Dysregulation Of Specific miRNAs Research Articles

The study of miRNA-200c expression and epithelial-to-mesenchymal transition-related transcription factors in the primary bladder urothelial carcinoma

Epithelial-mesenchymal transition (EMT) plays an important role in bladder carcinoma (BC) invasiveness and metastasis. Studies have shown that muscle-invasive BC (MIBC) and non-MIBC (NMIBC) are different at the molecular level owing to different EMT-related programming. Recent studies suggest that dysregulation of specific miRNAs is linked to EMT in BC. With this background, we aimed to study the immunoexpression of EMT-markers and its correlation with miRNA-200c expression in a series of MIBCs and NMIBCs. Quantitative real-time-polymerase chain reaction for the quantification of miR-200c expression was performed on 50 cases of urinary BC obtained from transurethral resection of bladder tumor (TURBT), cystectomy specimens, and ten peritumoral bladder tissue. Immunohistochemistry for ZEB1, ZEB2, TWIST, E-cadherin, and β-catenin was performed on tumor and peritumoral bladder tissue. Thirty-five TURBT and 15 cystectomy specimens were assessed. Among MIBC, loss of expression of E-cadherin (72.3%), β-catenin (66.7%), and ZEB1, ZEB2, and TWIST2 immunoreactivity was noted in 53.3%, 86.7%, and 73.3% of cases, respectively. Among NMIBC, loss of expression of E-cadherin (22.5%), β-catenin (17.1%) and ZEB1, ZEB2, and TWIST immunoreactivity was noted in 11.5%, 51.4%, and 91.4% of cases, respectively. Upregulation of miRNA-200c was noted in cases with retained E-cadherin and negative TWIST expression. Downregulation of miRNA-200c expression was noted in all the cases showing loss of E-cadherin, β-catenin, and in cases immunoreactive for ZEB1, ZEB2, and TWIST in MIBC. Downregulation of miRNA-200c expression was also noted in cases of MIBC with retained β-catenin and those immunonegative for ZEB1 and ZEB2. A similar trend was noted in NMIBC. Median miRNA-200c expression was low in both high-grade and low-grade NMIBC compared to peritumoral bladder tissue and was not statistically significant. This study for the first time explores the relation of miR200C with E-cadherin, b-catenin, and its direct transcriptional regulators, namely Zeb1, Zeb2, and Twist in the same cohort of BC. We observed that miRNA-200c is downregulated in both MIBC and NMIBC. We identified novel expression of TWIST in cases of BC showing downregulation of miR200Cs suggesting that it is one of the protein targets of altered miRNA-200c expression contributing to EMT and can serve as a promising diagnostic marker and therapeutic target. Loss of E-cadherin and ZEB1 immunoexpression in high-grade NMIBC suggests an aggressive clinical behavior. However, ZEB2 heterogeneous expression in BC limits its diagnostic and prognostic utility.

MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation.

MicroRNAs (miRNAs) are the small non-coding regulatory RNA molecules involved in gene regulation via base-pairing with complementary sequences in mRNAs. The dysregulation of specific miRNAs, such as miR-99b-5p (miR-99b), is associated with prostate cancer (PCa) progression. However, the mechanistic role of miR-99b in PCa remains to be determined. In this study, we aimed to investigate the functional and clinical significance of miR-99b in PCa. The expression of miR-99b and its downstream targets mTOR/AR in the PCa samples were analyzed by RT/qPCR. The effects of miR-99b overexpression/inhibition on PCa cell survival/proliferation, spheroid formation, and cell migration were examined by specific assays. Luciferase reporter assays were performed to determine the binding of miR-99b to 3' untranslated region (UTR) of the mTOR gene. The effects of miR-99b on the expression of mTOR, AR, and PSA proteins, as well as on AKT/mTOR signaling, autophagy, and neuroendocrine differentiation markers were analyzed by western blotting. The expression of miR-99b, mTOR, AR, PSA in AR-negative PC3 and AR-positive LNCaP cells was analyzed by RT/qPCR. The effect of miR-99b on global gene expression in PC3 cells was analyzed by RNA-seq. The expression of miR-99b was downregulated in tumor samples from PCa patients, whereas the expression of mTOR and AR was upregulated. In PCa cell lines, overexpression of miR-99b inhibited cell proliferation and cell colony/spheroid formation; induced apoptosis, and increased sensitivity towards docetaxel (DTX). In contrast, inhibition of miR-99b by miR-99b inhibitor resulted in increased cell growth in PCa cells. Mechanistically, miR-99b inhibited the expression of the mammalian target of the rapamycin (mTOR) gene by binding to its 3' UTR and induced autophagy. Furthermore, miR-99b inhibited androgen receptor (AR) activity in LNCaP cells and induced apoptosis. Activation of AR signaling by dihydrotestosterone (DHT) downregulated miR-99b expression and promoted cell PCa cell growth/survival, whereas inactivation of mTOR by rapamycin or AR by enzalutamide decreased miR-99b mediated PCa cell growth. Our data suggest that miR-99b functions as a tumor suppressor by targeting the mTOR/AR axis in PCa cells, implicating miR-99b as a novel biomarker and therapeutic target for PCa management.

