Selection and Characterization of a Novel DNA Aptamer, Apt-07S Specific to Hepatocellular Carcinoma Cells.

BACKGROUND: The efficacy of traditional therapeutic methods for liver cancer is unsatisfying because of the poor targeting, and inefficient drug delivery system. A recent study has proven that aptamers, developed through cell-SELEX, could specifically recognize cancer cells and show great potential in the development of a delivery system for anticancer drugs. PURPOSE: To develop a hepatocellular carcinoma specific aptamer using two kinds of hepatocellular carcinoma cell lines, HepG2 and SMMC-7721, as double targets and a normal hepatocyte, L02, as a negative control cell. METHODS: Hepatocellular carcinoma specific aptamer was developed via cell-SELEX. The enrichment of the library was monitored by flow cytometric analysis. The specificity, affinity, and distribution of the candidate aptamer were explored. Further study was carried to assess its potential in drug delivery. RESULTS: The library was enriched after 14 rounds of screening. Candidate aptamer Apt-07S can recognize four kinds of hepatocellular carcinoma cells and show little cell-binding ability to normal cells and four cell lines of different cancer types, revealing a high specificity of Apt-07S. Confocal imaging showed that Apt-07S distributed both on the surface and in the cytoplasm of the two target cells. Moreover, an anti-sense nucleotide to gene Plk1 (ASO-Plk1) was connected at the 3' end of Apt-07S to form an integrated molecule (Apt-07S-ASO-Plk1); the functional analysis indicated that the structure of Apt-07S may help ASO-Plk1 enter the cancer cells. CONCLUSION: The study indicates that Apt-07S can specifically target HCC and may have potential in the delivery of anticancer drugs.

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo.

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF-C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF-C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF-C expression, cleaved caspase-3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme-linked immunosorbent assay were used to measure the expression of VEGF-C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF-C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF-C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase-3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF-C expression and increased induction of apoptosis. The present study therefore indicated that VEGF-C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

Downregulation of survivin expression exerts antitumoral effects on mouse breast cancer cells in vitro and in vivo.

Metastasis constantly occurs in the majority of cases of primary breast cancer at late stage or following surgical treatment. Survivin, a member of the inhibitor of apoptosis protein family, has long been recognized as a promising anticancer target, but its antitumor effects remain largely unexplored. In order to elucidate the role of survivin in breast cancer metastasis, short interfering RNA (siRNA) was used in the present study to specifically downregulate survivin expression in the murine breast cancer cell line 4T1. The results demonstrated that blocking the expression of survivin by siRNA inhibited the proliferation, migration and invasion abilities of murine breast cancer cells in vitro. Vascular endothelial growth factor (VEGF)-C is a lymphatic endothelial cell-stimulating factor that may lead to the formation of lymphatic vessels in lymph nodes. In the present study, the inhibition of survivin by siRNA was able to reduce the overexpression of VEGF-C in 4T1 cells. Furthermore, intratumoral injections of the survivin-siRNA significantly inhibited the growth of orthotopically transplanted 4T1 tumors in vivo. In addition, the number of pulmonary metastases and the microlymphatic vessel density were significantly reduced in vivo, following transfection with survivin-siRNA. The results of the present study suggested that the Akt/hypoxia-inducible factor-1α signaling pathway participates in the survivin-mediated downregulation of VEGF-C expression observed in breast cancer cells treated with survivin-siRNA. Therefore, the use of siRNA specifically targeting survivin may be a potential anticancer method in the future.

[DUOX2 mutations in children with congenital hypothyroidism].

OBJECTIVE: To study the features of DUOX2 mutations and genotype-phenotype relationship in children with congenital hypothyroidism (CH), in order to provide evidence for gene diagnosis and gene treatment of CH. METHODS: Blood samples were collected from 10 CH children with thyromegaly. Genomic DNA was extracted from peripheral blood leukocytes. All exons of DUOX2 gene were analyzed using PCR and direct sequencing. RESULTS: G3632A mutation in the exon 28 of DUOX2 that may result in arginine to histidine substitution at codon 1211 was found in one patient. T2033C mutation in the exon 17 of DUOX2 that may result in histidine to arginine substitution at codon 678 was found in three patients. They were all heterozygous mutations. CONCLUSIONS: Heterozygous mutations in DUOX2 may affect protein function and cause CH. The relationship between DUOX2 genotypes and clinical phenotypes is unclear and needs further studies.

Downregulation of KDR expression induces apoptosis in breast cancer cells.

