Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.     

Prenatal Exposure to TCDD Triggers Significant Modulation of microRNA Expression Profile in the Thymus That Affects Consequent Gene Expression

Background

MicroRNAs (miRs) are a class of small RNAs that regulate gene expression. There are over 700 miRs encoded in the mouse genome and modulate most of the cellular pathways and functions by controlling gene expression. However, there is not much known about the pathophysiological role of miRs. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), an environmental contaminant is well known to induce severe toxicity (acute and chronic) with long-term effects. Also, in utero exposure of fetus to TCDD has been shown to cause thymic atrophy and alterations in T cell differentiation. It is also relevant to understand “the fetal basis of adult disease” hypothesis, which proposes that prenatal exposure to certain forms of nutritional and environmental stress can cause increased susceptibility to clinical disorders later in life. In the current study, therefore, we investigated the effects of prenatal exposure to TCDD on miR profile in fetal thymocytes and searched for their possible role in causing thymic atrophy and alterations in the expression of apoptotic genes.

Methodology/Principal Findings

miR arrays of fetal thymocytes post exposure to TCDD and vehicle were performed. Of the 608 mouse miRs screened, 78 miRs were altered more than 1.5 fold and 28 miRs were changed more than 2 fold in fetal thymocytes post-TCDD exposure when compared to vehicle controls. We validated the expression of several of the miRs using RT-PCR. Furthermore, several of the miRs that were downregulated contained highly complementary sequence to the 3′-UTR region of AhR, CYP1A1, Fas and FasL. Also, the Ingenuity Pathway Analysis software and database was used to analyze the 78 miRs that exhibited significant expression changes and revealed that as many as 15 pathways may be affected.

Conclusions/Significance

These studies revealed that TCDD-mediated alterations in miR expression may be involved in the regulation of its toxicity including cancer, hepatic injury, apoptosis, and cellular development.     

Regulation of proto-oncogene Orai3 by miR18a/b and miR34a

Store Operated Ca²⁺ Entry (SOCE) mediated by Orai channels is a ubiquitous Ca²⁺ influx pathway that regulates several cellular functions. We have earlier reported that Orai3, the mammalian specific Orai1 homolog, plays a critical role in breast cancer progression. More recently, Orai3 was demonstrated to regulate prostate and lung tumorigenesis. Although the tumorigenic potential of Orai3 is associated with increase in its expression, the molecular machinery regulating its expression remains largely unexplored. Here, by performing extensive bioinformatics analysis and functional studies, we identify and characterize micro-RNAs (miRNAs) that regulate Orai3 expression and function. We demonstrate that miR18a and miR18b positively regulate Orai3 whereas miR34a represses Orai3 expression and function. All these miRs exert their effect on Orai3 by virtue of their direct action on Orai3 3′UTR. These miRs provide novel opportunities for targeting Orai3 for better management of cancer. This study further opens up the possibility of targeting specific Orai homologs by different miRs in tissue and disease specific context.     

Defining lung cancer stem cells exosomal payload of miRNAs in clinical perspective

Since the first publication regarding the existence of stem cells in cancer [cancer stem cells(CSCs)] in 1994, many studies have been published providing in-depth information about their biology and function. This research has paved the way in terms of appreciating the role of CSCs in tumour aggressiveness, progression,recurrence and resistance to cancer therapy. Targeting CSCs for cancer therapy has still not progressed to a sufficient degree, particularly in terms of exploring the mechanism of dynamic interconversion between CSCs and non-CSCs. Besides the CSC scenario, the problem of cancer dissemination has been analyzed indepth with the identification and isolation of microRNAs(miRs), which are now considered to be compelling molecular markers in the diagnosis and prognosis of tumours in general and specifically in patients with non-small cell lung cancer.Paracrine release of miRs via "exosomes"(small membrane vesicles(30-100 nm),the derivation of which lies in the luminal membranes of multi-vesicular bodies)released by fusion with the cell membrane is gaining popularity. Whether exosomes play a significant role in maintaining a dynamic equilibrium state between CSCs and non-CSCs and their mechanism of activity is as yet unknown.Future studies on CSC-related exosomes will provide new perspectives for precision-targeted treatment strategies.     

MicroRNA signatures in tumor tissue related to angiogenesis in non-small cell lung cancer

Background

Angiogenesis is regarded as a hallmark in cancer development, and anti-angiogenic treatment is presently used in non-small cell lung cancer (NSCLC) patients. MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression. In this study we aimed to identify significantly altered miRs related to angiogenesis in NSCLC.

Methods

From a large cohort of 335 NSCLC patients, paraffin-embedded samples from 10 patients with a short disease specific survival (DSS), 10 with a long DSS and 10 normal controls were analyzed. The miRs were quantified by microarray hybridization and selected miRs were validated by real-time qPCR. The impacts of different pathways, including angiogenesis, were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). One of the most interesting candidate markers, miR-155, was validated by in situ hybridization (ISH) in the total cohort (n = 335) and correlation analyses with several well-known angiogenic markers were done.