Complexity in regulating microRNA biogenesis in cancer.

The discovery of microRNA (miRNA) significantly extends our knowledge on gene regulation and noncoding gene functions. MiRNAs are important post-transcriptional regulators involve in a wide range of biological functions and diseases, including cancer. MiRNAs are produced by a unique biogenesis pathway involving the two-step sequential nuclear and cytoplasmic RNase-dependent processing at post-transcriptional level. However, a specific (set) of miRNA(s) is (are) synthesized under certain circumstance or developmental/pathological stage to fine-tune the gene expression profile. In this minireview, we will discuss the mechanism of miRNA biogenesis in cancer, mainly focusing on how Drosha and Dicer, two critical molecules controlling miRNA biogenesis, are modulated and which factor contributes to the specificity of selected miRNA maturation. Impact statement The canonical maturation pathway of miRNAs is highly conserved, indicating the crucial roles of these mini-regulators in most cellular processes. Dysregulation of specific miRNAs or imbalance of miRNA abundance has been observed in cancers. Accumulating evidence has shown that the interplay between miRNA processing factors and regulatory proteins previously known as key players in cancer malignancy regulates the biogenesis of miRNAs, expression of target genes, and eventually the alteration of cellular phenotypes. This minireview summarizes the current findings in the modulation of miRNA biogenesis in cancer to advance the understanding of how noncoding RNA contributes to cancer development and malignancy.

MicroRNAs Change the Landscape of Cancer Resistance.

One of the major challenges in the cancer treatment is the development of drug resistance. It represents a major obstacle to curing cancer with constrained efficacy of both conventional chemotherapy and targeted therapies, even recent immune checkpoint blockade therapy. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in the resistance to various cancer treatments. MicroRNAs are a family of small noncoding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified in human genome. While one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance, thereby modulating the sensitivity of cancer cells to treatment. Therefore, manipulation of miRNAs may be an attractive strategy for more effective individualized therapies through reprograming resistant network in cancer cells.

Role of microRNAs in cardiac development and disease.

Heart disease-related deaths are the highest in most societies and congenital heart diseases account for approximately 40% of prenatal deaths and over 20% of mortality in the first few months after birth. Congenital heart disease affects approximately 1% of all newborns and is the causative factor for more deaths within the first year of life as compared to all other genetic defects. Advances in treatment approaches increased life expectancy and led to an expansion of adult population with clinical manifestation of congenital heart defects in up to 90% of the children born with congenital heart diseases. Regulation of cardiac gene expression involves multiple independent enhancers that play a critical role in maintaining a restricted and specific pattern of gene expression in the heart. Cardiac transcriptional pathways are intimately regulated by microRNAs (miRNAs), which are small, regulatory RNAs, approximately 22 nucleotides in length, also coded by specific genes. These miRNAs act as suppressors of gene expression by inhibiting translation and/or promoting degradation of target protein-coding mRNAs. There are several miRNAs involved in the development of heart and dysregulation of specific miRNAs is associated with congenital and other cardiac defects. Stress responsive cardiac hypertrophy is orchestrated among other factors, by specific miRNAs. miRNAs such as miR-499 are considered useful as biomarkers of a given heart disease. Therapeutic application of miRNAs is also envisaged considering the small size and specific effects of these molecules. In this review, we addressed different roles of miRNAs in the development and diseases of the heart.

