An in-silico approach to study the possible interactions of miRNA between human and SARS-CoV2

BackgroundThe progressive SARS-CoV2 outbreaks worldwide have evoked global investigation. Despite the numerousin-silico approaches, the virus-host relationship remains a serious concern. MicroRNAs are the small non-coding RNAs that help in regulating gene profiling. The current study utilized miRNA prediction tools along with the PANTHER classification system to demonstrate association and sequence similarities shared between miRNAs of SARS-CoV2 and human host.MethodAn in-silico approach was carried out using Vmir analyzer to predict miRNAs from SARS-CoV2 viral genomes. Predicted miRNAs from SARS-CoV2 viral genomes were used for effective hybridization sequence identification along the nucleotide similarities with human miRNAs from miRbase database. Further, it was proceeded to analyze the gene ontology using miRDB with PANTHER classification.ResultBased on the prediction and analysis, we have identified 22 potential miRNAs from five genomes of SARS-CoV2 linked with 12 human miRNAs. Analysis of human miRNAs hsa-mir-1267, hsa-mir-1-3p, hsa-mir-5683 were found shared between all the five viral SARS-CoV2 miRNAs. Further, PANTHER classification analyzed the gene-ontology being carried by these associations showed that 44 genes were involved in biological functions that includes genes specific for signaling pathway, immune complex generation, enzyme binding with effective role in the virus-host relationship.ConclusionOur analysis concludes that the genes identified in this study can be effective in analyzing the virus-host interaction. It also provides a new direction to understand viral pathogenesis with a probable new way to link, that can be used to understand and relate the miRNAs of the virus to the host conditions.     

A Target Recycling Amplification Process for the Digital Detection of Exosomal MicroRNAs through Photonic Resonator Absorption Microscopy

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis-ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub-attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT-PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low-cost, and minimally invasive point-of-care testing.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Polyphenol-Enriched Blueberry Preparation Controls Breast Cancer Stem Cells by Targeting FOXO1 and miR-145

Scientific evidence supports the early deregulation of epigenetic profiles during breast carcinogenesis. Research shows that cellular transformation, carcinogenesis, and stemness maintenance are regulated by epigenetic-specific changes that involve microRNAs (miRNAs). Dietary bioactive compounds such as blueberry polyphenols may modulate susceptibility to breast cancer by the modulation of CSC survival and self-renewal pathways through the epigenetic mechanism, including the regulation of miRNA expression. Therefore, the current study aimed to assay the effect of polyphenol enriched blueberry preparation (PEBP) or non-fermented blueberry juice (NBJ) on the modulation of miRNA signature and the target proteins associated with different clinical-pathological characteristics of breast cancer such as stemness, invasion, and chemoresistance using breast cancer cell lines. To this end, 4T1 and MB-MDM-231 cell lines were exposed to NBJ or PEBP for 24 h. miRNA profiling was performed in breast cancer cell cultures, and RT-qPCR was undertaken to assay the expression of target miRNA. The expression of target proteins was examined by Western blotting. Profiling of miRNA revealed that several miRNAs associated with different clinical-pathological characteristics were differentially expressed in cells treated with PEBP. The validation study showed significant downregulation of oncogenic miR-210 expression in both 4T1 and MDA-MB-231 cells exposed to PEBP. In addition, expression of tumor suppressor miR-145 was significantly increased in both cell lines treated with PEBP. Western blot analysis showed a significant increase in the relative expression of FOXO1 in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Furthermore, a significant decrease was observed in the relative expression of N-RAS in 4T1 and MDA-MB-231 cells exposed to PEBP and in MDA-MB-231 cells exposed to NBJ. Our data indicate a potential chemoprevention role of PEBP through the modulation of miRNA expression, particularly miR-210 and miR-145, and protection against breast cancer development and progression. Thus, PEBP may represent a source for novel chemopreventative agents against breast cancer.     

Suitable reference genes for relative quantification of miRNA expression in prostate cancer

Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa- miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa- miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.     

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.     

In-silico investigations of selective miRNA-gene targets and their validation studies in obstructive sleep apnea (OSA) patient cohorts

BackgroundObstructive sleep apnoea (OSA) is a prevalent form of sleep disordered breathing which results in sleep fragmentation and deprivation. Obesity and cardiovascular disorders are the major risk factors associated with OSA. Molecular analysis of the factors associated with OSA could demarcate the clinical analysis pattern in a population.ObjectiveThis study pertains to in-silico analyses of miRNA and their gene targets with validation for their potential role in OSA as putative biomarker candidates.MethodsmiRDB, TargetScan and miRanda databases were used to identify targets of miR-27 and let-7 that have documented role in OSA and co-related obesity and cardiovascular disorders. Quantitative PCR was used to analyze expression pattern of miR-27 and let-7 in obese and non-obese OSA patient cohorts with respective controls. In-silico analysis was done using PatchDoc to obtain atomic contact energy (ACE) scores that indicated the docked gene targets to the predicted miRNA structures. The docked structures were analysed using Maestro Suite 11 for the hydrogen and aromatic interactions.ResultsDownregulation of miR-27 and let-7 in OSA compared to controls was observed. In-silico data analysis was performed for gene targets (TGFBR1, TGFBR2, SMAD2, SMAD4, CRY2 and CNR1) of the selected miRNAs (miR-27 and let-7). Among all, CNR1 and CRY2 were found to be better targets for miR-27 and let-7 respectively as per ACE scores, ROC scores and expression fold change in OSA.ConclusionOur study gives insights to the expression profiling of miR-27 and let-7 and explore a set of potential target genes (CNR1 and CRY2) of these two miRNAs for a promising clinical relevance in OSA.     

