled by epigenetic modifications such as DNA methylation, and whether DNA methylation regulates macrophage efferocytosis. Data presented in Chapter 4 connect epigenetic modulation, mRNA expression and macrophage function. The results indicate that DNA methylation potentially regulates macrophage efferocytosis and is negatively correlated with the mRNA expression of S1P system components, in particular the S1PR5 receptor, suggesting epigenetic for COPD such as anti-oxidants or epigenome modifying agents.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015

Potential Impact of MicroRNA Gene Polymorphisms in the Pathogenesis of Diabetes and Atherosclerotic Cardiovascular Disease

MicroRNAs (miRNAs) are endogenous, small (18–23 nucleotides), non-coding RNA molecules. They regulate the posttranscriptional expression of their target genes. MiRNAs control vital physiological processes such as metabolism, development, differentiation, cell cycle and apoptosis. The control of the gene expression by miRNAs requires efficient binding between the miRNA and their target mRNAs. Genome-wide association studies (GWASs) have suggested the association of single-nucleotide polymorphisms (SNPs) with certain diseases in various populations. Gene polymorphisms of miRNA target sites have been implicated in diseases such as cancers, diabetes, cardiovascular and Parkinson’s disease. Likewise, gene polymorphisms of miRNAs have been reported to be associated with diseases. In this review, we discuss the SNPs in miRNA genes that have been associated with diabetes and atherosclerotic cardiovascular disease in different populations. We also discuss briefly the potential underlining mechanisms through which these SNPs increase the risk of developing these diseases

Clinical Utility of Amplification Refractory Mutation System-Based PCR and Mutation-Specific PCR for Precise and Rapid Genotyping of Angiotensin-Converting Enzyme 1 (ACE1-rs4646996 D>I) and Angiotensin-Converting Enzyme 2 (ACE2-rs4240157T>C) Gene Variations in Coronary Artery Disease and Their Strong Association with Its Disease Susceptibility and Progression

Background: Experimental clinical and research studies demonstrated that the renin–angiotensin system (RAS) affects the pathogenesis of atherosclerosis and the prognosis of coronary heart disease (CHD). The results show that ACE2 (angiotensin I-converting enzyme 2) might act as a protective protein for cardiovascular diseases; however, only a few studies in human populations have been carried out. The aim of this study was to develop, optimize, and validate a direct T-ARMS-based PCR assay for the precise and rapid genotyping of ACE1-rs4646996 D>I and ACE2-rs4240157T>C and study their association with coronary artery disease susceptibility and progression. Methodology: This study included 149 consecutive coronary artery disease patients and 150 healthy controls. We utilized T-ARMS for the precise and rapid genotyping of ACE2-rs4240157; rs4646994. Results: Our results indicated that the ACE1-rs4646996 D>I genotypes observed between CAD cases and controls were statistically significant (p < 0.008) and, similarly, the ACE2-rs4240157T>C genotypes observed were significant (p < 0.0001). Moreover, the frequency of the D allele (ACE1-D>I) and C allele (ACE2-rs4240157T>C) was found to be higher among CAD patients than the HC. Our results indicated that in the codominant model, the ACE2-ID genotype was strongly associated with increased CAD susceptibility in a codominant model with an OR of 2.37, (95%) CI = (1.023–5.504), and p < 0.04. Similarly, the ACE2-DD genotype was strongly associated with an increased CAD susceptibility with an OR of 3.48, (95%) CI = (1.49 to 8.117), and p < 0.003. Similarly, in allelic comparison, the D allele was strongly associated with CAD susceptibility with an OR of 1.59, (95%) CI = (1.12–2.24), and p < 0.003. Our results revealed that there was a significant correlation between ACE2-I/D genotypes and hypertension, T2D, and obesity (p < 0.05). The results of ACE2 rs4240157 genotyping indicated a strong association in the codominant model with an increased CAD susceptibility with an OR of 3.62, (95%) CI = (2.027 to 6.481), and p < 0.0001. Similarly, in a dominant inheritance model, a strong association is observed between the ACE2 rs4240157 (CT+CC) genotype with an OR of 6.34, (95%) CI = (3.741 to 10.749), and p < 0.0001. In allelic comparison, the T allele was strongly associated with CAD susceptibility with an OR of 5.56, (95% CI = (3.56 to 7.17), and p < 0.0001. Similarly, our results revealed that there was a significant association of the ACE2-rs4240157T>C genotypes with Triglycerides (mg/dL), HDL-C (mg/dL), total Cholesterol (mg/dL), and C-reactive protein (mg/L) in CAD. Conclusion: It was indicated that the ARMS technique and MS-PCR assay proved to be fast, accurate, and reliable for ACE2-rs4240157T>C and ACE1-rs4646996 D>I, respectively, and can be used as a potential molecular tool in the diagnosis of genetic diseases in undeveloped and developing countries—where there might be a shortage of medical resources and supplies. ACE1-I>D genotypes were strongly associated with T2D, hypertension, and obesity (p < 0.002). Besides the ACE2-rs4240157 CT heterozygosity genotype, the T allele was strongly associated with CAD susceptibility. Future longitudinal studies in different ethnic populations with larger sample sizes are recommended to validate these finding

