Enhanced Extracellular Vesicle Cargo Loading via microRNA Biogenesis Pathway Modulation.

Extracellular vesicles (EVs) are physiological vectors for the intercellular transport of a variety of molecules. Among these, small RNAs, and especially microRNAs (miRNAs), have been identified as prevalent components, and there has thus been a robust investigation of EVs for therapeutic miRNAs delivery. However, intrinsic levels of EV-associated miRNAs are generally too low to enable efficient and effective therapeutic outcomes. We hypothesized that miRNA localization to EVs could be improved by limiting competing interactions that occur throughout the miRNA biogenesis process. Using miR-146a-5p as a model, modulation of transcription, nuclear export, and enzymatic cleavage steps of miRNA biogenesis were tested for impact on EV miRNA loading. Working in HEK293T cells, various alterations in the EV biogenesis pathway were shown to impact miRNA localization to EVs. The system was then applied in induced pluripotent stem cells (iPSCs), a more promising substrate for therapeutic EV production, and EVs were separated and assessed for anti-inflammatory efficacy in vitro and in a murine colitis model, where the preservation of function was validated. Overall, the results highlight necessary considerations when designing a cell culture system for the devoted production of miRNA-loaded EVs.

The METHYLTRANSFERASE B-SERRATE interaction mediates the reciprocal regulation of microRNA biogenesis and RNA m6A modification.

In eukaryotes, RNA N6-methyladenosine (m6A) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The m6A methyltransferase complex (MTC) and the microprocessor complex both undergo liquid-liquid phase separation to form nuclear membraneless organelles. Although m6A methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive. Here, we demonstrate that the MTC and the microprocessor complex associate with each other through the METHYLTRANSFERASE B (MTB)-SERRATE (SE) interacting module. Knockdown of MTB impaired miRNA biogenesis by diminishing microprocessor complex binding to primary miRNAs (pri-miRNAs) and their respective MIRNA loci. Additionally, loss of SE function led to disruptions in transcriptome-wide m6A modification. Further biochemical assays and fluorescence recovery after photobleaching (FRAP) assay indicated that SE enhances the liquid-liquid phase separation and solubility of the MTC. Moreover, the MTC exhibited enhanced retention on chromatin and diminished binding to its RNA substrates in the se mutant background. Collectively, our results reveal the substantial regulatory interplay between RNA m6A modification and miRNA biogenesis.

Exposure to the extremely low-frequency electromagnetic field induces changes in the epigenetic regulation of gene expression in the endometrium.

Increasing technological development results in more sources of the extremely low-frequency electromagnetic field (ELF-EMF), which is recognized as an environmental risk factor. The results of the past study indicate that the ELF-EMF can affect the level of DNA methylation. The study aimed to determine whether the ELF-EMF induces changes in epigenetic regulation of gene expression in the endometrium of pigs during the peri-implantation period. Endometrial slices (100 ± 5 mg) collected on days 15-16 of pregnancy were exposed in vitro to the ELF-EMF at a frequency of 50 Hz for 2 h of treatment duration. To determine the impact of the ELF-EMF on elements of epigenetic regulations involved in DNA methylation, histone modification, and microRNA biogenesis in the endometrium, the DNMT1 and DNMT3a; EZH2, UHRF1, and MBD1; DICER1 and DGCR8 mRNA transcript and protein abundance were analyzed using Real-Time PCR and Western blot, respectively. Moreover, EED and SUZ12 mRNA transcript, global DNA methylation, and the activity of histone deacetylase (HDAC) were analyzed. The changes in the abundance of DNMT1 and DNMT3a, EZH2 mRNA transcript and protein, EED and SUZ12 mRNA transcript, global DNA methylation level, HDAC activity, and the abundance of proteins involved in microRNA biogenesis evoked by the ELF-EMF in the endometrium were observed. The ELF-EMF possesses the potential to alter epigenetic regulation of gene expression in the porcine endometrium. Observed alterations may be the reason for changes in the transcriptomic profile of the endometrium exposed to the ELF-EMF which in turn may disrupt biological processes in the uterus during peri-implantation.

Modulation of the Cellular microRNA Landscape: Contribution to the Protective Effects of High-Density Lipoproteins (HDL).

