Silencing of circular RNA circPDE5A suppresses neuroblastoma progression by targeting the miR-362-5p/NOL4L axis.

OBJECTIVE: Neuroblastoma (NB) is the most common extra-cranial solid tumour in early childhood. Circular RNAs (circRNAs) have been implicated in the development of NB. The purpose of the current study was to explore the molecular action of circRNA phosphodiesterase 5 A (circPDE5A) in NB malignant progression. MATERIALS AND METHODS: The expression levels of circPDE5A, miR-362-5p and nucleolar protein 4 like (NOL4L) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) assay. Cell migration and invasion were evaluated by transwell assay. The levels of glucose consumption and lactate production were measured using the commercial assay kits. Targeted correlations among circPDE5A, miR-362-5p and NOL4L were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assays were performed to examine the role of circPDE5A in tumour growth in vivo. RESULTS: Our results revealed that circPDE5A was up-regulated in NB tissues and cells. The silencing of circPDE5A suppressed NB cell proliferation, migration, invasion, and glycolysis in vitro and diminished tumour growth in vivo. Moreover, circPDE5A directly targeted miR-362-5p by binding to miR-362-5p. CircPDE5A silencing impeded NB malignant progression in vitro through up-regulating miR-362-5p. Furthermore, NOL4L was a direct target of miR-362-5p, and NOL4L mediated the regulation of miR-362-5p on NB malignant progression in vitro. Additionally, circPDE5A functioned as a regulator of NOL4L expression via targeting miR-362-5p. CONCLUSIONS: Our current findings identified that the knockdown of circPDE5A suppressed NB malignant progression at least in part by the regulation of the miR-362-5p/NOL4L axis, providing a novel rationale for developing circPDE5A as a potential target for NB management.

Identification and characterization of novel proteins associated with CHD4.

The CHD (chromodomain helicase DNA binding protein) family consists of nine chromatin remodeling factors that alter chromatin structure in an ATP-dependent manner. CHD4 contributes to the regulation of various cellular activities and processes including development through interaction with multiple proteins including formation of the NuRD (nucleosome remodeling and deacetylase activity) complex. Functions of CHD4 that appear not to be mediated by the NuRD complex or other known interactors have also been identified, however, suggesting the existence of unrecognized proteins that also associate with CHD4. We here generated HeLa-S3 and HEK293T cells with a knock-in allele for FLAG epitope-tagged CHD4 and used these cells to identify proteins that bind to CHD4 with the use of immunoprecipitation followed by liquid chromatography and tandem mass spectrometry. LCORL (ligand-dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as novel CHD4 interactors. Furthermore, RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, or NOL4L revealed consistent up-regulation of genes related to the Notch signaling pathway. Our results thus suggest that both LCORL and NOL4L may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.

Cancer Testis Antigen, NOL4, Is an Immunogenic Antigen Specifically Expressed in Small-Cell Lung Cancer.

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.

NOL4L, a novel nuclear protein, promotes cell proliferation and metastasis by enhancing the PI3K/AKT pathway in ovarian cancer.

Nucleolar protein 4-like (NOL4L) was first identified in acute myeloid leukaemia. Then, it was verified to be involved in cell progression in neuroblastoma. However, the functional role of NOL4L in tumor proliferation and metastasis and the underlying molecular mechanism(s) are not fully understood. Immunohistochemistry (IHC) assays were performed in patient tissues to reveal NOL4L expression profiles. Then, we knocked down NOL4L in two ovarian cancer cell lines (Skov3-ip1 and Hey), and cell-based in-vitro and in-vivo assays were subsequently conducted to gain insight into the underlying mechanism of NOL4L in ovarian cancer. We confirmed that the expression of NOL4L was higher in tumor tissues, especially in peritoneal metastatic tissues. Furthermore, we observed that NOL4L was related to prognosis in ovarian cancer patients. Next, we conducted CCK-8 assays, colony formation assays, migration and invasion experiments and wound healing assays and verified that NOL4L could promote proliferation and metastasis in ovarian cancer cells. In addition, NOL4L promoted tumor progression and metastasis in a nude mouse model. Mechanistically, we demonstrated that NOL4L influenced gene expression in the PI3K/AKT pathway. Overall, our study provides genetic and biochemical evidence that NOL4L is critical for tumor progression and metastasis in ovarian cancer cells. Thus, it could serve as a target for antimetastatic therapy in ovarian cancer.

Circ_0132817 facilitates cell proliferation, migration, invasion and glycolysis by regulating the miR-432-5p/NOL4L axis in neuroblastoma.

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children. Circular RNAs (circRNAs) have been shown to be involved in the development of NB. However, the function of circ_0132817 in NB is currently unclear. In this paper, the levels of circ_0132817 and NOL4L were induced in NB tissues and cells, and miR-432-5p expression was on the contrary. MiR-432-5p was verified as a target of circ_0132817 and miR-432-5p could bind to NOL4L. The inhibitory effects of miR-432-5p overexpression on cell proliferation, migration, invasion and glycolysis could be reversed by circ_0132817 facilitation. The suppression of NOL4L knockdown on NB cells progression could be rescued by miR-432-5p inhibition. Besides, knockdown of circ_0132817 repressed tumor growth in vivo. Thus, we came to a conclusion that circ_0132817 promoted the tumorigenesis of NB cells by up-regulating NOL4L and acting as a sponge for miR-432-5p.

LncRNA double homeobox A pseudogene 8 (DUXAP8) facilitates the progression of neuroblastoma and activates Wnt/ß-catenin pathway via microRNA-29/nucleolar protein 4 like (NOL4L) axis.

