ADAMTS9-AS1 Long Non­coding RNA Sponges miR­128 and miR-150 to Regulate Ras/MAPK Signaling Pathway in Glioma.

Glioma is a malignancy of the central nervous system with a poor prognosis. Therefore, the elaboration of its molecular features creates therapeutic opportunities. Looking for the regulatory non-coding RNAs (lncRNAs and miRNAs) that are involved in glioma incidence/progression, RNA-seq analysis introduced upregulated ADAMTS9-AS1 as a bona fide candidate that sponges miR-128 and miR-150 and shows the negative correlation of expression with them. Then, RT-qPCR verified the upregulation of ADAMTS9-AS1 in glioma tissues and cell lines. Furthermore, dual-luciferase assay supported that cytoplasmic ADAMTS9-AS1 is capable of sponging miR-128 and miR-150, which are known as regulators of Ras/MAPK, PI3K, and Wnt pathways. Following the overexpression of ADAMTS9-AS1 in 1321N1 and U87 glioma cells, tyrosine kinase receptors (IGF1R and TrkC), as well as Wnt receptors (Lrp6 and Fzd) were upregulated, detected by RT-qPCR. Furthermore, downstream genes of both Ras/MAPK and Wnt pathways were upregulated. Finally following the ADAMTS9-AS1 overexpression, upregulation of Ras/MAPK and Wnt signaling pathways was verified through western blotting and Top/Fop flash assay, respectively. At the cellular level, ADAMTS9-AS1 overexpression brought about reduced sub-G1 cell population, increased proliferation rate, reduced apoptosis level, increased migration rate, shortened Bax/Bcl2 ratio, induced EMT, and stemness characteristics of transfected cells, detected by flow cytometry, MTT assay, scratch test, and RT-qPCR. Overall, these results introduced ADAMTS9-AS1 as an oncogene that upregulates Ras/MAPK and Wnt pathways through sponging of the miR-128 and miR-150 in glioma cells. The outcome of ADAMTS9-AS1 expression is more aggression of the glioma cells through increased EMT and stemness characteristics. These features candidate ADAMTS9-AS1 locus for glioma therapy. As a result, we discovered the oncogenic properties of ADAMTS9-AS1 in glioma cancer. It sponges miR-128 and miR-150 and subsequently overstimulates RAS/MAPK and Wnt signaling pathways, particularly at the receptors level. Thus, ADAMTS9-AS1 increases proliferation, migration, and stemness in glioma cell lines. A schematic representation showing the functional effect of ADAMTS9-AS1.

Significance of microRNA-targeted ErbB signaling pathway genes in cardiomyocyte differentiation.

OBJECTIVE(S): Cardiomyocyte differentiation is a complex process that follows the progression of gene expression alterations. The ErbB signaling pathway is necessary for various stages of cardiac development. We aimed to identify potential microRNAs targeting the ErbB signaling pathway genes by in silico approaches. METHODS: Small RNA-sequencing data were obtained from GSE108021 for cardiomyocyte differentiation. Differentially expressed miRNAs were acquired via the DESeq2 package. Signaling pathways and gene ontology processes for the identified miRNAs were determined and the targeted genes of those miRNAs affecting the ErbB signaling pathway were determined. RESULTS: Results revealed highly differentially expressed miRNAs were common between the differentiation stages and they targeted the genes involved in the ErbB signaling pathway as follows: let-7g-5p targets both CDKN1A and NRAS, while let-7c-5p and let-7d-5p hit CDKN1A and NRAS exclusively. let-7 family members targeted MAPK8 and ABL2. GSK3B was targeted by miR-199a-5p and miR-214-3p, and ERBB4 was targeted by miR-199b-3p and miR-653-5p. miR-214-3p, miR-199b-3p, miR-1277-5p, miR-21-5p, and miR-21-3p targeted CBL, mTOR, Jun, JNKK, and GRB1, respectively. MAPK8 was targeted by miR-214-3p, and ABL2 was targeted by miR-125b-5p and miR-1277-5p, too. CONCLUSION: We determined miRNAs and their target genes in the ErbB signaling pathway in cardiomyocyte development and consequently heart pathophysiology progression.