High-density lipoproteins (HDL) play an established role in protecting against cellular dysfunction in a variety of different disease contexts; however, harnessing this therapeutic potential has proved challenging due to the heterogeneous and relative instability of this lipoprotein and its variable cargo molecules. The purpose of this study is to examine the contribution of microRNA (miRNA; miR) sequences, either delivered directly or modulated endogenously, to these protective functions. This narrative review introduces the complex cargo carried by HDL, the protective functions associated with this lipoprotein, and the factors governing biogenesis, export and the uptake of microRNA. The possible mechanisms by which HDL can modulate the cellular miRNA landscape are considered, and the impact of key sequences modified by HDL is explored in diseases such as inflammation and immunity, wound healing, angiogenesis, dyslipidaemia, atherosclerosis and coronary heart disease, potentially offering new routes for therapeutic intervention.

PLK1 Regulates MicroRNA Biogenesis through Drosha Phosphorylation.

Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3ß, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

Nuclear receptor RXRα binds the precursor of miR-103 to inhibit its maturation.

BACKGROUND: The maturation of microRNAs (miRNAs) successively undergoes Drosha, Dicer, and Argonaute -mediated processing, however, the intricate regulations of the individual miRNA maturation are largely unknown. Retinoid x receptor alpha (RXRα) belongs to nuclear receptors that regulate gene transcription by binding to DNA elements, however, whether RXRα binds to miRNAs to exert physiological functions is not known. RESULTS: In this work, we found that RXRα directly binds to the precursor of miR-103 (pre-miR-103a-2) via its DNA-binding domain with a preferred binding sequence of AGGUCA. The binding of RXRα inhibits the processing of miR-103 maturation from pre-miR-103a-2. Mechanistically, RXRα prevents the nuclear export of pre-miR-103a-2 for further processing by inhibiting the association of exportin-5 with pre-miR-103a-2. Pathophysiologically, the negative effect of RXRα on miR-103 maturation correlates to the positive effects of RXRα on the expression of Dicer, a target of miR-103, and on the inhibition of breast cancer. CONCLUSIONS: Our findings unravel an unexpected role of transcription factor RXRα in specific miRNA maturation at post-transcriptional level through pre-miRNA binding, and present a mechanistic insight regarding RXRα role in breast cancer progression.

[MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress].

MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal ß-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.

Altered MicroRNA Maturation in Ischemic Hearts: Implication of Hypoxia on XPO5 and DICER1 Dysregulation and RedoximiR State.

Ischemic cardiomyopathy (ICM) is associated with abnormal microRNA expression levels that involve an altered gene expression profile. However, little is known about the underlying causes of microRNA disruption in ICM and whether microRNA maturation is compromised. Therefore, we focused on microRNA maturation defects analysis and the implication of the microRNA biogenesis pathway and redox-sensitive microRNAs (redoximiRs). Transcriptomic changes were investigated via ncRNA-seq (ICM, n = 22; controls, n = 8) and mRNA-seq (ICM, n = 13; control, n = 10). The effect of hypoxia on the biogenesis of microRNAs was evaluated in the AC16 cell line. ICM patients showed a reduction in microRNA maturation compared to control (4.30 ± 0.94 au vs. 5.34 ± 1.07 au, p ˂ 0.05), accompanied by a deregulation of the microRNA biogenesis pathway: a decrease in pre-microRNA export (XPO5, FC = -1.38, p ˂ 0.05) and cytoplasmic processing (DICER, FC = -1.32, p ˂ 0.01). Both processes were regulated by hypoxia in AC16 cells (XPO5, FC = -1.65; DICER1, FC = -1.55; p ˂ 0.01; Exportin-5, FC = -1.81; Dicer, FC = -1.15; p ˂ 0.05). Patients displayed deregulation of several redoximiRs, highlighting miR-122-5p (FC = -2.41, p ˂ 0.001), which maintained a good correlation with the ejection fraction (r = 0.681, p ˂ 0.01). We evidenced a decrease in microRNA maturation mainly linked to a decrease in XPO5-mediated pre-microRNA export and DICER1-mediated processing, together with a general effect of hypoxia through deregulation of biogenesis pathway and the redoximiRs.

Unpaired nucleotides on the stem of microRNA precursor are important for precise cleavage by Dicer-like 1 in Arabidopsis.

Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

Small Non-coding RNAs in Embryonic Pre-implantation.

