PURPOSE: Breast cancer is one of the most common malignancies among females, and its prognosis is affected by a complex network of gene interactions. Weighted gene co-expression network analysis was used to construct free-scale gene co-expression networks and to identify potential biomarkers for breast cancer progression.
METHODS: The gene expression profiles of GSE42568 were downloaded from the Gene Expression Omnibus database. RNA-sequencing data and clinical information of breast cancer from TCGA were used for validation.
RESULTS: A total of ten modules were established by the average linkage hierarchical clustering. We identified 58 network hub genes in the significant module (R
CONCLUSIONS: AGO2, CDC20, CDCA5, MCM10, MYBL2, and TTK were identified as candidate biomarkers for further basic and clinical research on breast cancer based on co-expression analysis.

The expression of microRNA in eukaryotic cells is subject to tightly regulated processing. The altered expression of microRNAs in a number of cancers suggests their contribution to disease pathogenesis, where processing pathways may be involved in disease pathogenesis. In the present study, we evaluated changes in the profile of two main components of microRNA biogenesis, AGO2 and DICER, and assessed their correlation with disease progression in childhood acute lymphoblastic leukaemia (ALL). To achieve this aim, 25 patients afflicted with ALL were included in the study along with 25 healthy subjects as control. The expression level of AGO2 and DICER was evaluated by real-time PCR. The results revealed an increase in the expression of DICER and a decrease in AGO2 in patients. The correlation between the alteration levels of these genes with pathologic events was also studied. This increase or decrease proved to be directly correlated with the progression of the disease particularly in L1 to L2. According to the obtained results, it can be deduced that dysregulation in transcription of DICER and AGO2, involved in the formation of mature microRNAs in cytoplasm of ALL cancer cells, is a part of the pathological molecular mechanism implicated in the exacerbation of this malignancy. Therefore, the genes involved in microRNAs biogenesis that have been studied here could be considered as candidate prognostic markers especially in childhood ALL which will help towards a better understanding of the molecular basis of ALL.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

BACKGROUND: Bladder cancer (BC) is a common type of cancer that involves tumors of the urinary system and poses a serious threat to human health. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers. Novel lncRNA biomarkers in BC urgently need to be investigated in regard to its function and regulatory mechanisms.
METHODS: Identification of differentially expressed lncRNAs in BC tissue was performed via microarray analysis. To investigate the biological functions of LINC00612, loss-of-function and gain-of-function experiments were performed in vitro and in vivo. Bioinformatics analysis, dual-luciferase reporter assays, AGO2-RIP assays, RNA pull-down assays, real-time quantitative PCR (RT-qPCR) arrays, fluorescence in situ hybridization assays, and western blot assays were conducted to explore the underlying mechanisms of competitive endogenous RNAs (ceRNAs).
RESULTS: LINC00612 was upregulated in BC tissues and cell lines. Functionally, downregulation of LINC00612 inhibited cell proliferation and invasion in vitro and in vivo, whereas overexpression of LINC00612 resulted in the opposite effects. Bioinformatics analysis and luciferase assays revealed that miR-590 was a direct target of LINC0061, which was validated by dual-luciferase reporter assays, AGO2-RIP assays, RNA pull-down assays, RT-qPCR arrays, and rescue experiments. Additionally, miR-590 was shown to directly target the PHD finger protein 14 (PHF14) gene. LNIC00612 modulated the expression of E-cadherin and vimentin by competitively sponging miR-590 to elevate the expression of PHF14, thus affecting BC cellular epithelial-mesenchymal transition (EMT).
CONCLUSIONS: Our results indicate that LINC00612 enhances the proliferation and invasion ability of BC cells by sponging miR-590 to upregulate PHF14 expression and promote BC cellular EMT, suggesting that LINC00612 may act as a potential biomarker and therapeutic target for BC.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown.
METHODS: The expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay.
RESULTS: We revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p's target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells.
CONCLUSIONS: Our results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

PURPOSE: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development.
MATERIALS AND METHODS: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated
RESULTS: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells
CONCLUSION: miR-206 reduces OS cell malignancy