Two leading international congresses in Iran in the era of COVID-19: 21st royan international twin congress, 4th international and 16th Iranian genetics congress.

In this report, we look at the challenges posed by the outbreak of COVID-19 and how the Executive Board of these two congresses succeeded in overcoming those challenges and holding two congresses. The approach for a large festival with different virtual setting components provided a suitable solution that led to exemplary achievements and created an appropriate model for future virtual or combined virtual and face-to-face events. These events proved that pandemic problems could not limit the organizers, pushing them to make better use of the facilities and turning this threat into an opportunity.

ErbB4-encoded novel miRNAs act as tumor suppressors by regulating ErbB/PI3K signaling.

BACKGROUND: ErbB/PI3K signaling is widely recognized as a critical modulator of malignancy and miRNAs have been found to play a crucial role in the regulation of this pathway. OBJECTIVE: This study aimed to identify novel miRNAs related to the ErbBs loci and investigate the functional effects of these miRNAs on ErbB/PI3K signaling in cancer progression. MATERIALS AND METHODS: Bioinformatics tools and RNA-seq data were used to discover novel miRNAs in breast and colon cancer cells. Gene expression levels were determined using RT-qPCR. Western blotting and dual-luciferase assays were used to identify the regulatory mechanism between ErbB4-miR1/2 and related genes. The effects of ErbB4-miR1/2 on cell proliferation, viability, ROS production, and migration were assessed by PI-flow cytometry, colony formation, MTT, ROS, scratch, and transwell assays in SKBR3 and SW480 cells. RESULTS: MicroRNA prediction tools, RNA-seq data, RT-qPCR, and sequencing results identified ErbB4-miR1 and ErbB4-miR2 (ErbB4-miR1/2) as novel miRNAs encoded by ErbB4 gene. ErbB4-miR1/2 were downregulated in breast and colon tumor tissues and also in different cancerous cells. RT-qPCR and dual-luciferase assays revealed that ErbB2 and ErbB3 genes are regulated by ErbB4-miR1/2. Consistently, a decrease in the p-AKT/AKT protein ratio verified the suppressive effect of ErbB4-miR1/2 on ErbB/PI3K activity. Furthermore, ErbB4-miR1/2 overexpression suppressed cell proliferation, viability, and migration, and increased ROS production. CONCLUSIONS: ErbB4-miR1/2 are novel tumor suppressor miRNAs which attenuate ErbB/PI3K signaling in breast and colon cancer cells.

LINC02381-ceRNA exerts its oncogenic effect through regulation of IGF1R signaling pathway in glioma.

PURPOSE: LncRNAs play essential roles in the cellular and molecular biology of glioma. Some LncRNAs exert their role through sponging miRNAs and regulating multiple signaling pathways. LINC02381 is involved in several cancer types as either oncogene or tumor suppressor. Here, we intended to find the molecular mechanisms of the LINC02381 effect during the glioma progression in related cell lines. METHODS AND RESULTS: RNA-seq data analysis indicated the oncogenic characteristics of LINC02381, and RT-qPCR results confirmed its upregulation compared to normal tissues. Besides its expression was relatively stronger in invasive glioma cell lines. Furthermore, in silico analysis revealed LINC02381 is concentrated in the cytoplasm and predicted its sponging effect against miR-128 and miR-150, which was verified through dual luciferase assay. When LINC02381 was overexpressed in 1321N1, U87, and A172 cell lines, IGF1R and TrkC receptors as well as their downstream pathways (PI3K and RAS/MAPK), were upregulated, detected by RT-qPCR, and verified by western analysis. Consistently, LINC02381 overexpression was followed by an increased proliferation rate of transfected glioma cell lines, detected by flow cytometry and MTT assay, and RT-qPCR. It also resulted in elevated EMT and stemness markers expression level, increased migration rate, and reduced apoptosis rate, detected by RT-qPCR, western analysis, scratch test, and Annexin/PI flow cytometry analysis, respectively. CONCLUSION: The overall results indicated that LINC02381 exerts its oncogenic effect in glioma cells through sponging miR-128 and miR-150 to upregulate the IGF1R signaling pathway. Our results introduce LINC02381 and miR-128, and miR-150 as potential prognosis and therapy targets for the treatment of glioma.