The emerging role of microRNAs in resistance to lung cancer treatments.

One of the major challenges in the treatment of lung cancer is the development of drug resistance. This represents a major obstacle in the treatment of patients, limiting the efficacy of both conventional chemotherapy and biological therapies. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and in developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in resistance to various cancer treatments. MicroRNAs are a family of small non-coding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified to date. While as little as one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance to a number of cancer treatments, thereby modulating the sensitivity of cancer cells to such therapies. Therefore, targeting miRNAs may be an attractive strategy for developing novel and more effective individualized therapies, improving drug efficiency, and for predicting patient response to different treatments. In this review, we provide an overview on the role of miRNAs in resistance to current lung cancer therapies and novel biological agents.

Bcl-xL Silencing Induces Alterations in hsa-miR-608 Expression and Subsequent Cell Death in A549 and SK-LU1 Human Lung Adenocarcinoma Cells

Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.

Deep Sequencing of Small RNA Repertoires in Mice Reveals Metabolic Disorders-Associated Hepatic miRNAs

Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.

The function of miRNA in cardiac hypertrophy

Cardiac hypertrophy is an adaptive enlargement of the myocardium in response to altered stress or injury. The cellular responses of cardiomyocytes and non-cardiomyocytes to various signaling pathways should be tightly and delicately regulated to maintain cardiac homeostasis and prevent pathological cardiac hypertrophy. MicroRNAs (miRNAs) are endogenous, single-stranded, short non-coding RNAs that act as regulators of gene expression by promoting the degradation or inhibiting the translation of target mRNAs. Recent studies have revealed expression signatures of miRNAs associated with pathological cardiac hypertrophy and heart failure in humans and mouse models of heart diseases. Increasing evidence indicates that dysregulation of specific miRNAs could alter the cellular responses of cardiomyocytes and non-cardiomyocytes to specific signaling upon the pathological hemodynamic overload, leading to cardiac hypertrophy and heart failure. This review summarizes the cell-autonomous functions of cardiomyocyte miRNAs regulated by different pathways and the roles of non-cardiomyocyte miRNAs in cardiac hypertrophy. The therapeutic effects of a number of miRNAs in heart diseases are also discussed.

H. pylori eradication did not improve dysregulation of specific oncogenic miRNAs in intestinal metaplastic glands

Many microRNAs (miRNAs) are differentially expressed in Helicobacter pylori-infected gastric mucosa and in gastric cancer tissue. We aimed to compare the effect of H. pylori eradication on gastric mucosal miRNAs in subjects in a high-risk group for gastric cancer compared to controls. Patients with a recent history of endoscopic resection for early gastric cancer and sex- and age-matched non-cancer controls were enrolled. The expression of 21 miRNAs was examined using gastric mucosal biopsy specimens and microdissected gastric glands from the lesser and greater curvatures of the gastric corpus both before and one year after H. pylori eradication. Twenty patients and 14 controls were enrolled. The expression of oncogenic miRNAs (miR-17/92 and the miR-106b-93-25 cluster, miR-21, miR-194, and miR-196) was significantly higher in the gastric mucosa of the cancer group than in the controls. H. pylori eradication resulted in a significant fall in the expression of oncogenic miRNAs only in the controls, whereas miR-223 expression was decreased and let-7d expression was increased in both groups. miR-196 was expressed only in intestinal metaplastic glands. The expression of oncogenic miRNAs was significantly higher in the intestinal metaplastic glands than in the non-intestinal metaplastic glands irrespective of H. pylori eradication. In neither group did H. pylori eradication significantly change any miRNA expression in the intestinal metaplastic glands. Dysregulation of specific miRNAs is present in H. pylori-induced corpus gastritis. H. pylori eradication improved miRNA dysregulation, but not in intestinal metaplastic glands or in the gastric mucosa of patients in a high-risk group for gastric cancer.