BACKGROUND: Intensive investigations have identified a collection of microRNAs (miRNAs) and their functional machineries in cytoplasm. However, a comprehensive view of miRNAs and mRNAs in cytoplasm and nucleus has not been explored. This study aims to reveal the mechanisms of miRNA-RNA interactions in nucleus and cytoplasm.
METHODS: In this study, the miRNAs and their target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were characterized using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP). Then, the selected miRNAs were verified by Northern blot. The target mRNAs in the Argonaute2 (Ago2) complex of nucleus and cytoplasm of gastric cancer cells were analyzed through Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis.
RESULTS: The results revealed that there were 243 miRNAs and 265 miRNAs in the Ago2 complexes of nucleus and cytoplasm, respectively. The majority of mature miRNAs existed in cytoplasm. The analysis of miRNA targetome from the Ago2 complexes indicated that a lot of mRNAs with high expression level existed in nucleus. The target genes of miRNAs in the Ago2 complexes of nucleus and cytoplasm played important roles in cell proliferation, cell differentiation, innate immune response and tumorigenesis.
CONCLUSIONS: microRNA-mRNA interactions occur in nucleus and cytoplasm of gastric cancer cells. Therefore, our study demonstrated that miRNA-mRNA interactions not only took place in cytoplasm but also in nucleus.

Glioma invasion is a main cause of a poor prognosis and relapse in patients suffering from the disease. However, the molecular mechanisms responsible for glioma cell invasion remain poorly understood. In this study, the characteristics of exosomes were identified using electron microscope (TEM), and western blot analysis. The potential mechanism of long non‑coding RNA (lncRNA) activated by TGF‑β (lncRNA‑ATB) was demonstrated using luciferase reporter assays and RNA immunoprecipitation. We found that glioma cell‑derived exosomes promoted the activation of astrocytes and had the ability to shuttle long non‑coding RNA (lncRNA) activated by TGF‑β (lncRNA‑ATB) to astrocytes. More importantly, lncRNA‑ATB activated astrocytes through the suppression of microRNA (miRNA or miR)‑204‑3p in an Argonaute 2 (Ago2)‑dependent manner. Furthermore, astrocytes activated by lncRNA‑ATB in turn promoted the migration and invasion of glioma cells. Taken together, the findings of this study suggest that lncRNA‑ATB may play an important role in modulating glioma microenvironment through exosomes. Thus, a better understanding of this process may provide implications for the prevention of highly invasive glioma.

BACKGROUND/AIMS: Cellular senescence, or permanent growth arrest, is known as an effective tumor suppressor mechanism that can be induced by different stressors, such as oncogenes, chemotherapeutics or cytokine cocktails. Previous studies demonstrated that the growth-repressing state of oncogene-induced senescent cells depends on argonaute protein 2 (Ago2)-mediated transcriptional gene silencing and Ago2/Rb corepression of E2F-dependent cell cycle genes. Cytokine-induced senescence (CIS) likewise depends on activation of the p16Ink4a/Rb pathway, and consecutive inactivation of the E2F family of transcription factors. In the present study, we therefore analyzed the role of Ago2 in CIS.
METHODS: Human cancer cell lines were treated with interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) to induce senescence. Senescence was determined by growth assays and measurement of senescence-associated β-galactosidase (SA-β-gal) activity, Ago2 translocation by Ago2/ Ki67 immunofluorescence staining and western blot analysis, and gene transcription by quantitative polymerase chain reaction (qPCR).
RESULTS: IFN-γ and TNF permanently stopped cell proliferation and time-dependently increased SA-β-gal activity. After 24 - 48 h of cytokine treatment, Ago2 translocated from the cytoplasm into the nucleus of Ki67-negative cells, an effect which was shown to be reversible. Importantly, the proinflammatory cytokine cocktail suppressed Ago2-regulated cell cycle control genes, and siRNA-mediated depletion of Ago2 interfered with cytokine-induced growth inhibition.
CONCLUSION: IFN-γ and TNF induce a stable cell cycle arrest of cancer cells that is accompanied by a fast nuclear Ago2 translocation and repression of Ago2-regulated cell cycle control genes. As Ago2 downregulation impairs cytokine-induced growth regulation, Ago2 may contribute to tissue homeostasis in human cancers.