Tenascin-C as a noninvasive biomarker of coronary artery disease.

BACKGROUND: Coronary artery disease (CAD), is the leading cause of mortality and morbidity worldwide. Tenascin-C (TNC) with high expression levels in inflammatory and cardiovascular diseases, leads to the rupture of atherosclerotic plaques. The origin of plaque destabilization can be associated to endothelial dysfunction. Given the high prevalence of CAD, finding valuable biomarkers for its early detection is of great interest. Using serum samples from patients with CAD and individuals without CAD, we assessed the efficacy of TNC expression levels in serum exosomes and during endothelial cell differentiation as a noninvasive biomarker of CAD. METHODS: TNC expression was analyzed using the RNA-sequencing data sets of 6 CAD and 6 normal samples of blood exosomes and endothelial differentiation transitions. Additionally, TNC expression was investigated in the serum samples of patients with CAD and individuals without CAD via qRT-PCR. ROC curve analysis was employed to test the suitability of TNC expression alterations as a CAD biomarker. RESULTS: TNC exhibited higher expression in the exosomes of the CAD samples than in those of the non-CAD samples. During endothelial differentiation, TNC expression was upregulated and then consistently downregulated in mature endothelial cells. Moreover, TNC was significantly upregulated in the serum of the CAD group (P = 0.02), with an AUC of 0.744 for the expression level (95% confidence interval, 0.582 to 0.907; P = 0.011). Hence its expression level can be discriminated CAD from non-CAD samples. DISCUSSION: Our study is the first to confirm that altered TNC expression is associated with pathological CAD conditions in Iranian patients. The expression of TNC is involved in endothelial differentiation and CAD development. Accordingly, TNC can serve as a valuable noninvasive biomarker with potential application in CAD diagnosis.

Downregulation of Talin-1 is associated with the increased expression of miR-182-5p and miR-9-5p in coronary artery disease.

BACKGROUND: Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD). METHODS: Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction. RESULTS: Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples. CONCLUSIONS: Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis.

Linc-ROR has a Potential ceRNA Activity for OCT4A by Sequestering miR-335-5p in the HEK293T Cell Line.

Linc-ROR has a regulatory role in reprogramming, and the core stem cell transcription factors, OCT4, SOX2, and NANOG, regulate its expression. MicroRNAs (miRNAs) are also a critical constituent of pivotal posttranscriptional regulatory pathways. One of such interactions is a competing endogenous RNA interaction that connects small and long non-coding RNAs with coding transcripts. Here, we aimed to investigate the existence of such associations between OCT4A, Linc-ROR, hsa-miR-335-5p, and hsa-miR-544. Bioinformatic analysis was performed to evaluate the expression status of OCT4A, Linc-ROR, miR-335, and miR-544 throughout differentiation as well as in various differentiated cells. The complete lengths of OCT4A and Linc-ROR, and OCT4A 3'-UTR were cloned in the luciferase reporter vector, and the precursors of miR-335 and miR-544 were cloned in expression vectors. Following the overexpression of miR-335 and miR-544 in the 5637 cell line, the endogenous expression of OCT4A and Linc-ROR was evaluated. Afterward, the expression vectors of miRNAs and the reporter vectors of OCT4A/Linc-ROR were co-transfected in the HEK293T cell line. Via the Dual-Luciferase assay, the effect of the overexpression of miRNAs on their two possible targets (Linc-ROR and OCT4A) was investigated. The bioinformatic analysis demonstrated a relatively similar expression pattern for OCT4A and Linc-ROR, while miR-335 showed a different expression status. Both miR-335 and miR-544 inhibited the endogenous expression of OCT4A. The Dual-Luciferase assay likewise confirmed the inhibitory effect of miR-335 and miR-544 on OCT4A expression. In contrast, the miR-335 inhibitory effect was reversed in the presence of Linc-ROR, resulting in the upregulation of OCT4A. Such evidence suggests that Linc-ROR may compete with OCT4A to interact with miR-335.

