Glucose-induced RYBP suppresses tumor cell aerobic glycolysis and migration.

RYBP (Ring 1 and YY1 binding protein) has been frequently reported to play an important role during body development, stem cell differentiation, apoptosis and tumorigenesis, but whether RYBP carries out additional functions remains mysterious. Here, we demonstrated that RYBP protein levels elevate with increasing glucose concentration in cell culture medium in human tumorigenic cell lines, but an opposite trend was observed in non-tumorigenic cells. Mechanistic exploration disclosed that glucose inhibits polyubiquitination and proteasomal degradation, leading to RYBP stabilization in tumor cells. Further study showed that RYBP inhibits the glycolysis of tumor cells, as both extracellular acidification rate (ECAR) and lactate production increase when RYBP is knocked down, and decrease when RYBP is over-expressed, and this effect is unrelated to the glucose uptake ability of tumor cells. The functional study showed that RYBP is involved in the regulation of glucose on tumor cell migration. Compared to low glucose culture and their wildtypes, high glucose significantly enhanced tumor cell migration in RYBP knockdown or knockout tumor cells. Taken together, our current study uncovered a previously unknown function of RYBP in tumor metabolism, and this finding will enhance the exploration of the interplay between RYBP and nutrients during tumor cell metabolic reprogramming.

Re-expression of circ_0043610 contributes to trophoblast dysfunction through the miR-558/RYBP pathway in preeclampsia.

An increasing number of data have shown the pathogenesis of preeclampsia (PE) involves circular RNA (circRNA). The study aims to investigate the function and the potential mechanism of circ_0043610 in PE. The study was performed on two human placental trophoblastic cell lines (JEG-3 and HTR-8/SVneo). The expression of circ_0043610, microRNA-558 (miR-558), and RING1 and YY1 binding protein (RYBP) was detected by quantitative real-time polymerase chain reaction. The protein levels of N-cadherin, E-cadherin, and RYBP were assessed by Western blotting. Cell viability, proliferation, apoptosis, invasion, and migration were evaluated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine, flow cytometry analysis, transwell invasion assay, and wound-healing assay, respectively. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay were performed to identify the associations among circ_0043610, miR-558, and RYBP. Compared with normal placental controls, the increased expression of circ_0043610 and RYBP and the decreased miR-558 expression were detected in PE placental tissues. The overexpression of circ_0043610 led to decreased trophoblast cell proliferation, invasion, and migration but increased cell apoptosis. Mechanistically, circ_0043610 acted as a miR-558 sponge, and miR-558 bound to RYBP. Besides, miR-558 introduction remitted circ_0043610-mediated effects in JEG-3 and HTR-8/SVneo cells. Moreover, RYBP participated in the regulation of miR-558 on trophoblast cell behaviors. Further, the ectopic expression of circ_0043610 led to RYBP upregulation through miR-558. Circ_0043610 induced RYBP production to promote trophoblast dysfunction by binding to miR-558 in PE.

RYBP Sensitizes Cancer Cells to PARP Inhibitors by Regulating ATM Activity.

Ring1 and YY1 Binding Protein (RYBP) is a member of the non-canonical polycomb repressive complex 1 (PRC1), and like other PRC1 members, it is best described as a transcriptional regulator. Previously, we showed that RYBP, along with other PRC1 members, is also involved in the DNA damage response. RYBP inhibits recruitment of breast cancer gene 1(BRCA1) complex to DNA damage sites through its binding to K63-linked ubiquitin chains. In addition, ataxia telangiectasia mutated (ATM) kinase serves as an important sensor kinase in early stages of DNA damage response. Here, we report that overexpression of RYBP results in inhibition in both ATM activity and recruitment to DNA damage sites. Cells expressing RYBP show less phosphorylation of the ATM substrate, Chk2, after DNA damage. Due to its ability to inhibit ATM activity, we find that RYBP sensitizes cancer cells to poly-ADP-ribose polymerase (PARP) inhibitors. Although we find a synergistic effect between PARP inhibitor and ATM inhibitor in cancer cells, this synergy is lost in cells expressing RYBP. We also show that overexpression of RYBP hinders cancer cell migration through, at least in part, ATM inhibition. We provide new mechanism(s) by which RYBP expression may sensitize cancer cells to DNA damaging agents and inhibits cancer metastasis.

ETS1 targets RYBP transcription to inhibit tumor cell proliferation.

