Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus.

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

The Reprimo Gene Family: A Novel Gene Lineage in Gastric Cancer with Tumor Suppressive Properties.

The reprimo (RPRM) gene family is a group of single exon genes present exclusively within the vertebrate lineage. Two out of three members of this family are present in humans: RPRM and RPRM-Like (RPRML). RPRM induces cell cycle arrest at G2/M in response to p53 expression. Loss-of-expression of RPRM is related to increased cell proliferation and growth in gastric cancer. This evidence suggests that RPRM has tumor suppressive properties. However, the molecular mechanisms and signaling partners by which RPRM exerts its functions remain unknown. Moreover, scarce studies have attempted to characterize RPRML, and its functionality is unclear. Herein, we highlight the role of the RPRM gene family in gastric carcinogenesis, as well as its potential applications in clinical settings. In addition, we summarize the current knowledge on the phylogeny and expression patterns of this family of genes in embryonic zebrafish and adult humans. Strikingly, in both species, RPRM is expressed primarily in the digestive tract, blood vessels and central nervous system, supporting the use of zebrafish for further functional characterization of RPRM. Finally, drawing on embryonic and adult expression patterns, we address the potential relevance of RPRM and RPRML in cancer. Active investigation or analytical research in the coming years should contribute to novel translational applications of this poorly understood gene family as potential biomarkers and development of novel cancer therapies.

Reprimo, a Potential p53-Dependent Tumor Suppressor Gene, Is Frequently Hypermethylated in Estrogen Receptor α-Positive Breast Cancer.

Aberrant DNA methylation is a hallmark of many cancers. Currently, there are four intrinsic molecular subtypes in breast cancer (BC): Luminal A, B, Her2-positive, and triple negative (TNBC). Recently, The Cancer Genome Atlas (TCGA) project has revealed that Luminal subtypes have higher levels of genome-wide methylation that may be a result of Estrogen/Estrogen receptor α (E2/ERα) signaling pathway activation. In this study, we analyze promoter CpG-island (CGIs) of the Reprimo (RPRM) gene in breast cancers (n = 77), cell lines (n = 38), and normal breast tissue (n = 10) using a MBDCap-seq database. Then, a validation cohort (n = 26) was used to confirm the results found in the MBDCap-seq platform. A differential methylation pattern was found between BC and cell lines compared to normal breast tissue. In BC, a higher DNA methylation was observed in tissues that were ERα-positive than in ERα-negative ones; more precisely, subtypes Luminal A compared to TNBC. Also, significant reverse correlation was observed between DNA methylation and RPRM mRNA expression in BC. Our data suggest that ERα expression in BC may affect the DNA methylation of CGIs in the RPRM gene. This approach suggests that DNA methylation status in CGIs of some tumor suppressor genes could be driven by E2 availability, subsequently inducing the activation of the ERα pathway.

Downregulated Reprimo by LINC00467 participates in the growth and metastasis of gastric cancer.

Gastric cancer (GC) as an aggressive malignancy still causes a global health problem. It has been documented that long noncoding RNAs are involved in GC development. Therefore, this research was designed to explore the role of LINC00467 in the growth and metastasis of GC. The expression of LINC00467 and Reprimo in GC tissues and cells was detected. The binding relationship among LINC00467, DNA methyltransferase 1 (DNMT1) and Reprimo was assessed following. Reprimo promoter methylation was detected by methylation sequencing. GC cell lines overexpressing or knock downing LINC00467 were constructed for pinpointing the effect of LINC00467 on cell functions as well as growth and metastasis of GC cells in vivo. LINC00467 was highly expressed, whereas Reprimo was poorly expressed in GC tissues and cells. Mechanically, LINC00467 promoted the methylation and decreased the expression of Reprimo promoter by recruiting DNMT1 in GC cells. Knockdown of LINC00467 diminished the malignant properties of GC cells. Knockdown of LINC00467 reduced tumorigenesis and metastasis of GC cells in vivo. LINC00467 might exert oncogenic effects in GC via Reprimo downregulation by recruiting DNMT1.

Prediction of onset of remnant gastric cancer by promoter DNA methylation of CDO1/HOPX/Reprimo/E-cadherin.