BACKGROUND/AIMS: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells.
METHODS: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML.
RESULTS: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3'-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay.
CONCLUSION: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.

Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.

Argonaute2 (AGO2) is an effector of small RNA mediated gene silencing. Increasing evidence show that post-translational modifications of AGO2 can change miRNA activity at specific or global levels. Among the six mature miRNAs that are encoded by miR-17-92, miR-19b1 is the most powerful to exert the oncogenic properties of the entire cluster. Here we identify that AGO2 can be acetylated by P300/CBP and deacetylated by HDAC7, and that acetylation occurs at three sites K720, K493, and K355. Mutation of K493R/K720R, but not K355R at AGO2, inhibits miR-19b biogenesis. We demonstrate that acetylation of AGO2 specifically increases its recruiting pre-miR-19b1 to form the miPDC (miRNA precursor deposit complex), thereby to enhance miR-19b maturation. The motif UGUGUG in the terminal-loop of pre-miR-19b1, as a specific processing feature that is recognized and bound by acetylated AGO2, is essential for the assembly of miRISC (miRNA-induced silencing complex) loading complex. Analyses on public clinical data, xenograft mouse models, and IHC and ISH staining of lung cancer tissues, further confirm that the high levels of both AGO2 acetylation and miR-19b correlate with poor prognosis in lung cancer patients. Our finding reveals a novel function of AGO2 acetylation in increasing oncogenic miR-19b biogenesis and suggests that modulation of AGO2 acetylation has potential clinical implications.

BACKGROUND/AIMS: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach.
METHODS: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs.
RESULTS: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting.
CONCLUSION: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

PURPOSE: To evaluate indications/outcomes for open partial nephrectomy (OPN) when non-flank approaches are required, with comparison to patients managed with the flank approach. Outcomes with a non-flank approach are presumed less favorable yet there have been no previous reports on this topic.
METHODS: 2747 OPNs were performed (1999-2015) and 76 (2.8%) required a non-flank approach. We also reviewed all traditional flank OPNs performed during odd years in this timeframe yielding 1467 patients for comparison.
RESULTS: Overall, median tumor size was 3.5 cm and 274 patients (18%) had a solitary kidney. Non-flank patients were younger, and tumor size and clinical/pathologic stage were significantly increased for this cohort, but the groups were otherwise comparable. Indications for non-flank OPN included large tumor size/locally advanced disease (n = 21), need for simultaneous surgery (n = 25), previous flank incision or failed thermoablation (n = 13), or congenital/vascular abnormalities (n = 9). The most common non-flank approach was anterior subcostal (n = 39, 51%). Operative times, estimated blood loss, positive margins, and functional decline were all modestly increased for non-flank patients. Intraoperative and genitourinary complications were more common in non-flank patients (p < 0.05), although all were manageable, typically with conservative measures. There were no mortalities among non-flank patients and none required long-term dialysis.
CONCLUSIONS: Our series, the first to address this topic, suggests that outcomes with non-flank OPN are generally less advantageous likely reflecting increased tumor/operative complexity. However, complications in this challenging patient population are manageable and final dispositions are generally favorable. Our findings should be useful for counseling regarding potential outcomes when a non-flank incision is required.

It remains unknown whether microRNA (miRNA/miR) can target transfer RNA (tRNA) molecules. Here we provide evidence that miR-34a physically interacts with and functionally targets tRNA

In human cancers, histone methyltransferase SETDB1 (SET domain, bifurcated 1) is frequently overexpressed but its significance in carcinogenesis remains elusive. A recent study shows that SETDB1 downregulation induces de-repression of retroelements and innate immunity in cancer cells. The possibility of SETDB1 functioning as a surveillant of retroelement expression is discussed in this study: the cytoplasmic presence of retroelement-derived nucleic acids (RdNAs) drives SETDB1 into the nucleus by the RNA-interference route, rendering the corresponding retroelement transcriptionally inert. These RdNAs could, therefore, be signals of genome instability sent out for SETDB1 present in the cytoplasm to maintain genome integrity.