Role of MicroRNAs in the Regulation of Cytochrome P450s and Transcriptional Factors

MicroRNAs (miRNAs) are endogenous ~22-nucleotide non-coding RNAs that regulate gene expression through the translational repression or degradation of target mRNAs. The human genome contains over 1400 miRNAs and over 60% of human mRNAs are predicted to be targets of miRNAs. The miRNAs have roles in fine-tuning the expression of their target genes forming intricate networks. Research on miRNA is growing exponentially, and it is now clear that miRNAs can potentially regulate every aspect of cellular processes such as differentiation, proliferation and apoptosis as well as a large range of physiological processes such as development, immune response, metabolism, tumor formation, and disease development. The roles of miRNAs in the metabolism of xenobiotics and endobiotics have only recently been revealed. This review describes the current knowledge on the regulation of cytochrome P450s and transcriptional factors by miRNAs, and its physiological and clinical significance, which were disclosed in our studies. The miRNA expression is readily altered by chemicals, carcinogens, drugs, hormones, stress, or diseases, and the dysregulation of specific miRNAs might lead to changes in the drug metabolism potency or pharmacokinetics as well as pathophysiological changes. Utilizing miRNAs opens a new era in the fields of drug metabolism and pharmacokinetics as well as toxicology.

MicroRNAs target gene and signaling pathway by bioinformatics analysis in the cardiac hypertrophy

Cardiac hypertrophy is a physiological adaptive response of the heart to diverse pathophysiological stimuli. Initially, it may be adaptive to normalize wall stress and to preserve contractile performance. This adaptive process may gradually progress to dilated cardiomyopathy, fibrotic diseases, arrhythmia, heart failure and even sudden death. Although various molecular pathways responsible for the coordinated control of the hypertrophic program, little is known about their underlying molecular mechanisms. Very recently, increasing evidence showed that miRNAs are key modulators of both cardiovascular development and function, which govern the process of cardiac hypertrophy and heart failure. MicroRNAs (miRNAs) act in a complex functional network in which each single miRNAs might control thousands of distinct target genes, and each single protein-coding gene can be regulated by many different miRNAs. Identifying the roles of miRNAs, their target genes and signaling pathways in cardiac hypertrophy by bioinformatic analysis will provide more insight into the molecular mechanisms underlying this disease process. Currently, bioinformatics resource such as GO and KEGG was applied to describe the miRNAs target genes function and identify the mRNA interaction networks that are responsible for various cellular processes. It provides a useful approach to observe the function of microRNA in physiological and pathological conditions. In this review, we will give a discussion on the dysregulation of specific miRNAs in cardiac hypertrophy and signaling pathways linking the hypertrophy-regulating miRNAs to the pathological process of cardiac hypertrophy. Finally, we place special emphasis on the essential role of bioinformatics analysis to predict the target genes and miRNAs gene networks.

MicroRNA profiling of Barrett's oesophagus and oesophageal adenocarcinoma

The genetic changes that drive metaplastic progression from squamous oesophageal mucosa toward intestinal metaplasia and adenocarcinoma are unclear. The aberrant expression of microRNAs (miRNAs) is involved in the development of cancer. This study examined whether miRNAs play a role in the development of oesophageal adenocarcinoma. RNA was extracted from mucosa of normal oesophageal squamous epithelium, normal gastric epithelium, Barrett's oesophagus with intestinal metaplasia and oesophageal adenocarcinoma obtained from 16 individuals. Expression profiles of 377 human miRNAs were determined by microarray analysis and selected miRNAs were analysed further using real-time reverse transcription-polymerase chain reaction (RT-PCR) in tissues from 32 individuals. Microarray analyses identified 44 miRNAs likely to have altered expression between various mucosal samples. Of these, miR-21, miR-143, miR-145, miR-194, miR-203, miR-205 and miR-215 were chosen for validation by real-time RT-PCR. Tissue-specific expression profiles were observed, with miR-21, miR-143, miR-145, miR-194 and miR-215 significantly upregulated in columnar tissues compared with normal squamous epithelium. Expression of miR-143, miR-145 and miR-215 was lower in oesophageal adenocarcinoma than in Barrett's oesophagus. Levels of miR-203 and miR-205 were high in normal squamous epithelium and low in columnar epithelia. MiR-205 levels were lower in gastric epithelium than in both Barrett's oesophagus and adenocarcinoma. Expression of miRNA might define disease states in oesophageal epithelium. Dysregulation of specific miRNAs could contribute to metaplastic and neoplastic processes in the oesophageal mucosa.

