MicroRNA - Volume 7, Issue 2, 2018

Background: RNA interference (RNAi) and related pathways involving small interfering RNAs (siRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs) regulate processes such as antiviral defense, genome surveillance, heterochromatin formation, and gene expression in animals, plants, and fungi. Studies on RNAi have revealed a two-step mechanism: (i) Degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. (ii) The siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the mRNA and degrades it. Objective: Molecular structures of Dicer, Argonaute proteins, and RNA-bound complexes have offered insights into the underlying mechanisms of RNA-silencing pathways. Methods: Sequence specific gene silencing using small interfering RNA (siRNA) is now being evaluated as a novel therapeutic strategy. Results: Recently, promising data have been obtained from clinical trials for the treatment of respiratory syncytial virus and age-related macular degeneration. The exact mechanism of the RNAi pathways is still unclear. Conclusion: Our review summarizes the RNAi pathways and the known functions of siRNAs, miRNAs, and piRNAs in lower and higher organisms (mostly focusing on mammals) and discusses the potential applications of RNAi.

Background: High-risk HPV subtypes are driving forces for human cancer development: HPV-16 and HPV-18 are responsible for most HPV-caused cancers. Objective: This review describes the present knowledge on HR-HPV genomes coding potential for viral miRNAs. Methods: HPV subtypes miRNA database, VIRmiRtar, has been constructed applying bioinformatics and a computational method, ViralMir, exploiting structural features, the presence of hairpins, and validation by comparison with RNA sequencing datasets. Results: Several miRNA candidates have been localised in the genomes of high-risk HPV subtypes. Among these, HPV-16 miR-1, miR-2 and miR-3. The database contains a list of host candidate gene targets that may be responsible for the oncogenesis in the various cellular environments. Conclusion: miRNA silencing therapies, based on specific cellular uptake of miRNA mimics and antagomiRs, directed towards HPV encoded miRNAs and/or microRNAs deregulated in the host cells, could be a valuable approach to support pharmaceutical interventions in the treatment of HPV dependent cancers.

Background: Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death in the world and considered as one of the most susceptible cancers in humans. The microRNA molecule, hsa-miR122, considered as a potential biological marker linked with the injury of hepatocellular tissue, is the most common microRNA in human liver cancer. Understanding the expression profile of hsa-miR122 plays an important role in the diagnosis of HCC. Objective: Identification and comparison of cut-off values of plasma hsa-miR122 expression were conducted in blood samples of healthy control, HBV infected and HBV-related HCC Vietnamese patients. Methods and Result: Fifty-two blood samples of healthy control and HBV-related HCC cases, collected between 2015 and 2017 were obtained from Ho Chi Minh City Oncology Hospital, Vietnam. Written informed consent was attained from all patients and the Human Research Ethics Committee, Oncology Hospital (#08/BVUB-HDDD) approved the research protocol. Total RNA was isolated from blood samples with TrizolTM Reagent (Thermo Fisher Scientific, USA). To analyze the expression level of hsa-miR122, miRNA specific reverse transcription was performed using Sensi- FASTTM cDNA Synthesis Kit (Bioline, UK) as described by the manufacturer, followed by running RT-qPCR with SensiFASTTMSYBR No-ROX Kit (Bioline, UK). The housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used for normalization. The presence of hsamiR122 and HBV-DNA was identified in human blood using RT-PCR and LAMP techniques. Downregulation of plasma hsa-miR122 was observed in HBV-related HCC patients with a ΔCt value of 7.9 ± 2.1 which was significantly lower than found in healthy control (p<0.01). The loss of hsa-miR122 expression was observed in HBV infected patients. We also identified the difference of diagnostic values of this microRNA in different populations and provided a high diagnostic accuracy of HCC (AUC = 0.984 with sensitivity and specificity of 96% and 94%, respectively). Conclusion: hsa-miR122 was downregulated in HBV-related HCC patients and found to be lower by approximately 10 fold than in healthy control, resulting in a potential biomarker for microRNA based diagnosis of HCC in human blood.