ETS1 (E26 transformation specific-1) is the founding member of ETS transcriptional factor family. It transcriptionally modulates numerous gene expressions, and is involved in cellular differentiation, tissue remodeling, angiogenesis, drug resistance and tumorigenesis. ETS1 is usually regarded as an oncogene. However, its apoptosis-inducing activity was also frequently reported. Here, we identified RYBP (Ring1 and YY1 binding protein), a critical epigenetic regulator and apoptosis enhancer, as a novel transcriptional target of ETS1. Specifically, we found that overexpression of ETS1 up-regulates the promoter activity of RYBP in HEK293T and tumor cells from different tissue origins, indicating a universal transcriptional regulatory effect. Subsequently, both overexpression and RNA interfering experiments demonstrated that ETS1 positively modulates RYBP expression from both mRNA and protein levels. Bioinformatics analysis combined with site-directed mutagenesis suggested that there probably exist a multiple of ETS1 binding sites in RYBP promoter region, and chromatin immunoprecipitation assay validates the physical association between ETS1 protein and RYBP promoter region. Functional studies showed that ectopic expression of ETS1 significantly suppresses tumor cell proliferation. However, this inhibitory effect was partially compromised when RYBP was concomitantly knocked down by its specific short hairpin RNA. Meanwhile, we provide evidence to demonstrate that cyclin-dependent kinase inhibitor p21 is possibly involved in this regulatory loop. Taken together, our current study identified RYBP as a new transcriptional target which is utilized by ETS1 to carry out its tumor cell growth inhibitory effect.

Overexpression of RYBP inhibits proliferation, invasion, and chemoresistance to cisplatin in anaplastic thyroid cancer cells via the EGFR pathway.

Ring1 and YY1 binding protein (RYBP), a new member of the polycomb group protein family, has been reported to play an important role in various biological processes. Recently, more and more studies have demonstrated an implication of RYBP in cancer development. However, the specific role of RYBP in anaplastic thyroid cancer (ATC) remains unknown. In this study, we investigated for the first time the expression pattern and biological functions of RYBP in ATC. We showed that RYBP was lowly expressed in ATC tissues and cell lines. We also found that overexpression of RYBP inhibited ATC cell proliferation, invasion, and cisplatin resistance. Furthermore, we observed that upregulation of RYBP decreased the phosphorylation of EGFR and ERK1/2 in ATC cells. Taken together, our data indicated that RYBP might be considered as a promising therapeutic target for the treatment of ATC.

Multiple roles of Ring 1 and YY1 binding protein in physiology and disease.

Ring 1 and YY1 binding protein (RYBP) was first identified in 1999, and its structure includes a conserved Npl4 Zinc finger motif at the N-terminus, a central region that is characteristically enriched with arginine and lysine residues and a C-terminal region enriched with serine and threonine amino acids. Over nearly 20 years, multiple studies have found that RYBP functions as an organ developmental adaptor. There is also evidence that RYBP regulates the expression of different genes involved in various aspects of biological processes, via a mechanism that is dependent on interactions with components of PcG complexes and/or through binding to different transcriptional factors. In addition, RYBP interacts directly or indirectly with apoptosis-associated proteins to mediate anti-apoptotic or pro-apoptotic activity in both the cytoplasm and nucleus of various cell types. Furthermore, RYBP has also been shown to act as tumour suppressor gene in different solid tumours, but as an oncogene in lymphoma and melanoma. In this review, we summarize our current understanding of the functions of this multifaceted RYBP in physiological and pathological conditions, including embryonic development, apoptosis and cancer, as well as its role as a component of polycomb repressive complex 1.

RYBP Expression Is Regulated by KLF4 and Sp1 and Is Related to Hepatocellular Carcinoma Prognosis.

The expression of Ring1- and YY1-binding protein (RYBP) is reduced in several human cancers, but the molecular mechanism(s) have remained elusive. In this study, we used human hepatocellular carcinoma (HCC) cell lines and tissue specimens to study the mechanism and herein report several new findings. First, we cloned and characterized the basal promoter region of the human RYBP gene. We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation. We identified two transcription factors, KLF4 and Sp1, which directly bind the promoter region of RYBP to induce and suppress RYBP transcription, respectively. We mapped the binding sites of KLF4 and Sp1 on the RYBP promoter. Studies in vitro showed that KLF4 suppresses whereas Sp1 promotes HCC cell growth through modulating RYBP expression. Deregulated KLF4 and Sp1 contributed to decreased expression of RYBP in HCC tumor tissues. Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor (in association with altered KLF4 and Sp1 expression) was statistically associated with a larger tumor size, poorer differentiation, and an increased susceptibility to distant metastasis. These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4, Sp1, and RYBP may lead to a poorer prognosis. Our findings support the idea that RYBP may represent a target for cancer therapy and suggest that it may be useful as a prognostic biomarker for HCC, either alone or in combination with KLF4 and Sp1.