Bladder cancer still requires improvements in diagnosis and prognosis, because many of the cases will recur and/or metastasize with bad outcomes. Despite ongoing research on bladder biomarkers, the clinicopathological impact and diagnostic function of miRNA maturation regulators Drosha and Argonaute proteins AGO1 and AGO2 in urothelial bladder carcinoma remain unclear. Therefore, we conducted immunohistochemical investigations of a tissue microarray composed of 112 urothelial bladder carcinomas from therapy-naïve patients who underwent radical cystectomy or transurethral resection and compared the staining signal with adjacent normal bladder tissue. The correlations of protein expression of Drosha, AGO1 and AGO2 with sex, age, tumor stage, histological grading and overall survival were evaluated in order to identify their diagnostic and prognostic potential in urothelial cancer. Our results show an upregulation of AGO1, AGO2 and Drosha in non-muscle-invasive bladder carcinomas, while there was increased protein expression of only AGO2 in muscle-invasive bladder carcinomas. Moreover, we were able to differentiate between non-muscle-invasive and muscle-invasive bladder carcinoma according to AGO1 and Drosha expression. Finally, despite Drosha being a discriminating factor that can predict the probability of overall survival in the Kaplan⁻Meier analysis, AGO1 turned out to be independent of all clinicopathological parameters according to Cox regression. In conclusion, we assumed that the miRNA processing factors have clinical relevance as potential diagnostic and prognostic tools for bladder cancer.

Long noncoding RNAs (lncRNAs) are a type of noncoding RNA transcript that are characterized by lack of protein-coding capacity. The vital role of lncRNAs in non-small cell lung cancer (NSCLC) is attracting increasingly more attention. In the present study, we investigate the role of lncRNA antisense RNA of the TP73 gene (TP73-AS1) in NSCLC carcinogenesis. The results demonstrate that TP73-AS1 is markedly upregulated in NSCLC tissues, and functional experiments revealed that TP73-AS1 is significantly increased in NSCLC tissue and cell lines, indicating a possible oncogenic role. In loss-of-function assays, the knockdown of TP73-AS1 inhibited NSCLC cell proliferation, tumor growth and cycle progression in vivo and in vitro. Bioinformatic tools predicted that miR-449a both targeted the 3'-UTR of TP73-AS1 and EZH2, which was confirmed using luciferase reporter assay and AGO2-dependent RNA immunoprecipitate (RIP). TP73-AS1 and miR-449a were in the same RNA-induced silencing complex (RISC). In summary, the results indicate an explicit oncogenic role of TP73-AS1 in the NSCLC tumorigenesis, suggesting a TP73-AS1-miR-449a-EZH2 axis and providing new insight for NSCLC tumorigenesis.

OBJECTIVE: To determine the expression profile of small noncoding RNAs (sncRNAs) in leiomyoma, which has not been investigated to date.
DESIGN: Laboratory-based investigation.
SETTING: Academic center.
PATIENT(S): Women undergoing hysterectomy for benign indications.
INTERVENTION(S): Next-generation sequencing and screening of an sncRNA database with confirmatory analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).
MAIN OUTCOME MEASURE(S): Expression profile of sncRNAs in leiomyoma and matched myometrium.
RESULT(S): Screening our previously determined RNA sequencing data with the sncRNA database resulted in identification of 15 small nuclear (sn) RNAs, 284 small nucleolar (sno) RNAs, 98 Piwi-interacting (pi) RNAs, 152 transfer (t) RNAs, and 45 ribosomal (r) RNAs, of which 15 snoRNAs, 24 piRNAs, 7 tRNAs, and 6 rRNAs were differentially expressed at a 1.5-fold change cutoff in leiomyoma compared with myometrium. We selected 5 snoRNAs, 4 piRNAs, 1 tRNA, and 1 rRNA that were differentially expressed and confirmed their expression in paired tissues (n = 20) from both phases of the menstrual cycle with the use of qRT-PCR. The results indicated up-regulation of the snoRNAs (SNORD30, SNORD27, SNORA16A, SNORD46, and SNORD56) and down-regulation of the piRNAs (piR-1311, piR-16677, piR-20365, piR-4153), tRNA (TRG-GCC5-1), and rRNA (RNA5SP202) expression in leiomyoma compared with myometrium (P<.05). The pattern of expression of these sncRNAs was similar to RNA sequencing analysis, with no menstrual cycle-dependent differences detected except for SNORD30. Because Argonaute 2 (AGO2) is required for sncRNA-mediated gene silencing, we determined its expression and found greater abundance in leiomyoma.
CONCLUSION(S): Our results provide the first evidence for the differential expression of additional classes of sncRNAs and AGO2 in leiomyoma, implicating their roles as a gene regulatory mechanism.