Modulations of obesity-related microRNAs after exercise intervention: a systematic review and bioinformatics analysis.

Obesity is one of the prevalent health-threatening conditions; however, it is preventable by lifestyle interventions such as exercise. The molecular mechanisms underlying physiological adaptation to physical activity are not fully understood. It has been documented that both intracellular and extracellular (circulating) microRNAs (miRNAs) are involved in both obesogenic and exercise adaptation mechanisms. We aimed to conduct a systematic review of publications that examined the effect of exercise on the expression of miRNAs in individuals with obesity. In addition, bioinformatics analysis was performed on most repetitive miRNAs. PubMed, Scopus, and Google Scholar were searched with relevant keywords. We only included studies that utilized exercise as a modality for the health management of human subjects with obesity to evaluate the changes in expression of obesity-related miRNAs. Through checking of 211 retrieved articles, we reached 12 eligible studies. Some studies reported a statistically significant correlation between the change of miRNAs and clinical parameters such as body mass index and fasting glucose. In silico analysis of most repetitive miRNAs i.e. miR-126, miR-21, miR-146a, miR-221, and miR-223 resulted in the molecular signaling pathways that potentially involve in cellular adaption to exercise in people with obesity. miRNAs partake in health-related benefits of physical activity on obesity-associated cellular and molecular phenomena. However, our understanding of the exact mechanism is still in its infancy. Consistently, the clinicians waiting for the result of more integrated experiments to develop a miRNAs panel as a predictive biomarker of exercise in patients with obesity.

Expression and Function of C1orf132 Long-Noncoding RNA in Breast Cancer Cell Lines and Tissues.

miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.

Bioinformatics prediction and experimental validation of a novel microRNA: hsa-miR-B43 within human CDH4 gene with a potential metastasis-related function in breast cancer.

As a class of short noncoding RNAs, microRNAs (miRNAs) play a key role in the modulation of gene expression. Although, the regulatory roles of currently identified miRNAs in various cancer types including breast cancer have been well documented, there are many as yet undiscovered miRNAs. The aim of the current study was to bioinformatically reanalyze a list of 189 potentially new miRNAs introduced in a previously published paper (PMID: 21346806) and experimentally explore the existence and function of a candidate one: hsa-miR-B43 in breast cancer cells. The sequences of 189 potential miRNAs were re-checked in the miRbase database. Genomic location and conservation of them were assessed with the University of California Santa Cruz (UCSC) genome browser. SSC profiler, RNAfold, miRNAFold, MiPred, and FOMmiR bioinformatics tools were furthermore utilized to explore potential hairpin structures and differentiate real miRNA precursors from pseudo ones. hsa-miR-B43 was finally selected as one of the best candidates for laboratory verification. The expression and function of hsa-miR-B43 were examined by real-time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and wound-healing assays. DIANA-microT, RNAhybrid and Enrichr tools were used to predict the miRNA target genes and for further enrichment analysis. We could detect the exogenous and endogenous expression of hsa-miR-B43, as a real novel miRNA, in cancer cell lines. Gene Ontology enrichment, pathway analysis and wound-healing assay results furthermore confirmed that a metastasis-related function may be assigned to hsa-miR-B43. Our results introduced hsa-miR-B43, as a novel functional miRNA, which might play a role in the metastatic process. Further studies will be necessary to completely survey the existence and function of hsa-miR-B43 in other cancer types.

Hsa-miR-6165 downregulates insulin-like growth factor-1 receptor (IGF-1R) expression and enhances apoptosis in SW480 cells.