Role of microRNAs in the regulation of drug metabolism and disposition

Background: The important role of nuclear receptors in transcriptional gene regulation of drug-metabolizing enzymes (DMEs) and drug transporters (DTs) has been well documented. In contrast, there is limited understanding of post-transcriptional gene regulation of DMEs and DTs. MicroRNAs (miRNAs) represent a large group of non-coding RNAs that control the post-transcriptional expression of target genes. Currently, > 800 miRNAs have been identified in human genome, which are believed to regulate thousands of human protein-coding genes. Objective: This review aims to discuss the potential role of miRNAs in drug metabolism and disposition, following an introduction of miRNA biogenesis, regulatory mechanisms, and target identification and validation. Results/conclusions: Some miRNAs have been shown to directly or indirectly control the expression of DMEs, DTs or nuclear receptors, and consequently, affect the capacity of drug metabolism and disposition, and influence the sensitivity of cells to xenobiotic agents. Furthermore, the expression of some miRNAs is readily altered in cells after an acute or chronic exposure to medications, toxins or carcinogens. Therefore, dysregulation of specific miRNAs by a xenobiotic agent, which control the expression of DMEs or DTs, might lead to considerable change in the pharmacokinetic and pharmacodynamic property of a concomitant drug or the agent itself. Improved understanding of the regulatory pathways of DMEs and DTs shall provide novel insight into multi-drug resistance and potential drug–drug interaction in clinical pharmacotherapy as well as in drug discovery and development.

Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis

Acute myocardial infarction (MI) due to coronary artery occlusion is accompanied by a pathological remodeling response that includes hypertrophic cardiac growth and fibrosis, which impair cardiac contractility. Previously, we showed that cardiac hypertrophy and heart failure are accompanied by characteristic changes in the expression of a collection of specific microRNAs (miRNAs), which act as negative regulators of gene expression. Here, we show that MI in mice and humans also results in the dysregulation of specific miRNAs, which are similar to but distinct from those involved in hypertrophy and heart failure. Among the MI-regulated miRNAs are members of the miR-29 family, which are down-regulated in the region of the heart adjacent to the infarct. The miR-29 family targets a cadre of mRNAs that encode proteins involved in fibrosis, including multiple collagens, fibrillins, and elastin. Thus, down-regulation of miR-29 would be predicted to derepress the expression of these mRNAs and enhance the fibrotic response. Indeed, down-regulation of miR-29 with anti-miRs in vitro and in vivo induces the expression of collagens, whereas over-expression of miR-29 in fibroblasts reduces collagen expression. We conclude that miR-29 acts as a regulator of cardiac fibrosis and represents a potential therapeutic target for tissue fibrosis in general.

Overlapping expression of microRNAs in human embryonic colon and colorectal cancer

MicroRNAs (miRNAs) are essential for regulating cell differentiation and maintaining the pluripotent state of stem cells. Although dysregulation of specific miRNAs has been associated with certain types of cancer, to date no evidence has linked miRNA expression in embryonic and tumor tissues. We assessed the expression of mature miRNAs in human embryonic colon tissue, and in colorectal cancer and paired normal colon tissue. Overlapping miRNA expression was detected between embryonic colonic mucosa and colorectal cancer. We have found that the miR-17-92 cluster and its target, E2F1, exhibit a similar pattern of expression in human colon development and colonic carcinogenesis, regulating cell proliferation in both cases. In situ hybridization confirmed the high level of expression of miR-17-5p in the crypt progenitor compartment. We conclude that miRNA pathways play a major role in both embryonic development and neoplastic transformation of the colonic epithelium.