TGF-β/Activin induces epithelial-to-mesenchymal transition and stemness in pancreatic ductal adenocarcinoma (PDAC). However, the microRNAs (miRNAs) regulated during this response have remained yet undetermined. Here, we show that TGF-β transcriptionally induces MIR100HG lncRNA, containing miR-100, miR-125b and let-7a in its intron, via SMAD2/3. Interestingly, we find that although the pro-tumourigenic miR-100 and miR-125b accordingly increase, the amount of anti-tumourigenic let-7a is unchanged, as TGF-β also induces LIN28B inhibiting its maturation. Notably, we demonstrate that inactivation of miR-125b or miR-100 affects the TGF-β-mediated response indicating that these miRNAs are important TGF-β effectors. We integrate AGO2-RIP-seq with RNA-seq to identify the global regulation exerted by these miRNAs in PDAC cells. Transcripts targeted by miR-125b and miR-100 significantly overlap and mainly inhibit p53 and cell-cell junctions' pathways. Together, we uncover that TGF-β induces an lncRNA, whose encoded miRNAs, miR-100, let-7a and miR-125b play opposing roles in controlling PDAC tumourigenesis.

Circular RNAs (circRNAs) are a group of non-protein-coding RNAs that are generated from back-splicing. Recent evidence indicates that circRNAs play important roles in tissue development, gene regulation, and carcinogenesis. It was recently demonstrated that circular RNAs can function as sponges for miRNAs. In our study, the clinical implications of circRNF13 were assessed in 50 pathologically diagnosed lung adenocarcinoma samples and their paired peripheral normal lung tissues by using quantitative polymerase chain reaction. We validated that circRNF13 was almost 2.98-fold down-regulated in cancer tissues. The expression level of circRNF13 was significantly negatively correlated with TNM staging and lymph node metastasis. In vitro experiments indicated that circRNF13 repressed the invasion and metastasis of lung adenocarcinoma cell lines. Cell fraction analyses and fluorescence in situ hybridization detected that circRNF13 was mostly located in the cytoplasm. Bioinformatic analyses and RIP experiments revealed that circRNF13 could interact with Ago2, an RNA binding protein, and could function as sponge for miR-93-5p. Our data suggest that circRNF13 represents a potential novel biomarker and a therapeutic target of lung adenocarcinoma.

BACKGROUND/AIMS: CircRNAs regulate gene expression in different malignancies. However, the role of Cdr1as in the tumourigenesis of bladder cancer and its potential mechanisms remain unknown.
METHODS: qRT-PCR was used to detect Cdr1as and target miRNA expression in bladder cancer tissues and cell lines. Biological functional experiments were performed to detect the effects of Cdr1as on the biological behaviour of bladder cancer cells in vivo and in vitro. Bioinformatic analysis was utilised to predict potential miRNA target sites on Cdr1as. Ago2 RNA binding protein immunoprecipitation assay, RNA antisense purification assay, biotin pull down assay and RNA FISH were performed to detect the interaction between Cdr1as and target miRNAs. Western blot was used to determine the expression level of p21 in bladder cancer cells.
RESULTS: Cdr1as was significantly down-regulated in bladder cancer tissues compared with adjacent normal tissues. Overexpression of Cdr1as inhibited the proliferation, invasion and migration of bladder cancer cells in vitro and slowed down tumour growth in vivo. Cdr1as sponged multiple miRNAs in bladder cancer. Moreover, Cdr1as directly bound to miR-135a and inhibited its activity in bladder cancer.
CONCLUSION: Cdr1as is down-regulated and sponges multiple miRNAs in bladder cancer. It exerts anti-oncogenic functions by sponging microRNA-135a.