Failure of embryo implantation has been introduced as an important limiting parameter in early assisted reproduction and pregnancy. The embryo-maternal interactions, endometrial receptivity, and detections of implantation consist of the embryo viability. For regulating the implantation, multiple molecules may be consistent; however, their specific regulatory mechanisms still stand unclear. MicroRNAs (miRNAs) have attracted a lot of attention due to their important effect on human embryo implantation. MicroRNA (miRNA), which acts as the transcriptional regulator of gene expression, is consisted of embryo implantation. Recent studies indicated that miRNAs not only act inside the cells but also can be secreted by cells into the extracellular environment via multiple packaging forms, facilitating intercellular communication and providing indicative information related to various conditions. The detection of extracellular miRNAs provided new information in cases of implantation studies. For embryo-maternal communication, MiRNAs offered novel approaches. In addition, in assisted reproduction, for embryo choice and prediction of endometrial receptivity, they can act as non-invasive biomarkers and can enhance the accuracy in the process of reducing the mechanical damage for the tissue.

Association of MicroRNA Biogenesis Genes Polymorphisms with Risk of Large Artery Atherosclerosis Stroke.

Convincing evidence has shown that microRNAs (miRNAs) are involved in the pathogenesis of stroke. This study aimed to examine whether miRNA biogenesis genes polymorphisms are associated with risk of large artery atherosclerosis (LAA) stroke. Three polymorphisms (DROSHA rs10719 T>C, RAN rs3803012 A>G, and PIWIL1 rs10773771 C>T) were screened by certain criteria. A total of 1,785 (710 cases and 1,075 controls) study subjects were included in this study. We found that rs10773771 CC genotype was associated with a decreased risk of LAA stroke (CC vs. TT/CT: OR 0.63, 95% CI 0.46-0.86, P = 3 × 10-3). In silico analysis suggested that rs10773771 can change the mRNA secondary structure of PIWIL1 and affect the binding of the miRNAs and regulatory motifs to the 3'-UTR of PIWIL1. Expression quantitative trait loci analysis showed that rs10773771 could change the expression of PIWIL1 in human skin (P = 1.534 × 10-10) and thyroid tissues (P = 4.869 × 10-6). These findings suggested that PIWIL1 rs10773771 may be associated with a decreased risk of LAA stroke.

Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins.

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Multifaceted Regulation of MicroRNA Biogenesis: Essential Roles and Functional Integration in Neuronal and Glial Development.

MicroRNAs (miRNAs) are small, non-coding RNAs that function as endogenous gene silencers. Soon after the discovery of miRNAs, a subset of brain-enriched and brain-specific miRNAs were identified and significant advancements were made in delineating miRNA function in brain development. However, understanding the molecular mechanisms that regulate miRNA biogenesis in normal and diseased brains has become a prevailing challenge. Besides transcriptional regulation of miRNA host genes, miRNA processing intermediates are subjected to multifaceted regulation by canonical miRNA processing enzymes, RNA binding proteins (RBPs) and epitranscriptomic modifications. Further still, miRNA activity can be regulated by the sponging activity of other non-coding RNA classes, namely circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). Differential abundance of these factors in neuronal and glial lineages partly underlies the spatiotemporal expression and function of lineage-specific miRNAs. Here, we review the continuously evolving understanding of the regulation of neuronal and glial miRNA biogenesis at the transcriptional and posttranscriptional levels and the cooperativity of miRNA species in targeting key mRNAs to drive lineage-specific development. In addition, we review dysregulation of neuronal and glial miRNAs and the detrimental impacts which contribute to developmental brain disorders.

MicroRNAs in Cancer: From Gene Expression Regulation to the Metastatic Niche Reprogramming.

By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors' opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.

The MicroRNA Family Gets Wider: The IsomiRs Classification and Role.

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases.

Long non-coding RNA RP11-283G6.5 confines breast cancer development through modulating miR-188-3p/TMED3/Wnt/ß-catenin signalling.