The potential mechanism of neuroblastoma (NB) progression remains elusive. We intended to uncover the role and network of long noncoding RNA (lncRNA) double homeobox A pseudogene 8 (DUXAP8) in NB. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to detect the levels of DUXAP8, microRNA-29 (miR-29) and nucleolar protein 4 like (NOL4L). The proliferation, colony formation, cell cycle and metastasis of NB cells were examined by (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, plate colony formation assay, flow cytometry and transwell assays. Western blot was conducted to detect the expression of metastasis and proliferation-associated proteins and NOL4L. The target relationship was predicted by StarBase software and was confirmed by dual-luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay. Nude mice bearing tumors were used to verify the role of DUXAP8 in vivo. We found the expression of DUXAP8 was positively related to the stage of NB tumors, and it was negatively associated with the survival rate of NB patients. DUXAP8 knockdown inhibited the proliferation, colony formation, cycle and motility of NB cells. MiR-29 could interact with DUXAP8, and DUXAP8 exacerbated NB via sponging miR-29. MiR-29 could bind to NOL4L, and the influence of NOL4L intervention on the functions of NB cells could be alleviated by the transfection of miR-29 inhibitor. NOL4L was regulated by DUXAP8/miR-29 axis in NB cells. DUXAP8 knockdown blocked the progression of NB in vivo. Collectively, DUXAP8 deteriorated NB through serving as a sponge for miR-29 to up-regulate the expression of NOL4L in vitro and in vivo.

NOL4 is Downregulated and Hyper-Methylated in Papillary Thyroid Carcinoma Suggesting Its Role as a Tumor Suppressor Gene.

BACKGROUND: Thyroid cancer is the fourth most common cancer in the world. Papillary thyroid carcinoma (PTC) accounts for 80% of all types of thyroid neoplasm. Epigenetic alterations such as DNA methylation are known as the main cause of different types of cancers through inactivation of tumor suppressor genes. OBJECTIVES: In the present study, the expression and methylation of suggested gene namely nucleolar protein 4 (NOL4) in PTC in comparison to multi nodular goiter (MNG) have been studied. METHODS: Forty-one patients with PTC and 38 patients affected by MNG were recruited. Thyroid tissues were obtained during thyroidectomy. RNA and DNA were extracted from thyroid tissues. Quantitative RT-PCR assay was performed for determining the mRNA level of NOL4 while methylation-sensitive high resolution methylation was applied for assessing the methylation status with designing six pairs primers for six regions on gene promoter which were named from NOL4 (a) to NOL4 (f). RESULTS: Methylation assessment of 81 CpG islands in the promoter region of NOL4 gene revealed that NOL4 (f), the nearest region to the start codon, was significantly hypermethylated in PTC cases compared to MNG cases. NOL4 level in PTC cases in comparison with MNG cases were downregulated. The methylation status and mRNA level of NOL4 (f) were associated with age of diagnosis (Age of the patient at the time of diagnosis), lymph node metastasis, and advanced stages of disease. CONCLUSIONS: These data suggested an aberrant promoter hyper-methylation of NOL4 in PTC cases may be linked with its downregulation. Therefore, NOL4 gene can be proposed as a potential tumor suppressor gene in PTC tissues.

Cancer/Testis Antigens: "Smart" Biomarkers for Diagnosis and Prognosis of Prostate and Other Cancers.

A clinical dilemma in the management of prostate cancer (PCa) is to distinguish men with aggressive disease who need definitive treatment from men who may not require immediate intervention. Accurate prediction of disease behavior is critical because radical treatment is associated with high morbidity. Here, we highlight the cancer/testis antigens (CTAs) as potential PCa biomarkers. The CTAs are a group of proteins that are typically restricted to the testis in the normal adult but are aberrantly expressed in several types of cancers. Interestingly, >90% of CTAs are predicted to belong to the realm of intrinsically disordered proteins (IDPs), which do not have unique structures and exist as highly dynamic conformational ensembles, but are known to play important roles in several biological processes. Using prostate-associated gene 4 (PAGE4) as an example of a disordered CTA, we highlight how IDP conformational dynamics may regulate phenotypic heterogeneity in PCa cells, and how it may be exploited both as a potential biomarker as well as a promising therapeutic target in PCa. We also discuss how in addition to intrinsic disorder and post-translational modifications, structural and functional variability induced in the CTAs by alternate splicing represents an important feature that might have different roles in different cancers. Although it is clear that significant additional work needs to be done in the outlined direction, this novel concept emphasizing (multi)functionality as an important trait in selecting a biomarker underscoring the theranostic potential of CTAs that is latent in their structure (or, more appropriately, the lack thereof), and casts them as next generation or "smart" biomarker candidates.

Mblk-1 Transcription Factor Family: Its Roles in Various Animals and Regulation by NOL4 Splice Variants in Mammals.

Transcription factors play critical roles in regulation of neural development and functions. A transcription factor Mblk-1 was previously reported from a screen for factors possibly important for the higher brain functions of the honeybee. This review first summarizes how Mblk-1 was identified, and then provides an overview of the studies of Mblk-1 and their homologs. Mblk-1 family proteins are found broadly in animals and are shown to affect transcription activities. Studies have revealed that the mammalian homologs can interact with several cofactors and together regulate transcription. Interestingly, a recent study using the mouse homologs, Mlr1 and Mlr2, showed that one of their cofactor proteins, NOL4, have several splice variants with different effects on the transactivation activities of Mlr proteins. These findings suggest that there is an additional layer of the regulation of Mblk-1 family proteins by cofactor splice variants and provide novel insights into our current understanding of the roles of the conserved transcription factor family.