MicroRNAs are small non-coding RNAs that are implicated in various biological processes. Hsa-miR-6165 (miR-6165), located in the p75NTR gene, is known to induce apoptosis in human cell lines, but its mechanism of action is not fully understood yet. Here, we predicted the insulin-like growth factor 1 receptor (IGF-1R) gene as a bona fide target for miR-6165. The overexpression of miR-6165 in SW480 cells resulted in significant downregulation of IGF-1R expression as detected by real time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Also, it resulted in reduced transcript levels of AKT2, AKT3, PI3KR3, PI3KR5, CCND1, c-MYC and P21 genes detected by RT-qPCR analysis. In addition, a direct interaction between miR-6165 and a 3'UTR sequence of the IGF-1R gene was verified through a dual luciferase assay. Furthermore, miR-6165 and IGF-1R showed opposite patterns of expression during the neural differentiation process of NT2 cells. Annexin V analysis and MTT assay showed that miR-6165 overexpression was followed by increased apoptosis and reduced the viability rate of SW480 cells. Moreover, a lower expression level of miR-6165 was detected in high-grade colorectal tumors compared with low-grade tumors. Taken together, the results of our study suggest a tumor suppressive role of miR-6165 in colorectal cancer, which seems to take place by regulating IGF-1R gene expression.

MicroRNA profiling reveals important functions of miR-125b and let-7a during human retinal pigment epithelial cell differentiation.

Retinal pigment epithelial (RPE) cells are indispensable for eye organogenesis and vision. To realize the therapeutic potential of in vitro-generated RPE cells for cell-replacement therapy of RPE-related retinopathies, molecular mechanisms of RPE specification and maturation need to be investigated. So far, many attempts have been made to decipher the regulatory networks involved in the differentiation of human pluripotent stem cells into RPE cells. Here, we exploited a highly-efficient RPE differentiation protocol to determine global expression patterns of microRNAs (miRNAs) during human embryonic stem cell (hESC) differentiation into RPE using small RNA sequencing. Our results revealed a significant downregulation of pluripotency-associated miRNAs along with a significant upregulation of RPE-associated miRNAs in differentiating cells. Our functional analyses indicated that two RPE-enriched miRNAs (i.e. miR-125b and let-7a) could promote RPE fate at the expense of neural fate during RPE differentiation. Taken together, these mechanistic interrogations might shed light on a better understanding of RPE cell development and provide insights for the future application of these cells in regenerative medicine.

A panel of noncoding RNAs in non-small-cell lung cancer.

Non-small-lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC could pave the way for effective therapies. Analysis of molecular genetic biomarkers in biological fluids has been proposed as a useful tool for cancer diagnosis. Here, we aimed to develop a panel of noncoding RNAs (ncRNAs) in sputum for NSCLC early detection. Expression of 11 ncRNAs were analyzed by real-time polymerase chain reaction in sputum samples of 30 NSCLC patients and 30 sex- and age-matched cancer-free controls. Stability of endogenous microRNAs (miRNAs) in sputum was evaluated after 3 and 6 days at 4°C, 6 months, and 1 year at -80°C. Nine ncRNAs showed significant differences of their expression in sputum between NSCLC patients and controls. A logistic regression model with the best prediction was built based on miR-145, miR-126, and miR-7. The composite of the three miRNAs produced 90% sensitivity and specificity in distinguishing NSCLC patients from the controls. Results indicate that miRNAs could be useful biomarkers based on their stability under various storage conditions and maintain differential changes between cancer and control groups. Moreover, measurement of miRNAs in sputum could be a noninvasive approach for detection of lung cancer.

miR-21-5p, miR-141-3p, and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer.

BACKGROUND: Early detection of cancers improves patients' survival and decreases the treatment cost. Unfortunately, the current methods for diagnosis of bladder and prostate cancers, two most common urothelial malignancies, suffer from a low sensitivity and specificity. MicroRNAs, as a group of endogenously produced non-coding RNAs, regulate gene expression and their expression is observed to be altered in many cancers and cancer progression phenomena. The remarkable stability of microRNAs in biofluids and their unique expression pattern in different pathological conditions make them an appealing, noninvasive diagnostic method in cancer diagnosis. Our objective is to identify microRNAs as biomarkers in urine samples of bladder and prostate cancers to improve the existing diagnostic methods in this field. MATERIALS AND METHODS: In this study, urine samples from 110 men with either bladder (n = 45) or prostate (n = 23) cancer, benign prostatic hyperplasia (n = 22) and healthy controls (n = 20) were collected. qPCR was used to evaluate the expression level of miR-21-5p, miR-141-3p, and miR-205-5p in these samples. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. RESULTS: The analysis of the data revealed that miR-21-5p, miR-141-3p, and miR-205-5p are differentially expressed in urine of bladder and prostate cancer patients. All these three microRNAs were upregulated in these samples and they were also able to differentiate benign prostatic hyperplasia from malignant cases. The statistical analyses revealed a good specificity for each individual microRNA. CONCLUSION: The results show that these three urine-based microRNAs might be a good choice to implement a specific and non-invasive diagnostic tool for bladder and prostate cancer. The expression pattern of all three microRNAs was particularly useful to distinguish benign and invasive tumors in prostate cases. From the patients' perspective the improvement of the diagnostic situation is awaited eagerly.