Electrochemical Biosensors for Cancer Biomarkers Detection: Recent Advances and Challenges

Biomarkers are described as characteristics that provide information about biological conditions whether normal or pathological. Detection of biomarkers at the earliest stage of the cancer is of utmost importance for clinical diagnosis. Electrochemical biosensors allow detecting the low levels of specific analytes in blood, urine or saliva and providing a sensitive approach for direct measurement for cancer biomarker detection. Moreover, the integration of electrochemical devices with nanomaterials, such as carbon nanotubes, gold and magnetic particles offer amplification and multiplexing capabilities for simultaneous measurements of cancer biomarkers very sensitively. This review summarizes the recent developments of electrochemical biosensors systems for the detection of cancer biomarkers with emphasis on voltammetric, amperometric and impedimetric biosensors. A special attention is paid to aptamers and miRNAs that are very promising for the ultra‐sensitive and specific cancer biomarker detection.     

Expression of miRNAs Targeting <Emphasis Type="Italic">mTOR</Emphasis> and <Emphasis Type="Italic">S6K1</Emphasis> Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3′-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.     

Bioinformatic Analysis and in Vitro Validation of Let-7b and Let-7c in Breast Cancer

Members of the let-7 family of miRNAs are well-known with their tumor suppressor properties as they are expressed at low levels in several types of human malignancies. Among them, let-7b and let-7c have gained special attention due their broad significance. Although the role of let-7b and let-7c have been widely reported in various types of cancers, their functional importance and role in oncogenic signaling of breast cancer is poorly investigated. Therefore, in the present study, prognostic and diagnostic significance of let-7b and let-7c in breast cancer and the effects these miRNAs on genes involved in cancer progression were determined by using several bioinformatics analysis and validated in vitro mimic assays, respectively. Using data of TCGA, OncomiR and dbDEMC 2.0, overall expression analysis of let-7b and let-7c was performed. The effect of let-7b and let-7c on genes involved in cancer progression was investigated by mimic transfection assays. We found that both let-7b and let-7c were significantly altered in breast cancer and associated with the clinicopathological findings of patients. Additionally, both let-7b and let-7c significantly altered oncogenic signaling in breast cancer cells. Consequently, both miRNAs might have fundamental roles in breast cancer progression and can be considered as potential targets for breast cancer therapy and diagnosis.     

Os(VI)bipy-based electrochemical assay for detection of specific microRNAs as potential cancer biomarkers

Recently, altered expression levels of microRNAs (miRNAs) – short noncoding RNA molecules which bind to mRNAs and thus regulate gene expression – were observed in many cancer cells. miRNA expression profiling is therefore of great interest, but current standard methods are still considered relatively laborious and expensive. Electrochemistry has a potential to become quick and inexpensive alternative. Here, we describe modification of miRNA with an electroactive complex composed of six-valent osmium and 2,2′-bipyridine, Os(VI)bipy, specifically binding to the 3′-end of the ribose, which is detectable at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of a resulting signal. By combining miRNA labeling step with magnetic beads-based hybridization assay, detection of specific miRNA sequence from a mixture of other noncomplementary miRNAs was possible.     

A Two‐Stage Dissociation System for Multilayer Imaging of Cancer Biomarker‐Synergic Networks in Single Cells

The monitoring of cancer biomarkers is crucial to the early detection of cancer. However, a limiting factor in biomarker analysis is the ability to obtain the multilayered information of various biomarker molecules located at different parts of cells from the plasma membrane to the cytoplasm. A two‐stage dissociation nanoparticle system based on multifunctionalized polydopamine‐coated gold nanoparticles (Au@PDA NPs) is reported, which allows for the two‐stage imaging of cancer biomarkers in single cells. We demonstrate the feasibility of this strategy on sialic acids (SAs), p53 protein, and microRNA‐21 (miRNA‐21) in MCF‐7 breast cancer cells by two custom‐built probes. Furthermore, the multicolor fluorescence information extracted is used for the monitoring of biomarker expression changes under different drug combinations, which allows us to investigate the complex interactions between various cancer biomarkers and to describe the cancer biomarker‐synergic networks in single cells.     

Comprehensive analysis of microRNA-mRNA co-expression in circadian rhythm

To investigate the potential role of microRNA (miRNA) in the regulation of circadian rhythm, we performed microarray-based expression profiling study of both miRNA and mRNA in mouse liver for 48 h at 4-hour intervals. Circadian miRNA-mRNA target pair is defined as the pair both elements of which show circadian expression patterns and the sequence-based target relationship of which can be predicted. Circadian initiators, Clock and Bmal1, showed inversely correlated circadian expression patterns against their corresponding miRNAs, miR-181d and miR-191, targeting them. In contrast, circadian suppressors, Per, Cry, CKIe and Rev-erba, exhibited positively correlated circadian expression patterns to their corresponding miRNAs. Genomic location analysis revealed that intronic region showed higher abundance of cyclic than non-cyclic miRNAs targeting circadian genes while other (i.e., 3''-UTR, exon and intergenic) regions showed no difference. It is suggested that miRNAs are involved in the regulation of peripheral circadian rhythm in mouse liver by modulating Clock:Bmal1 complex. Identifying specific miRNAs and their targets that are critically involved in circadian rhythm will provide a better understanding of the regulation of circadian-clock system.