Genetic Determinants of Cardiovascular Disease: The Endothelial Nitric Oxide Synthase 3 (eNOS3), Krüppel-Like Factor-14 (KLF-14), Methylenetetrahydrofolate Reductase (MTHFR), MiRNAs27a and Their Association with the Predisposition and Susceptibility to Coronary Artery Disease

Coronary artery disease (CAD) is an important cause of death worldwide. CAD is caused by genetic and other factors including hypertension, hyperlipidemia, obesity, stress, unhealthy diet, physical inactively, smoking and Type 2 diabetes (T2D). The genome wide association studies (GWASs) have revealed the association of many loci with risk to diseases such as cancers, T2D and CAD. Nitric oxide (NO) is a potent vasodilator and is required for normal vascular health. It is produced in the endothelial cells in a reaction catalyzed by the endothelial NO synthase (eNOS). Methylenetetrahydrofolate reductase (MTHFR) is a very important enzyme involved in metabolism of folate and homocysteine, and its reduced function leads to cardiovascular disease. The Krüppel-like factor-14 (KLF-14) is an important transcriptional regulator that has been implicated in metabolic syndrome. MicroRNA (MiRNAs) are short non-coding RNAs that regulate the gene expression of proteins involved in important physiological processes including cell cycle and metabolism. In the present study, we have investigated the potential impact of germline pathogenic variants of endothelial eNOS, KLF-14, MTHFR, MiRNA-27a and their association with risk to CAD in the Saudi population. Methods: Amplification Refractory Mutation System (ARMS) PCR was used to detect MTHFR, KLF-14, miRNA-27a and eNOS3 genotyping in CAD patients and healthy controls. About 125 CAD cases and 125 controls were enrolled in this study and statistical associations were calculated including p-value, risk ratio (RR), and odds ratio (OD). Results: There were statistically significant differences (p T, KLF-14 rs972283 G>A, miRNAs27a rs895819 A>G and eNOS3 rs1799983 G>T between CAD patients and controls. In addition, our results indicated that the MTHFR-TT genotype was associated with increased CAD susceptibility with an OR 2.75 (95%) and p p p = 0.016. Our results also indicated that eNOS3 -GT genotype is associated with CAD susceptibility with an OR = 2.65, and p T, KLF14 rs972283 G>A, miRNAs27a A>G, and eNOS3 rs1799983 G>T genotypes were associated with CAD susceptibility (p < 0.05). These findings require verification in future large-scale population based studies before these loci are used for the prediction and identification of individuals at risk to CAD. Weight control, physical activity, and smoking cessation are very influential recommendations given by clinicians to the at risk individuals to reduce or delay the development of CAD

Correlations between relative mRNA expression of various components of the S1P signalling system in alveolar macrophages.

<p><b>(A)</b> correlation between <i>SPHK1 and</i> 2, p<0.05; <b>(B)</b> correlation between <i>S1PR1 and S1PR5</i>, p<0.05; <b>(C)</b> correlation between <i>S1PR2 and S1PR4</i>, p<0.05; <b>(D)</b> correlation between <i>SGPP1 and SGPP2</i> p<0.05; <b>(E)</b> correlation between <i>SGPL1</i> and <i>SGPP1</i> p<0.05; <b>(F)</b><i>SGPL1</i> and <i>SGPP2</i>. The correlation coefficients are listed in the results.</p

Relative mRNA expression of S1P degradation enzymes in alveolar macrophages.

<p><b>(A)</b> Significantly higher mRNA expression of <i>SGPL1</i> in COPD patients (4.5-fold increase) versus control subjects (*p<0.05 vs control). No significant increase of <i>SGPL1</i>was found in current smoker COPD, ex-smoker COPD or healthy current smokers.<b> (B)</b> Significantly higher mRNA expression levels of <i>SGPP2</i> in current smoker COPD patients (6.1-fold increase) versus control subjects (*p<0.05 vs control) was noted. No significant increase of <i>SGPP2</i> was found in ex-smoker COPD subjects or healthy current smokers Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122771#pone.0122771.g001" target="_blank">Fig 1</a>. The Ct values were subtracted from 20 so that higher values mean higher mRNA expression levels.</p

Correlations between the relative mRNA expression of various components of the S1P signalling system and lung function (fev₁).

<p>Significant (p<0.05) correlations were found between fev₁ and <b>(A)</b><i>SPHK1</i><b>(B)</b><i>S1PR1</i><b>(C)</b><i>S1PR3</i>, <b>(D)</b><i>S1PR5</i>, p<0.05. The correlation coefficients are listed in the results.</p