Overexpression of the Rybp Gene Inhibits Differentiation of Bovine Myoblasts into Myotubes.

RING1 and YY1 binding protein (Rybp) genes inhibit myogenesis in mice, but there are no reports on the effects of these genes in cattle. The aim of this study is to investigate the roles of the Rybp gene on bovine skeletal muscle development and myoblast differentiation. In the present study, the Rybp gene was overexpressed in bovine myoblasts via adenovirus. RNA-seq was performed to screen differentially expressed genes (DEGs). The results showed that overexpressing the Rybp gene inhibits the formation of myotubes. The morphological differences in myoblasts began on the second day and were very significant 6 days after adenovirus induction. A total of 1311 (707 upregulated and 604 downregulated) DEGs were screened using RNA-seq between myoblasts with added negative control adenoviruses (AD-NC) and Rybp adenoviruses (AD-Rybp) after 6 days of induction. Gene ontology (GO) and KEGG analysis revealed that the downregulated DEGs were mainly involved in biological functions related to muscle, and, of the 32 pathways, those associated with muscle development were significantly enriched for the identified DEGs. This study can not only provide a theoretical basis for the regulation of skeletal muscle development in cattle by exploring the roles of the Rybp gene in myoblast differentiation, but it can also lay a theoretical foundation for molecular breeding of beef cattle.

Recurrent deletion of 3p13 targets multiple tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion-positive prostate cancers.

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1-specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG(+) cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG(+) tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG(+) cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over-expression of FOXP1, RYBP and SHQ1 resulted in decreased colony-formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG(+) prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors.

RYBP contributes to improved prognosis in colorectal cancer via regulation of cell cycle, apoptosis and oxaliplatin sensitivity.

Ring1 and YY­1 binding protein (RYBP) is a member of the polycomb repressive complex 1 and serves as a transcriptional suppressor via epigenetic modification. RYBP has a tumour­suppressive role in solid tumours, but its function in colorectal cancer (CRC) remains unknown. The present study evaluated the expression of RYBP using immunohistochemistry in 140 cases of primary CRC and 11 patient­matched cases of liver metastases. Using CRC cell lines with different TP53 gene status such as HCT116 (TP53wt/wt), HCT116 (TP53­/­), SW48 and DLD­1 cells, proliferation, cell cycle progression and apoptosis, as well as the effect of RYBP on oxaliplatin sensitivity, were assessed. Clinical data showed that low RYBP expression was significantly associated with risk of distant metastasis and recurrence, and patients with high RYBP expression demonstrated significantly better cancer­specific and disease­free survival. In vitro experiments revealed that RYBP suppressed cell proliferation by inducing cell cycle arrest and apoptosis in TP53 wild­type cells. In addition, endogenous RYBP overexpression enhanced sensitivity to oxaliplatin. Therefore, RYBP may contribute to improved prognosis in CRC by regulating the cell cycle, apoptosis and oxaliplatin sensitivity via the p53­mediated pathway.

Polycomb protein RYBP activates transcription factor Plagl1 during in vitro cardiac differentiation of mouse embryonic stem cells.

RING1 and YY1 binding protein (RYBP) is primarily known to function as a repressor being a core component of the non-canonical polycomb repressive complexes 1 (ncPRC1s). However, several ncPRC1-independent functions of RYBP have also been described. We previously reported that RYBP is essential for mouse embryonic development and that Rybp null mutant embryonic stem cells cannot form contractile cardiomyocytes (CMCs) in vitro. We also showed that PLAGL1, a cardiac transcription factor, which is often mutated in congenital heart diseases (CHDs), is not expressed in Rybp-null mutant CMCs. However, the underlying mechanism of how RYBP regulates Plagl1 expression was not revealed. Here, we demonstrate that RYBP cooperated with NKX2-5 to transcriptionally activate the P1 and P3 promoters of the Plagl1 gene and that this activation is ncPRC1-independent. We also show that two non-coding RNAs residing in the Plagl1 locus can also regulate the Plagl1 promoters. Finally, PLAGL1 was able to activate Tnnt2, a gene important for contractility of CMCs in transfected HEK293 cells. Our study shows that the activation of Plagl1 by RYBP is important for sarcomere development and contractility, and suggests that RYBP, via its regulatory functions, may contribute to the development of CHDs.