Background: Single Nucleotide Polymorphisms (SNPs) in microRNA (miRNA) networks may serve as diagnostic and prognostic biomarkers of a variety of diseases such as cancer. Some studies have been performed to examine associations between miR-149 and miR-608 polymorphisms and susceptibility to colorectal cancer, but the results remain controversial and race-dependent. Objective: The aim of our study was to investigate the association of miR-608 (rs4919510) and miR- 149 (rs2292832) with colorectal cancer and its clinical features in a sample of Iranian population. Methods: This retrospective study was conducted on 76 CRC cases and 70 controls. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) method. To confirm the RFLP process, 10% of the PCR products were validated by direct sequencing. Results: Our findings showed significant correlation between adjusted data of rs2292832 with sex and age in TT genotype (OR= 5.148, 95% CI=1.081 ± 24.511, P=0.04). Distribution of rs4919510 polymorphism was not significantly different between controls and patients (CG, adjusted OR= 1.243, 95% CI=0.546 ± 2.831; P=0.604 and GG, adjusted OR= 0.249, 95% CI=0.063 ± 0.959; P=0.05). On the other hand, our results showed that a significant correlation was present between metastatic clinicopathological features and miR-608 (rs4919510) polymorphism (P=0.044). Conclusion: Our findings reveal that genotypes of rs2292832 and rs4919510 are not associated with risk of colorectal cancer in Iranian population. Moreover, the CC genotype of rs4919510 contributes to the metastatic features of the colorectal cancer.

Background: Human Milk (HM) is a biological fluid representing the first nutrient for newborns. It directly impacts the development of the infant's immune system. In this concern, specific microRNAs (miRNAs) such as hsa-miR-21, hsa-miR-181a, hsa-miR-150 and hsa-miR-223 are known to be involved in the innate and acquired immune response. Objective: Herein, these miRNAs were evaluated in frozen and pasteurized samples of human colostrum and HM in order to elucidate the distribution and the expression profile of these biological mediators in both biological fluids. Methods: Using quantitative approach qRT-PCR, we analyzed immune-related microRNAs in both, colostrum and HM. Results: Our study provided evidence of a comparable profile of immune specific miRNAs in colostrum and HM. Although we detected all the four miRNAs tested, we point out the prevalence of hsamiR- 181a and hsa-miR-223 indicative to act on T and granulocytes cell populations as selective targets. Therefore, these biomolecules could affect newborn's immune homeostasis at early stages of life. While, variation in immune-related miRNAs was found in HM amongst lactating women, it was not evidenced in colostrum. Of interest, pasteurization procedure did not alter the distribution or the expression profile of the miRNAs tested in both colostrum and HM. Herein, we also proposed a simple method to determine the quantity of these biomolecules in biological fluids. Conclusion: Considering, this evidence the variation in immune-related miRNAs should be take into account and could be relevant for preterm and hospitalized infants who usually received pasteurized HM from donors.

Background: Circulating cell-free miRNAs hold great promise as a new class of biomarkers due to their high stability in body fluids and association with disease stages. However, even using sensitive and specific methods, technical challenges are associated with miRNA analysis in body fluids. A major source of variation in plasma and serum is the potential cell-derived miRNA contamination from hemolysis. Objectives: The study aimed to evaluate the effect of the delayed whole blood processing time on the concentrations of miR-1 and -423-5p. Methods: Ten blood samples were incubated for 0, 3 and 24 hours at room temperature prior to processing into plasma. For each time point, hemolysis was assessed in plasma by UV spectrophotometry at 414nm wavelength (λ414). Circulating levels of miR-1 and -423-5p were measured by RT-qPCR; miR-23a and -451 were also analyzed as controls. Results: A significant hemolysis was observed only after 24h (λ414 0.3 ± 0.02, p < 0.001). However, only small changes in miR-1 and -423-5p levels were observed up to 24h of storage at room temperature (Ct 31.5 ± 0.5 to 31.8 ± 0.6for miR-1, p = 0.989; and 29.01 ± 0.3 to 29.04 ± 0.3, p = 0.614 for - 423-5p). No correlation was observed between hemolysis and the levels of miR-1 and -423-5p. Conclusion: Our data indicate that the storage of whole blood samples at room temperature for up to 24h prior to their processing into plasma does not appear to have a significant impact on miR-1 and - 423-5p concentrations.