Alternative splicing of the OCC-1 gene generates three splice variants and a novel exonic microRNA, which regulate the Wnt signaling pathway.

The Wnt signaling pathway is hyperactivated in most colorectal cancers (CRC). Finding new regulators of this pathway represents the potential for cancer diagnosis or treatment. OCC-1 was initially reported as an up-regulated gene in colon carcinoma, without knowing its mechanism of action. Here, two novel transcript variants and an exonic microRNA that originated from the OCC-1 gene are reported, showing positive effects on Wnt activity. Up-regulation of the known OCC-1 variant (assigned as OCC-1A/B) was limited to CRC, and its overexpression increased survival of CRC-originated SW480 cells (Wnt+), while resulting in apoptosis of Wnt-suppressed SW480 cells or HeLa cells (Wnt-) detected by PI staining. Immunocytochemistry showed that the OCC-1A/B-encoded peptide was localized to the nucleus, where its overexpression resulted in Wnt signaling up-regulation, detected by TOP/FOPflash assay. The noncoding portion of the OCC-1A/B transcript had a suppressive effect on Wnt activity and had a negative correlation with APPL2 neighboring gene expression. Unlike OCC-1A/B, the novel OCC-1C splice variant had no expression alteration in CRC, and it seemed to encode a smaller peptide with cytoplasmic localization. A 60-nucleotide (nt) fragment containing an AUG start codon is spliced out to produce an OCC-1D noncoding RNA variant. The 60-nt RNA was validated as the precursor of a novel microRNA, which we named miR-ex1 Both OCC-1D and miR-ex1 were coordinately up-regulated in CRC. MiR-ex1 functional analysis revealed that it is targeting the APC2 tumor suppressor gene and is an activator of the Wnt signaling pathway. Overall, the OCC-1 gene is now introduced as a novel Wnt signaling regulator and as a potential therapeutic target.

HDAC Inhibitors Induce BDNF Expression and Promote Neurite Outgrowth in Human Neural Progenitor Cells-Derived Neurons.

Besides its key role in neural development, brain-derived neurotrophic factor (BDNF) is important for long-term potentiation and neurogenesis, which makes it a critical factor in learning and memory. Due to the important role of BDNF in synaptic function and plasticity, an in-house epigenetic library was screened against human neural progenitor cells (HNPCs) and WS1 human skin fibroblast cells using Cell-to-Ct assay kit to identify the small compounds capable of modulating the BDNF expression. In addition to two well-known hydroxamic acid-based histone deacetylase inhibitors (hb-HDACis), SAHA and TSA, several structurally similar HDAC inhibitors including SB-939, PCI-24781 and JNJ-26481585 with even higher impact on BDNF expression, were discovered in this study. Furthermore, by using well-developed immunohistochemistry assays, the selected compounds were also proved to have neurogenic potential improving the neurite outgrowth in HNPCs-derived neurons. In conclusion, we proved the neurogenic potential of several hb-HDACis, alongside their ability to enhance BDNF expression, which by modulating the neurogenesis and/or compensating for neuronal loss, could be propitious for treatment of neurological disorders.