Cataracts in Havanese: genome wide association study reveals two loci associated with posterior polar cataract.

BACKGROUND: Cataract is considered an important health issue in Havanese, and studies indicate a breed predisposition. Possible consequences of cataracts include lens induced uveitis, reduced eyesight, and blindness in severe cases. Reducing the prevalence of cataracts could therefore improve health and welfare significantly. The most frequently diagnosed forms of cataract in Havanese are cortical- and anterior suture line cataract, but cases of posterior polar cataract are also regularly reported. Out of the three, posterior polar- and cortical cataracts are considered the most clinically relevant. RESULTS: We performed a genome wide association study that included 57 controls and 27 + 23 + 7 cases of cortical-, anterior suture line- and posterior polar cataract, respectively. An association analysis using a mixed linear model, revealed two SNPs on CFA20 (BICF2S23632983, p = 7.2e-09) and CFA21 (BICF2G630640490, p = 3.3e-09), that were significantly associated with posterior polar cataract, both of which are linked to relevant candidate genes. The results suggest that the two variants are linked to alleles with large effects on posterior polar cataract formation, possibly in a dominant fashion, and identifies regions that should be subject to further sequencing. Promising regions on CFA4 and CF30 were also identified in the association analysis of cortical cataract. The top SNPs on each chromosome, chr4_12164500 (p = 4.3e-06) and chr30_28836339 (p = 5.6e-06), are located within, or in immediate proximity to, potential cataract candidate genes. The study shows that age at examination is strongly associated with sensitivity of cataract screening. Havanese in Norway are on average 3.4 years old when eye examinations are performed: an age where most dogs that are genetically at risk have not yet developed clinically observable changes. Increasing the average age of breeding animals could increase accuracy of selection, leading to improved health. CONCLUSIONS: The study identified two loci, on CFA20 and CFA21, that were significantly associated with posterior polar cataract in Havanese. SNPs that showed putative association with cortical cataracts, were observed on CFA4 and CFA30. All the top SNPs are located in close proximity to cataract candidate genes. The study also show that sensitivity of cataract screening is highly dependent on age at examination.

Upregulation of circRNA hsa_circ_0008726 in Pre-eclampsia Inhibits Trophoblast Migration, Invasion, and EMT by Regulating miR-345-3p/RYBP Axis.

Accumulating evidence shows that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration contribute to the etiology and pathogenesis of pre-eclampsia (PE). circRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many diseases, including PE. This study aims to investigate the role of circRNA hsa_circ_0008726 in regulating the migration and invasion of extravillous trophoblast cells. RNase R assay was performed to confirm that circ_0008726 was a circular transcript. The expression of circ_0008726, RYBP, and miR-345-3p was examined by qRT-PCR. The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Cell migration and invasion ability was analyzed by Transwell assays. Western blot was used for the quantification of RYBP protein level. Circ_0008726 expression was significantly increased in PE placenta tissues as compared with normal placenta tissues. Circ_0008726 was resistant to RNase R digestion and was predominately located in the cytoplasm of HTR-8/SVneo cells. Silencing circ_0008726 promoted cell migration and EMT (epithelial-mesenchymal transition), while circ_0008726 overexpression suppressed these processes. Mechanistically, circ_0008726 sponged miR-345-3p to negatively regulate its expression, and miR-345-3p negatively modulated the expression of RYBP. In PE samples, the expression level of circ_0008726 was negatively correlated with miR-345-3p level, but was positively correlated with RYBP expression. Transfection of miR-345-3p mimic or RYBP knockdown counteracted the effects of circ_0008726 overexpression on cell migration and EMT. Our data demonstrate the upregulation of circ_0008726 in PE placenta, which inhibits the migration, invasion, and EMT of HTR-8/SVneo cells by targeting miR-345-3p/RYBP axis. These data suggest that circ_0008726 could be a potential biomarker and therapeutic target for PE.

The nuclear localization sequence of the epigenetic factor RYBP binds to human importin α3.