The oncofetal IGF2 mRNA binding proteins (IGF2BPs) are upregulated in most cancers but their paralogue-specific roles in tumor cells remain poorly understood. In a panel of five cancer-derived cell lines, IGF2BP1 shows highly conserved oncogenic potential. Consistently, the deletion of IGF2BP1 impairs the growth and metastasis of ovarian cancer-derived cells in nude mice. Gene expression analyses in ovarian cancer-derived cells reveal that the knockdown of IGF2BPs is associated with the downregulation of mRNAs that are prone to miRNA regulation. All three IGF2BPs preferentially associate upstream of miRNA binding sites (MBSs) in the 3'UTR of mRNAs. The downregulation of mRNAs co-regulated by miRNAs and IGF2BP1 is abrogated at low miRNA abundance or when miRNAs are depleted. IGF2BP1 associates with these target mRNAs in RISC-free complexes and its deletion enhances their association with AGO2. The knockdown of most miRNA-regulated target mRNAs of IGF2BP1 impairs tumor cell properties. In four primary cancers, elevated synthesis of these target mRNAs is largely associated with upregulated IGF2BP1 mRNA levels. In ovarian cancer, the enhanced expression of IGF2BP1 and most of its miRNA-controlled target mRNAs is associated with poor prognosis. In conclusion, these findings indicate that IGF2BP1 enhances an aggressive tumor cell phenotype by antagonizing miRNA-impaired gene expression.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19

Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. β-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.

Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences by a mechanism termed RNA activation (RNAa). In the present study, we designed 4 candidate saRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these saRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, leading to inhibition of cell growth and induction of apoptosis, and suggest that targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.

This study proposes that a novel developmental hierarchy of breast cancer (BC) cells (BCCs) could predict treatment response and outcome. The continued challenge to treat BC requires stratification of BCCs into distinct subsets. This would provide insights on how BCCs evade treatment and adapt dormancy for decades. We selected three subsets, based on the relative expression of octamer-binding transcription factor 4 A (Oct4A) and then analysed each with Affymetrix gene chip. Oct4A is a stem cell gene and would separate subsets based on maturation. Data analyses and gene validation identified three membrane proteins, TMEM98, GPR64 and FAT4. BCCs from cell lines and blood from BC patients were analysed for these three membrane proteins by flow cytometry, along with known markers of cancer stem cells (CSCs), CD44, CD24 and Oct4, aldehyde dehydrogenase 1 (ALDH1) activity and telomere length. A novel working hierarchy of BCCs was established with the most immature subset as CSCs. This group was further subdivided into long- and short-term CSCs. Analyses of 20 post-treatment blood indicated that circulating CSCs and early BC progenitors may be associated with recurrence or early death. These results suggest that the novel hierarchy may predict treatment response and prognosis.