Background: Evidence is increasing that microRNAs (miR) are particularly important in lung homeostasis and development and have been shown to be involved in many pulmonary diseases such as asthma, chronic obstructive pulmonary disease, sarcoidosis, Lung Cancer (LC) and other smoking-related diseases. Objective: The objective of this study was to investigate the expression of miR-155 and miR-486-5p in tissues from LC patients and healthy endobronchial mucosa as prognostic biomarkers for diagnosing LC. Methods: Bronchoscopic and thoracoscopic tissue biopsies were taken from 50 LC patients and other 50 control subjects without lung mass, who were planned for a clinical bronchoscopy. The expressions of miR-155 and miR-486-5p in both tumor tissue and healthy mucosa were detected by quantitative real-time polymerase chain reaction. Results: Histopathology showed that 72% of LC patients were in advanced stages III and IV, with non-small cell lung carcinoma and adenocarcinoma being the most common diagnosis. miR-155 was significantly overexpressed while, miR-486-5p was underexpressed, in LC patients as compared to controls. Area under receiver operating characteristic curves of miR-155 (<-0.9) and miR-486 (>-0.62) had sensitivity of 92 and 96% and specificity of 80 and 84%, respectively, in discriminating LC patients from controls with benign solitary pulmonary nodules. Conclusion: miR-155 was highly overexpressed, yet it did not correlate with stages, while miR-486- 5p was extremely underexpressed and significantly correlated with stages of LC. Thus, their detection represents an excellent diagnostic/prognostic tool to support more established techniques linked to LC spread locally and systemically.

Background: The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events. Methods: This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs. Results: The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs. Conclusion: Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).

Background: The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material. Objective: Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction. Methods: Three μl of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays. Results: Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies. Conclusion: We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.

Background: Increased cardiovascular disease risk and prevalence associated with overweight and obesity is due, in part, to heightened inflammatory burden. The mechanisms underlying adiposity-related amplification of inflammation are not fully understood. Alterations in regulators of inflammatory processes such as microRNAs (miRs), however, are thought to play a pivotal role. Objective: The aim of this study was to determine the influence of overweight and obesity, independent of other cardiovascular risk factors, on circulating expression of miR-34a, miR-126, miR-146a, miR-150 and miR-181b. Methods: Forty-five sedentary, middle-aged (47-64 years) adults were studied: 15 were normal weight (8M/7F; BMI: 23.3 ± 0.3 kg/m2); 15 were overweight (8M/7F; 28.2 ± 0.3 kg/m2); and 15 were obese (7M/8F; 32.3 ± 0.5 kg/m2). All subjects were non-smokers, normotensive and free of overt cardiometabolic disease. Circulating levels of the following inflammation-related miRs: miR-34a, miR-126, miR-146a, miR-150 and miR-181b were determined in plasma using standard RT-PCR techniques. miR expression was normalized to exogenous C. elegans miR-39 and reported as relative expression (AU). Results: Circulating miR-34a was ~200% higher (P < 0.05) in the obese as compared with normal weight and overweight groups. Whereas, miR-126, miR-146a and miR-150 were significantly lower (~65%) in both the obese and overweight groups than the normal weight group. There were no significant group differences in circulating expression of miR-181b. miR-34a was positively related (r = 0.43; P < 0.05); whereas, miR-126 (r = -0.48), miR-146a (r = -0.33) and miR-150 (r = -0.43) levels were significantly inversely related to BMI. Conclusion: Overweight and obesity, independent of other cardiometabolic risk factors, negatively influences circulating inflammation-related miRs. Dysregulation of circulating miRs may contribute mechanistically to the heightened inflammatory state associated with overweight and obesity.