BACKGROUND: Early detection of remnant gastric cancer (RGC) is required to reduce the risk of death, but long-term endoscopic surveillance is difficult after gastrectomy. In this study, data for the methylation status of 4 methylation genes (CDO1, HOPX, Reprimo, and E-cadherin) to predict the onset of RGC are presented. RESULTS: The 4 genes showed hypermethylation in RGC tumors in contrast to the corresponding non-cancerous mucosa tissues. The methylation level in the non-cancerous mucosa tissues of the initial surgery was obviously high in initial malignant disease for CDO1 (P = 0.0001), while in initial benign one for E-cadherin (P = 0.003). Promoter DNA methylation status in the remnant non-cancerous mucosa tissues together with the basic clinical data in turn predicted either initial malignant disease or initial benign disease with a high AUC score of 0.94, suggesting that methylation events are differentially recognized between the initial malignant and benign disease. We then finally confirmed that 4 genes hypermethylation of the non-cancerous tissues by biopsy prior to onset of RGC could predict terms until RGC occurred (P < 0.0001). METHODS: A total of 58 RGC patients were used to establish the model. The 4 genes promoter methylation were analyzed for DNA obtained from the patient's specimens using quantitative methylation specific polymerase chain reaction. CONCLUSIONS: This risk model would help provide guidance for endoscopic surveillance plan of RGC after gastrectomy.

Studying the expression rate and methylation of Reprimo gene in the blood of patients suffering from gastric cancer.

As gastric cancer has no exclusive signals in its initial phases, it is usually diagnosed in advanced phases. Although many researches have been conducted on methylation and diagnosis of cancer's markers, the methylation and expression of Reprimo gene and its correlation with gastric cancer has not been thoroughly studied. Methylation of Reprimo promoter is a repetitive procedure exclusive to cancer which nullifies its expression and performance. The present research seeks to study the expression and methylation of Reprimo among people suffering with gastric cancer so that it may be used as a biomarker for early diagnosis. Fifty blood samples taken from healthy people (normal samples) and 50 blood samples obtained from gastric cancer patients were analyzed using Real-Time PCR. The methylation status of the promoter of Reprimo was studied using Methylation Specific PCR technique in normal samples and in gastric cancer Iranian patients. We observed reduction in expression rate of Reprimo in the blood samples of patients suffering with gastric cancer in comparison to normal blood samples. A significant correlation was also observed between the expression rate of this gene, age and methylation of its promoter among patients suffering with gastric cancer and various analysis points to a correlation between reduced expressions of Reprimo gene in gastric cancer patients. In conclusion, reduced expression of Reprimo gene and greater levels of methylation of its promoter seems to be promising biomarkers for early diagnosis of gastric cancer.

Inverse expression of survivin and reprimo correlates with poor patient prognosis in gastric cancer.

BACKGROUND: The objective of the study was to determine the relationship between Survivin and Reprimo transcript/protein expression levels, and gastric cancer outcome. METHODS: In silico correlations between an agnostic set of twelve p53-dependent apoptosis and cell-cycle genes were explored in the gastric adenocarcinoma TCGA database, using cBioPortal. Findings were validated by regression analysis of RNAseq data. Separate regression analyses were performed to assess the impact of p53 status on Survivin and Reprimo. Quantitative reverse-transcription PCR (RT-qPCR) and immunohistochemistry confirmed in silico findings on fresh-frozen and paraffin-embedded gastric cancer tissues, respectively. Wild-type (AGS, SNU-1) and mutated p53 (NCI-N87) cell lines transfected with pEGFP-Survivin or pCMV6-Reprimo were evaluated by RT-qPCR and Western blotting. Kaplan-Meier method and Long-Rank test were used to assess differences in patient outcome. RESULTS: cBioPortal analysis revealed an inverse correlation between Survivin and Reprimo expression (Pearson's r= -0.3, Spearman's ρ= -0.55). RNAseq analyses confirmed these findings (Spearman's ρ= -0.37, p<4.2e-09) and revealed p53 dependence in linear regression models (p<0.05). mRNA and protein levels validated these observations in clinical samples (p<0.001). In vitro analysis in cell lines demonstrated that increasing Survivin reduced Reprimo, while increasing Reprimo reduced Survivin expression, but only did so in p53 wild-type gastric cells (p<0.05). Survivin-positive but Reprimo-negative patients displayed shorter overall survival rates (p=0.047, Long Rank Test) (HR=0.32; 95%IC: 0.11-0.97; p=0.044). CONCLUSIONS: TCGA RNAseq data analysis, evaluation of clinical samples and studies in cell lines identified an inverse relationship between Survivin and Reprimo. Elevated Survivin and reduced Reprimo protein expression correlated with poor patient prognosis in gastric cancer.

Expression of RPRM/rprm in the Olfactory System of Embryonic Zebrafish (Danio rerio).

The Reprimo (RPRM) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (Danio rerio) embryogenesis, and primarily locates in the nervous system. Its most characterized member, RPRM, which duplicated to give rise rprma and rprmb in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three rprm genes (rprma, rprmb, and rprml) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish, rprma mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE), rprmb is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas rprml is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of RPRM has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of RPRM gene products during zebrafish and mouse development as a first step to approach the physiological role of the RPRM gene family.