RYBP (Ring1 and YY1 binding protein, UniProt ID: Q8N488) is an epigenetic factor with a key role during embryonic development; it does also show an apoptotic function and an ubiquitin binding activity. RYBP is an intrinsically disordered protein (IDP), with a Zn-finger domain at its N-terminal region, which folds upon binding to DNA. It is predicted that RYBP has a nuclear localization sequence (NLS), comprising residues Asn58 to Lys83, to allow for nuclear translocation. We studied in this work the ability of intact RYBP to bind Impα3 and its truncated species, ΔImpα3, without the importin binding domain (IBB), by using fluorescence and circular dichroism (CD). Furthermore, the binding of the peptide matching the isolated NLS region of RYBP (NLS-RYBP) was also studied using the same methods and isothermal titration calorimetry (ITC), and in silico molecular docking. Moreover, we carried out experiments with NLS-RYBP in the absence or in the presence of NaCl (140 mM). Our results show that RYBP interacted with Impα3 and ΔImpα3, causing protein precipitation. The NLS-RYBP also interacted with both importin species (dissociation constant in the low micromolar range), at low or high ionic strength, as shown by intrinsic fluorescence and ITC. These findings indicate that the NLS region, which was mainly unfolded in isolation in solution, was essentially responsible for the binding of RYBP to each of the importin species. Furthermore, the molecular simulations predict that the anchoring of NLS-RYBP takes place in the major binding site for the NLS of cargo proteins bound to Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in RYBP and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

Knockdown of miR-125b-5p inhibits the proliferation and invasion of gastric carcinoma cells by targeting RYBP.

Gastric carcinoma, one of the most aggressive and lethal human malignancies, is associated with poor prognosis despite progress in therapeutic strategies. This study examined the potential function and mechanism of action of microRNA-125b-5p (miR-125b-5p) in the pathogenesis of gastric carcinoma. We recognized that miR-125b-5p was elevated in gastric carcinoma, and its decreased expression was associated with a better prognosis. Loss-of-function assays showed that miR-125b-5p suppression inhibited the proliferative and invasive abilities of gastric cancer cells. Furthermore, RING1 and YY1-binding protein (RYBP) was found to be target gene for miR-125b-5p action; miR-125b-5p negatively regulates RYBP expression. According to the results of rescue experiments, RYBP downregulation partially counteracted the miR-125b-5p silence-mediated inhibitory function in gastric cancer progression. Collectively, these data elucidated the molecular mechanisms of the miR-125b-5p/RYBP axis in gastric cancer invasion and growth.

RYBP modulates embryonic neurogenesis involving the Notch signaling pathway in a PRC1-independent pattern.

RYBP (Ring1 and YY1 binding protein), an essential component of the Polycomb repressive complex 1 (PRC1), plays pivotal roles in development and diseases. However, the roles of Rybp in neuronal development remains completely unknown. In the present study, we have shown that the depletion of Rybp inhibits proliferation and promotes neuronal differentiation of embryonic neural progenitor cells (eNPCs). In addition, Rybp deficiency impairs the morphological development of neurons. Mechanistically, Rybp deficiency does not affect the global level of ubiquitination of H2A, but it inhibits Notch signaling pathway in eNPCs. The direct interaction between RYBP and CIR1 facilitates the binding of RBPJ to Notch intracellular domain (NICD) and consequently activated Notch signaling. Rybp loss promotes CIR1 competing with RBPJ to bind with NICD, and inhibits Notch signaling. Furthermore, ectopic Hes5, Notch signaling downstream target, rescues Rybp-deficiency-induced deficits. Collectively, our findings show that RYBP regulates embryonic neurogenesis and neuronal development through modulating Notch signaling in a PRC1-independent manner.

Rybp orchestrates spermatogenesis via regulating meiosis and sperm motility in mice.

Ring1 and Yin Yang 1-Binding Protein (RYBP) is a member of non-canonical polycomb repressive complex 1 to mediate monoubiquitination of histone H2A at lysine 119. It plays an important role in development, but its role in reproduction remains illusive. In this study, we used Rybp conditional knockout mouse model to genetically ablate Rybp in male germ cells. We found that Rybp deficiency during spermatogenesis led to smaller testes, loss of germline cells, disturbed meiosis, increased apoptosis of spermatocytes, decreased sperm motility, and reduced global H3K9me3, without impacting retrotransposon expression. Meanwhile, we depleted Rybp during oogenesis, but oocyte maturation and preimplantation development were normal. Our findings demonstrate that RYBP plays important roles in spermatogenesis through regulating meiosis and sperm motility.

Ring 1 and YY1 Binding Protein Expressed in Murine Spermatocytes but Dispensable for Spermatogenesis.