Results

128 miRs were significantly up- or down-regulated; normal versus long DSS (n = 68) and/or normal versus short DSS (n = 63) and/or long versus short DSS (n = 37). The pathway analysis indicates angiogenesis-related miRs to be involved in NSCLC. There were strong significant correlations between the array hybridization and qPCR validation data. The significantly altered angiogenesis-related miRs of high interest were miR-21, miR-106a, miR-126, miR-155, miR-182, miR-210 and miR-424. miR-155 correlated significantly with fibroblast growth factor 2 (FGF2) in the total cohort (r = 0.17, P = 0.002), though most prominent in the subgroup with nodal metastasis (r = 0.34, P<0.001).

Conclusions

Several angiogenesis-related miRs are significantly altered in NSCLC. Further studies to understand their biological functions and explore their clinical relevance are warranted.     

A systematic screen for micro-RNAs regulating the canonical Wnt pathway

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of a-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.     

Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer

microRNAs (miRs) modulate the expression levels of mRNAs and proteins and can thus contribute to cancer initiation and progression. In addition to their intracelluar function, miRs are released from cells and shed into the circulation. We postulated that circulating miRs could provide insight into pathways altered during cancer progression and may indicate responses to treatment. Here we focus on pancreatic cancer malignant progression. We report that changes in miR expression patterns during progression of normal tissues to invasive pancreatic adenocarcinoma in the p48-Cre/LSL-Kras(G12D) mouse model mirrors the miR changes observed in human pancreatic cancer tissues. miR-148a/b and miR-375 expression were found decreased whereas miR-10, miR-21, miR-100 and miR-155 were increased when comparing normal tissues, premalignant lesions and invasive carcinoma in the mouse model. Predicted target mRNAs FGFR1 (miR-10) and MLH1 (miR-155) were found downregulated. Quantitation of nine microRNAs in plasma samples from patients distinguished pancreatic cancers from other cancers as well as non-cancerous pancreatic disease. Finally, gemcitabine treatment of control animals and p48-Cre/LSL-Kras(G12D) animals with pancreatic cancer caused distinct and up to 60-fold changes in circulating miRs that indicate differential drug effects on normal and cancer tissues. These findings support the significance of detecting miRs in the circulation and suggests that circulating miRs could serve as indicators of drug response.     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

Lung cancer cells and their sensitivity/resistance to cisplatin chemotherapy: Role of microRNAs and upstream mediators

Cisplatin (CP) is a well-known chemotherapeutic agent with excellent clinical effects. The anti-tumor activity of CP has been demonstrated in different cancers such as breast, cervical, reproductive, lung, brain, and prostate cancers. However, resistance of cancer cells to CP chemotherapy has led to its failure in eradication of cancer cells, and subsequent death of patients with cancer. Fortunately, much effort has been put to identify molecular pathways and mechanisms involved in CP resistance/sensitivity. It seems that microRNAs (miRs) are promising candidates in mediating CP resistance/sensitivity, since they participate in different biological aspects of cells such as proliferation, migration, angiogenesis, and differentiation. In this review, we focus on miRs and their regulation in CP chemotherapy of lung cancer, as the most malignant tumor worldwide. Oncogenic miRs trigger CP resistance in lung cancer cells via targeting various pathways such as Wnt/β-catenin, Rab6, CASP2, PTEN, and Apaf-1. In contrast, onco-suppressor miRs inhibit oncogene pathways such as STAT3 to suppress CP resistance. These topics are discussed to determine the role of miRs in CP resistance/sensitivity. We also describe the upstream modulators of miRs such as lncRNAs, circRNAs, NF-κB, SOX2 and TRIM65 and their association with CP resistance/sensitivity in lung cancer cells. Finally, the effect of anti-tumor plant-derived natural compounds on miR expression during CP sensitivity of lung cancer cells is discussed.     

Downregulated NOX4 underlies a novel inhibitory role of microRNA-137 in prostate cancer