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation, and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs embedded within the gene. Using bioinformatics approaches, a novel microRNA named TrkC-miR2 was predicted within the TrkC gene capable of regulating the Wnt pathway. For experimental verification of this microRNA, the predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 after the treatment of HCT116 cells with 5-azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-quantitative PCR and luciferase assays indicated that the APC2 gene is targeted by TrkC-miR2, and Wnt signaling is up-regulated. Also, Wnt inhibition by using small molecules along with TrkC-miR2 overexpression and TOP/FOP flash assays confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, which was detected by flow cytometry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly up-regulated (∼70 times) in colorectal tumor tissues compared with their normal pairs. Moreover, the TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29, and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and the APC2 gene in colorectal cancer specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling, which might be a candidate oncogenic colorectal cancer biomarker.

Hsa-miR-5582-3P regulatory effect on TGFß signaling through targeting of TGFß-R1, TGFß-R2, SMAD3, and SMAD4 transcripts.

Transforming growth factor ß (TGFß) signaling pathway which is regulated by factors such as microRNAs (miRNAs) has pivotal roles in various cellular processes. Here, we intended to verify bioinformatics predicted regulatory effect of hsa-miR-5582-3P against TGFß/SMAD signaling pathway components. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis indicated a negative correlation of expression between hsa-miR-5582-3P against TGFß-R1, TGFß-R2, SMAD3, and SMAD4 putative target genes in all of tested cell lines. Also, hsa-miR-5582-3P was significantly downregulated in glioma, breast, and ovarian tumor tissues compared with their normal pairs, detected by RT-qPCR. Then dual luciferase assay supported direct interaction between this miRNA and TGFß-R1, TGFß-R2, SMAD3, and SMAD4, 3' untranslated region sequences. Western blot analysis confirmed negative effect of hsa-miR-5582-3P overexpression on at least TGFß-R1 expression. Consistently, hsa-miR-5582-3P overexpression brought about downregulation of TGFß-R1, TGFß-R2, SMAD3, and SMAD4 expression in HCT-116 cell line, followed by cell cycle arrest in sub-G1 phase, detected by flow cytometry. Altogether, our data suggest that hsa-miR-5582-3P reduces the TGFß/SMAD signaling pathway through downregulation of TGFß-R1, TGFß-R2, SMAD3, and SMAD4 transcripts. These data introduce hsa-miR-5582-3P as a potential tumor suppressors-miR and a therapy candidate to be tested in cancers in which TGFß/SMAD is deregulated.

Two long non-coding RNAs, Prcat17.3 and Prcat38, could efficiently discriminate benign prostate hyperplasia from prostate cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have recently appeared as new players in cancer biology. Recently, a number of new prostate cancer-associated lncRNAs has been listed via RNA-seq approach by Mitranscriptome project. By analyzing this data we chose four lncRNAs (Prcat17.3, Prcat38, Prcat47, and Cat2184.4) and evaluated their expressions and their abilities to discriminate prostate tumors from benign prostate hyperplasia (BPH). METHODS: Fresh Prostate tissue samples (30 BPH, and 30 tumor samples) and urine samples (19 BPH, and 19 tumor samples) were collected and their total RNA extracted for cDNA syntheses. The expression of candidate lncRNAs was assessed by the real-time PCR technique. RESULTS: Our data revealed that the expression levels of PRCAT17.3 (P < 0.0001) and PRCAT38 (P < 0.0002) were significantly upregulated in human prostate cancer tissues, compared to BPH ones. Moreover, the altered expression was much higher for PRCAT17.3 (∼2000 folds) than PRCAT38 (∼50 folds). In contrast, the expression of Cat2184.4 showed a significant down-regulation in tumor samples (P < 0.0001), compared to BPH ones. While the expression level of PRCAT47 was increased in cancer samples, the changes were not statistically significant. In discriminating prostate tumors from BPH samples, Prcat17.3 (AUC-ROC, 0.927) demonstrated a better diagnostic efficacy than Prcat38 (AUC-ROC, 0.778). Moreover, real-time RT-PCR analyses on urine samples of prostate cancer patients revealed that prcat17.3 level is significantly elevated, (P < 0.0197; AUC-ROC value of 0.72), compared to that of BPH patients. CONCLUSION: We introduce here two novel lncRNAs with a potential application in diagnosis of prostate cancer.