Spermatogenesis is a complex cellular-differentiation process that relies on the precise regulation of gene expression in spermatogonia, meiotic, and postmeiotic germ cells. The Ring 1 and YY1 binding protein (Rybp) is a member of the mammalian polycomb-group (PcG) protein family that plays multifunctional roles in development. Previous findings indicate that Rybp may function as an important regulator of meiosis. However, its expression in the testes and function in spermatogenesis have not been examined. In this study, we investigated Rybp expression in postnatal mouse testes using qRT-PCR and immunohistochemistry. We also examined the function of Rybp in spermatogenesis by using a conditional-knockout approach. Results showed that the relative expression of Rybp mRNA was significantly upregulated in the testes of postnatal day (PD) 6 mice. Immunofluorescent staining revealed that Rybp was enriched in the spermatocytes. Surprisingly, a conditional deletion of Rybp in fetal germ cells did not affect the fertility or normal development of spermatogenic cells. Further analysis revealed that Rybp deletion resulted in a decreased expression of meiosis-related genes, but that meiosis progression was normal. Together, these findings suggest that Rybp expression was enriched in spermatocytes, but that it was not required for spermatogenesis.

RYBP inhibits esophageal squamous cell carcinoma proliferation through downregulating CDC6 and CDC45 in G1-S phase transition process.

AIMS: RING1 and YY1-binding protein (RYBP) is an epigenetic regulator and plays crucial roles in embryonic development. The anti-tumor effect of RYBP has been reported in several cancers recently, but the role of RYBP in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. The present study aimed to investigate the biological function and the underlying molecular mechanisms of RYBP in ESCC. MATERIALS AND METHODS: We detected the expression of RYBP in ESCC tissue microarrays (TMA) by immunohistochemistry. Cell proliferation was assessed by CCK8 and colony formation assays. Cell cycle was analyzed by flow cytometry. Gene expression was determined by transcriptome arrays, quantitative real-time PCR (qRT-PCR) and Western blot. Four-week-old male nude mice were used to evaluate the effect of RYBP in ESCC growth. KEY FINDINGS: We found that RYBP was downregulated in ESCC compared with adjacent normal tissues. A high level of RYBP expression predicted a better outcome of ESCC patients. Furthermore, overexpression of RYBP inhibited ESCC growth both in vitro and in vivo. Transcriptome arrays and functional studies showed that RYBP decreased the expression of genes related to cell cycles, especially CDC6 and CDC45, which were essential to initiate the DNA replication and G1-S transition. SIGNIFICANCE: Taken together, our study suggests that RYBP suppresses ESCC proliferation by downregulating CDC6 and CDC45, thus inhibiting the G1-S transition.

MicroRNA-769-5p contributes to the proliferation, migration and invasion of hepatocellular carcinoma cells by attenuating RYBP.

Hepatocellular carcinoma (HCC) is the commonest primary liver cancer with highly aggressive features. MicroRNAs (miRNAs) are demonstrated to play important roles in the tumorigenesis and progression of HCC. miR-769-5p is a recently identified cancer-associated miRNA. But, the expression level of miR-769-5p and its function in HCC are unexplored. In this study, we found that miR-769-5p expression was obviously increased in HCC samples compared to adjacent noncancerous liver tissues. Additionally, we revealed that miR-769-5p was over-expressed in HCC cells as compared with LO2 cells. Notably, HCC tissues from patients with tumor size ≥5 cm, venous infiltration and advanced tumor stages showed higher levels of miR-769-5p compared to those from corresponding controls. Interestingly, our data indicated that HCC patients highly expressing miR-769-5p had significant shorter survivals. Next, functional experiments verified that miR-769-5p knockdown markedly suppressed HCC cell proliferation, migration and invasion. Conversely, ectopic expression of miR-769-5p promoted these biological behaviors of Hep3B cells. Furthermore, depletion of miR-769-5p repressed the growth and metastasis of HCCLM3 cells in vivo. Importantly, miR-769-5p inversely modulated RING1 and YY1 binding protein (RYBP) by directly binding to 3' untranslated region (UTR) in HCC cells. The expression of RYBP mRNA was down-regulated in HCC tissues and negatively correlated with miR-769-5p level. RYBP overexpression remarkably inhibited the proliferation, migration and invasion of HCCLM3 cells. Accordingly, knockdown of RYBP partially abolished miR-769-5p silencing-induced tumor suppressive effects on HCCLM3 cells. In summary, our study revealed the up-regulated expression of miR-769-5p, which contributed to HCC progression possibly by targeting RYBP.