Prostate cancer is the second highest caused by cancer-related death among males. microRNAs (miRs) have been reported to participate in carcinogenesis, yet their roles in prostate cancer are rarely studied or investigated. Therefore, the present study attempted to explore the effect of miR-137 in prostate cancer via regulating NADPH oxidase 4 (NOX4). Initially, microarray analysis was performed to obtain prostate cancer-related differentially expressed genes and miRs that regulated NOX4, followed by detecting the expression of miR-137 and NOX4 and its target relationship. Moreover, PC-3 cells were transfected with small interfering RNA (siNOX4) and miR-137 mimic for exploring the effect of miR-137 on glycolysis, cell proliferation, and apoptosis in prostate cancer by evaluating lactate production, glucose uptake, adenosine triphosphate (ATP) production, viability rate, and expression of cleaved caspases 3, 8, and 9, cytochrome c, cleaved poly ADP ribose polymerase (PARP), Bax, and Bcl-2. miR-137 was vital to prostate cancer progression via regulating NOX4. Besides, miR-137 expressed poorly while NOX4 expressed highly in prostate cancer. NOX4 was the target gene of miR-137. Additionally, overexpression of miR-137 and silencing of NOX4 were observed to decrease NOX4 and Bcl-2 protein expression, but increase cleaved caspases 3, 8, and 9, cytochrome c, cleaved-PARP, and Bax protein expression. Furthermore, miR-137 overexpression and NOX4 silencing contributed to decreased lactate production, glucose uptake, ATP production, and cell proliferation, but increased apoptosis rate. Collectively, the present study showed that miR-137 repressed glycolysis in prostate cancer through knockdown of NOX4, which might be a potential theoretical target for prostate cancer treatment.     

Search for signatures in miRNAs associated with cancer

Since the first discovery in the early 1990''s, the predicted and validated population of microRNAs (miRNAs or miRs) has grown significantly. These small (~22 nucleotides long) regulators of gene expression have been implicated and associated with several genes in the cancer pathway as well. Globally, the identification and verification of microRNAs as biomarkers for cancer cell types has been the area of thrust for most miRNA biologists. However, there has been a noticeable vacuum when it comes to identifying a common signature or trademark that could be used to demarcate a miR to be associated with the development or suppression of cancer. To answer these queries, we report an in silico study involving the identification of global signatures in experimentally validated microRNAs which have been associated with cancer. This study has thrown light on the presence of significant common signatures, viz., - sequential and hybridization, which may distinguish a miR to be associated with cancer. Based on our analysis, we suggest the utility of such signatures in the design and development of algorithms for prediction of miRs involved in the cancer pathway.     

Recurrence of Early Stage Colon Cancer Predicted by Expression Pattern of Circulating microRNAs

Systemic treatment of patients with early-stage cancers attempts to eradicate occult metastatic disease to prevent recurrence and increased morbidity. However, prediction of recurrence from an analysis of the primary tumor is limited because disseminated cancer cells only represent a small subset of the primary lesion. Here we analyze the expression of circulating microRNAs (miRs) in serum obtained pre-surgically from patients with early stage colorectal cancers. Groups of five patients with and without disease recurrence were used to identify an informative panel of circulating miRs using quantitative PCR of genome-wide miR expression as well as a set of published candidate miRs. A panel of six informative miRs (miR-15a, mir-103, miR-148a, miR-320a, miR-451, miR-596) was derived from this analysis and evaluated in a separate validation set of thirty patients. Hierarchical clustering of the expression levels of these six circulating miRs and Kaplan-Meier analysis showed that the risk of disease recurrence of early stage colon cancer can be predicted by this panel of miRs that are measurable in the circulation at the time of diagnosis (P = 0.0026; Hazard Ratio 5.4; 95% CI of 1.9 to 15).     

GSK3 and miRNA in neural tissue: From brain development to neurodegenerative diseases

MicroRNAs (miRs) are small RNAs modulating gene expression and creating intricate regulatory networks that are dysregulated in many pathological states, including neurodegenerative disorders. In silico analyses denote a multifunctional kinase glycogen synthase kinase-3 (GSK3) as a putative target of numerous miRs identified in neural tissue. GSK3 is engaged in almost all aspects of neuronal development and functioning. Moreover, there is an autoregulatory feedback between GSK3 and miRNAs as the kinase can influence biogenesis of miRs. Members of the miR-GSK3 axes might thus represent convenient therapeutic targets in neuropathologies that display its abnormal regulation. This review summarizes the present knowledge about direct interactions of GSK3 and miRs in brain, and their putative roles in pathogenesis of neurodegenerative and neuropsychiatric disorders. This article is part of a Special Issue entitled: GSK-3 and related kinases in cancer, neurological and other disorders edited by James McCubrey, Agnieszka Gizak and Dariusz Rakus.     

The Role of Exosomes in Pancreatic Cancer Microenvironment

Exosomes are nanovesicles shed by cells as a means of communication with other cells. Exosomes contain mRNAs, microRNAs (miRs) and functional proteins. In the present paper, we develop a mathematical model of tumor–immune interaction by means of exosomes shed by pancreatic cancer cells and dendritic cells. Cancer cells’ exosomes contain miRs that promote their proliferation and that inhibit immune response by dendritic cells, and by CD4+ and CD8+ T cells. Dendritic cells release exosomes with proteins that induce apoptosis of cancer cells and that block regulatory T cells. Simulations of the model show how the size of the pancreatic cancer can be determined by measurement of specific miRs (miR-21 and miR-203 in the case of pancreatic cancer), suggesting these miRs as biomarkers for cancer.