In the rapidly expanding era of cancer target therapy, regulators of apoptosis are emerging as attractive therapeutic targets. X-linked inhibitor of apoptosis (XIAP) is of specific interest owing to its characteristic overexpression in a wide variety of neoplasms, with a resultant survival advantage for tumor cells and treatment resistance. In this study, we examined three pyrazolo [3,4-d] pyridazine derivatives (PPDs) through molecular modeling and studied their modes of interaction with XIAP-BIR3 domain. PPD-1, which possessed the highest binding affinity with XIAP, was tested on A549 (lung cancer cell line); HCT-116 (colorectal carcinoma cell line); HEPG2 (liver carcinoma cell line), HFB4 (normal human skin melanocyte cell line) and WI-38 (human embryonic lung fibroblasts). In comparison to cisplatin as a positive control, PPD-1 yielded remarkable cytotoxicity on all cancer cell lines, with the highest anti-tumor activity on A549 and a favorable therapeutic ratio. Flow cytometry studies concluded that PPD-1 treatment induces Sub G1 and G2/M cell cycle arrest and apoptosis. The percentage of apoptotic cells in PPD-1 treated A549 cells was considerably higher than that in untreated cells (10.06% vs 0.57%, respectively). To further investigate the mechanism of induction of apoptosis by PPD-1, Real time-PCR was used to quantify the expression levels of key apoptotic regulators. Significant overexpression of the effector capsase-3, pro-apoptotic bax and tumor suppressor gene p53 were noted as compared to untreated cells (7.19 folds, 7.28 folds, and 5.08 folds, respectively). Moreover, PPD-1 inhibited the expression of the anti-apoptotic bcl-2 gene to 0.22 folds. These findings demonstrate that PPD-1 treatment disrupts the Bcl-2/BAX balance in lung cancer cell lines, leading to apoptosis induction possibly through intrinsic mitochondria-dependent pathway. These novel insights elucidate the mechanism of PPD-1 cytotoxicity in lung cancer cell lines and offer a promising therapeutic approach that needs further study.

There is an urgent need for an effective treatment for metastatic prostate cancer (PC). Prostate tumors invariably overexpress prostate surface membrane antigen (PSMA). We designed a nonviral vector, PEI-PEG-DUPA (PPD), comprising polyethylenimine-polyethyleneglycol (PEI-PEG) tethered to the PSMA ligand, 2-[3-(1, 3-dicarboxy propyl)ureido] pentanedioic acid (DUPA), to treat PC. The purpose of PEI is to bind polyinosinic/polycytosinic acid (polyIC) and allow endosomal release, while DUPA targets PC cells. PolyIC activates multiple pathways that lead to tumor cell death and to the activation of bystander effects that harness the immune system against the tumor, attacking nontargeted neighboring tumor cells and reducing the probability of acquired resistance and disease recurrence. Targeting polyIC directly to tumor cells avoids the toxicity associated with systemic delivery. PPD selectively delivered polyIC into PSMA-overexpressing PC cells, inducing apoptosis, cytokine secretion, and the recruitment of human peripheral blood mononuclear cells (PBMCs). PSMA-overexpressing tumors in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with partially reconstituted immune systems were significantly shrunken following PPD/polyIC treatment, in all cases. Half of the tumors showed complete regression. PPD/polyIC invokes antitumor immunity, but unlike many immunotherapies does not need to be personalized for each patient. The potent antitumor effects of PPD/polyIC should spur its development for clinical use.

BACKGROUND: MicroRNAs (miRNAs) exhibit essential regulatory functions related to cell growth, apoptosis, development and differentiation. Dysregulated expression of miRNAs is associated with a wide variety of human diseases. As such miRNA signatures are valuable as biomarkers for disease and for making treatment decisions. Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Here we screened for miRNAs in chronic HBV associated HCC.
METHODS: To determine the miRNAs in HCC occurrence associated with HBV infection, we analyzed global miRNA expression profiles in 12 pairs of HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients using microarray analyses. The microarray result was validated by real-time PCR in 32 HBV-positive and 24 HBV-negative patient HCC samples. The potential candidate target genes of the miRNAs were predicted by miRWalk software. Genes simultaneously predicted as targets by two or more miRNAs were subjected to GO and KEGG pathway analysis. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software.
RESULTS: Eight miRNAs (miR-223, miR-98, miR-15b, miR-199a-5p, miR-19b, miR-22, miR-451, and miR-101) were involved in HBV-unrelated HCC, 5 miRNAs (miR-98, miR-375, miR-335, miR-199a-5p, and miR-22) were involved in HBV infection, and 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) were specifically altered in HBV-related HCC. Gene Ontology and KEGG analyses predict that these HBV-related HCC miRNAs are involved in the regulation of: transcription, RNA polymerase II promoter, phosphorylation of proteins through MAPK signaling pathway, focal adhesion, and actin cytoskeleton. IPA analysis also suggest that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that significantly influence HCC occurrence and HBV infection.
CONCLUSION: Our data indicates that the unique 7 miRNAs expression signature could be involved in the development HBV- related HCC.