The relationship between DNA methylation and Reprimo gene expression in gastric cancer cells.

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.

p53 Configures the G2/M Arrest Response of Nucleostemin-Deficient Cells.

Nucleostemin (NS) protects the genome from replication-induced DNA damage and plays an indispensable role in maintaining the continuous proliferation of both p53-wildtype and mutant cells. Yet, some outcomes of NS-deficient cells appear to be shaped by their p53 status, which stimulates conflicting claims on the role of p53 in executing the NS function. This disparity was conveniently attributed to the usual suspect of cell-type variations. To provide a definitive resolution, we investigated the interplay between NS and p53 in two pairs of isogenic cells, i.e. genetically modified mouse embryonic fibroblast (MEF) cells and HCT116 human colon cancer cells. In MEF cells, p53 deletion further compromises rather than rescues the proliferative potential of NS-depleted cells without changing their G2/M arrest fate before prophase entry. The detrimental effect of p53 loss in NS-depleted MEF cells correlates with a dramatic increase of polyploid giant cells (PGCs) (up to 24%), which indicates aberrant mitosis. To determine how p53 shapes the response of cells to NS depletion at the molecular level, we showed that p53 turns on the expression of reprimo and MDM2 in NS-deficient MEF cells. In the absence of p53, NS-deficient MEF cells exhibit increased levels of phosphorylated cdc2 (Y15) protein and cyclin B1. In cancer (HCT116) cells, NS loss leads to G2/M arrest under both p53wt and p53ko conditions and increases phosphorylated cdc2 more in p53ko than in p53wt cells, as it does in MEF cells. Unlike its effect in MEF cells, NS depletion decreases tumor growth and increases the expression of reprimo and cyclin B1 in a p53-independent manner in HCT116 cells. Our data indicate that the p53 status of NS-deficient cells orchestrates how they respond to G2/M arrest in a normal vs. cancer cell distinct fashion.

tp53-dependent G2 arrest mediator candidate gene, Reprimo, is down-regulated by promoter hypermethylation in pediatric acute myeloid leukemia.

Reprimo (RPRM) is a novel tumor suppressor. However, the expression and molecular function of RPRM in pediatric acute myeloid leukemia (AML) is still unknown. We observed hypermethylation of the RPRM promoter in 8/11 leukemia cell lines and in 44.8% (47/105) of pediatric AML samples compared with 6.7% (2/30) of control samples. Bisulfite genomic sequencing analysis showed that the RPRM promoter was methylated in the majority of AML samples (66.2-83.1%), whereas RPRM was almost unmethylated in normal bone marrow samples (20.0-27.7%). Kaplan-Meier survival analysis revealed poor survival outcomes in samples with RPRM promoter methylation (p < 0.001). Proliferation of AML cells was inhibited in a dose-dependent manner (p < 0.05) after RPRM overexpression with lentivirus transfection. Apoptosis was up-regulated in RPRM-overexpressing AML cells. Real-time polymerase chain reaction array analysis revealed 50 dysregulated genes that might be implicated in apoptosis of RPRM-induced AML cells. RPRM may be a putative tumor suppressor in pediatric AML.

Implication of Reprimo and hMLH1 gene methylation in early diagnosis of gastric carcinoma.

DNA methylation has been recently recognized as a novel tumor marker. This study investigated the methylation status of Reprimo and hMLH1 gene in both plasma and tissue samples from gastric cancer patients, in an attempt to investigate their diagnostic implications in gastric cancer. A total of 180 tissue and plasma samples (including 50 cases of gastric cancer, 50 dysplasia, 50 chronic atrophic gastritis with intestinal metaplasia and 30 normal controls) were collected for detecting DNA methylation status of Reprimo and hMLH1 genes using MSP method. Tissue protein expression levels were further tested by immunohistochemical (IHC) staining. The positive rate of DNA methylation rate was, in ascending sequence, gastritis tissue, dysplasia tissue and gastric carcinoma tissue. All those tissues had significantly elevated DNA methylation level compared to normal group (P < 0.05). Expression level of Reprimo and hMLH1 proteins were, however, decreased in pathological tissues compared to normal ones (P < 0.05). A significantly negative relationship existed between protein level and promoter region methylation level. The DNA methylation occurred in promoter regions of both Reprimo and hMLH1 genes depressed the protein expression, and may participate in the occurrence and progression and gastric cancer. The combined assay of serum Reprimo and hMLH1 DNA methylation levels thus had critical importance in the early diagnosis and gastric cancer.

Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line

BACKGROUND: Reprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. METHODS: The correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5'-Aza-2'-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion. RESULTS: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERa(+) tumors showed higher methylation intensity than ERa(-). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition. CONCLUSION: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.