The contributions of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) to breast cancer are critical areas of investigation. In this study, we identified a novel lncRNA RP11-283G6.5 which was lowly expressed in breast cancer and whose low expression was correlated with poor overall survival and disease-free survival of breast cancer patients. Functional experiments revealed that ectopic expression of RP11-283G6.5 confined breast cancer cellular growth, migration, and invasion, and promoted cellular apoptosis. Conversely, RP11-283G6.5 silencing facilitated breast cancer cellular growth, migration, and invasion, and repressed cellular apoptosis. Moreover, RP11-283G6.5 was found to confine breast cancer tumour growth and metastasis in vivo. Mechanistically, RP11-283G6.5 competitively bound to ILF3, reduced the binding of ILF3to primary miR-188 (pri-miR-188), abolished the suppressive effect of ILF3 on pri-miR-188 processing, and therefore promoted pri-miR-188 processing, leading to the reduction of pri-miR-188 and the upregulation of mature miR-188-3p. The expression of RP11-283G6.5 was significantly positively correlated with that of miR-188-3p in breast cancer tissues. Through increasing miR-188-3p, RP11-283G6.5 decreased TMED3, a target of miR-188-3p. RP11-283G6.5 further suppressed Wnt/ß-catenin signalling via decreasing TMED3. Rescue assays revealed that inhibition of miR-188-3p, overexpression of TMED3 or blocking Wnt/ß-catenin signalling all attenuated the roles of RP11-283G6.5 in breast cancer. Collectively, these findings demonstrated that RP11-283G6.5 is a tumour suppressive lncRNA in breast cancer via modulating miR-188-3p/TMED3/Wnt/ß-catenin signalling. This study indicated that RP11-283G6.5 might be a promising prognostic biomarker and therapeutic target for breast cancer.

A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor.

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.

Upregulated long non­coding RNA LincIN promotes tumor progression via the regulation of nuclear factor 90/microRNA­7/HOXB13 in esophageal squamous cell carcinoma.

Long non­coding RNA LincIN has been reported to be overexpressed and to be involved in the metastasis of breast cancer. However, the expression and role of LincIN in esophageal squamous cell carcinoma (ESCC) remain unsolved. In the present study, LincIN expression was examined in ESCC by RT­qPCR, and the roles of LincIN in ESCC were determined using cell growth, migration and invasion assays. In addition, the effects of LincIN on nuclear factor 90 (NF90) and microRNA/miR (miR)­7 were examined by RNA immunoprecipitation assay, RT­qPCR, dual­luciferase reporter assay and western blot analysis. The results revealed that LincIN expression was significantly increased in ESCC tissues and cell lines. The increased expression of LincIN was positively associated with invasion depth, lymph node metastasis, TNM stage and a poor prognosis. Functional assays revealed that the overexpression of LincIN promoted ESCC cell growth, migration and invasion. Mechanistic analysis revealed that LincIN physically bound to NF90, enhanced the binding between NF90 and primary miR­7 (pri­miR­7), and further enhanced the inhibitory effects of NF90 on miR­7 biogenesis. Therefore, LincIN downregulated miR­7 expression in ESCC. The expression of miR­7 inversely correlated with that of LincIN in ESCC tissues. By downregulating miR­7, LincIN increased the expression of HOXB13, a target of miR­7. The overexpression of miR­7 or the depletion of HOXB13 both attenuated the tumor­promoting roles of LincIN in ESCC cell growth, migration and invasion. On the whole, the findings of the present study suggest that LincIN is overexpressed and plays an oncogenic role in ESCC via the regulation of the NF90/miR­7/HOXB13 axis. Thus, LincIN may prove to be a promising prognostic biomarker and therapeutic target for ESCC.

Hypoxia-induced Nur77 activates PI3K/Akt signaling via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition.

Background: Colorectal cancer (CRC) and the associated metastatic lesions are reported to be hypoxic. Hypoxia is a common feature in the tumor microenvironment and a potent stimulant of CRC. We have identified a regulatory role of Nur77 on Akt activation to enhance ß-catenin signaling essential for CRC progression under hypoxic conditions. Methods: The functional role of Nur77 in hypoxia-induced EMT was examined by scattering assays to monitor the morphologies of CRC cell lines under 1% O2. Sphere formation assays were performed to investigate whether Nur77 induced cancer stem cell-like properties in hypoxic CRC cells. The expression of various epithelial-to-mesenchymal transition (EMT) and stemness markers was analyzed by qPCR and Western blotting. Finally, Nur77 function and signaling in vivo was ascertained in subcutaneous tumor xenograft or liver metastasis model in nude mice using CRC cells stably transfected with appropriate constructs. Results: Herein, we show, for the first time, that Nur77 is a novel regulator of microRNA biogenesis that may underlie its significant tumor-promoting activities in CRC cells under hypoxia. Mechanistically, Nur77 interacted with the tumor suppressor protein p63, leading to the inhibition of p63-dependent transcription of Dicer, an important miRNA processor and subsequent decrease in the biogenesis of let-7i-5p which targeted the 3'UTR of p110α mRNA and regulated its stability. Knockdown of Nur77 or overexpression of let-7i-5p inhibited the tumor metastasis in vivo. Conclusion: Our data uncovered a novel mechanistic link connecting Nur77, Akt, and invasive properties of CRC in the hypoxic microenvironment.