Angiogenesis plays a crucial role in the growth, invasion and metastasis of breast cancer. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the key regulators of tumor angiogenesis. VEGFR-2, known as the kinase insert domain receptor (KDR), is a key receptor involved in malignant angiogenesis. We previously showed that knocking down KDR with short interference RNA (KDR-siRNA) markedly decreased KDR expression and suppressed tumor growth in a xenograft model. However, the mechanisms underlying the anti-cancer effects of KDR-siRNA are not clearly understood. This study aimed to elucidate the molecular mechanisms that induce apoptosis in human breast cancer MCF-7 cells after transfection with KDR-siRNA. We studied the effects of KDR-siRNA on proliferation, apoptosis, antiapoptotic and pro-apoptotic proteins, mitochondrial membrane permeability, cytochrome c release and caspase-3 activity. The results indicated that KDR-siRNA treatment significantly inhibited the proliferation and induced the apoptosis of MCF-7 cells, reduced the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, and increased the level of the pro-apoptotic protein Bax, resulting in a decreased Bcl-2/Bax ratio. KDR-siRNA also enhanced the mitochondrial membrane permeability, induced cytochrome c release from the mitochondria, upregulated apoptotic protease-activating factor-1 (Apaf-1), cleaved caspase-3, and increased caspase-3 activity in MCF-7 cells. Furthermore, KDR-siRNA-induced apoptosis in MCF-7 cells was blocked by the caspase inhibitor Z-VAD-FMK, suggesting a role of caspase activation in the induction of apoptosis. These results indicate that the Bcl-2 family proteins and caspase-related mitochondrial pathways are primarily involved in KDR-siRNAinduced apoptosis in MCF-7 cells and that KDR might be a potential therapeutic target for human breast cancer treatments.

Expression of gastrointestinal nesfatin-1 and gastric emptying in ventromedial hypothalamic nucleus- and ventrolateral hypothalamic nucleus-lesioned rats.

AIM: To determine the expression levels of gastrointestinal nesfatin-1 in ventromedial hypothalamic nucleus (VMH)-lesioned (obese) and ventrolateral hypothalamic nucleus (VLH)-lesioned (lean) rats that exhibit an imbalance in their energy metabolism and gastric mobility. METHODS: Male Wistar rats were randomly divided into a VMH-lesioned group, a VLH-lesioned group, and their respective sham-operated groups. The animals had free access to food and water, and their diets and weights were monitored after surgery. Reverse transcription-polymerase chain reaction and immunostaining were used to analyse the levels of NUCB2 mRNA and nesfatin-1 immunoreactive (IR) cells in the stomach, duodenum, small intestine, and colon, respectively. Gastric emptying was also assessed using a modified phenol red-methylcellulose recovery method. RESULTS: The VMH-lesioned rats fed normal chow exhibited markedly greater food intake and body weight gain, whereas the VLH-lesioned rats exhibited markedly lower food intake and body weight gain. NUCB2/nesfatin-1 IR cells were localised in the lower third and middle portion of the gastric mucosal gland and in the submucous layer of the enteric tract. Compared with their respective controls, gastric emptying was enhanced in the VMH-lesioned rats (85.94% ± 2.27%), whereas the VLH lesions exhibited inhibitory effects on gastric emptying (29.12% ± 1.62%). In the VMH-lesioned rats, the levels of NUCB2 mRNA and nesfatin-1 protein were significantly increased in the stomach and duodenum and reduced in the small intestine. In addition, the levels of NUCB2 mRNA and nesfatin-1 protein in the VLH-lesioned rats were decreased in the stomach, duodenum, and small intestine. CONCLUSION: Our study demonstrated that nesfatin-1 level in the stomach and duodenum is positively correlated with body mass. Additionally, there is a positive relationship between gastric emptying and body mass. The results of this study indicate that gastrointestinal nesfatin-1 may play a significant role in gastric mobility and energy homeostasis.

Aberrant NDRG1 methylation associated with its decreased expression and clinicopathological significance in breast cancer.

BACKGROUND: Cancer cell differentiation is an important characteristic of malignant tumor and has a great impact on prognosis and therapeutic decision for patients. The N-myc downstream regulated gene 1 (NDRG1), a putative tumor suppression gene, is involved in the regulation of human cell differentiation and metastasis in various cancers. Changes in the status of methylation of the NDRG1 gene have not been studied in detail in human breast cancer. RESULTS: The MDA-MB-231 breast tumor cell line could express NDRG1. However, it was only expressed after treatment with 5-Aza-2'-deoxycytidine (AZA) in T47D cell line, which revealed that NDRG1 expression could modulated by DNA methylation. Therefore, the fragment surrounding the transcript start site of NDRG1 gene promoter was cloned after sodium bisulfite DNA treatment. A high density (66%) of methylation for human NDRG1 gene promoter was detected in T47D; however, there was only 16% of methylated CpG dinucleotides in the NDRG1 gene promoter in MDA-MB-231. DNA methylation in the NDRG1 promoter was detected in 31.1% of primary breast cancer samples. Furthermore, the NDRG1 promoter methylation correlated with the Tumor Node Metastasis (TNM) at stage III/IV, metastasis, lymph invasion, moderate and poor histological grade in the breast cancer patients. CONCLUSION: These findings suggest that the DNA methylation status of NDRG1 gene may play an important role in the pathogenesis and/or development of breast cancer, and the expression could be regulated by aberrant DNA methylation.

The knockdown of c-myc expression by RNAi inhibits cell proliferation in human colon cancer HT-29 cells in vitro and in vivo.

We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.

The mechanisms on apoptosis by inhibiting VEGF expression in human breast cancer cells.

This study investigated apoptotic mechanisms of down-expression vascular endothelial growth factor (VEGF) by short interfering RNA (siRNA) in human breast cancer MCF-7 cells. Human breast cancer cells were evaluated for the expression of VEGF and VEGF receptor 2 (VEGFR-2). siRNA targeting VEGF mRNA were chemically synthesized and transfected into cells with Lipofectamine2000. In vitro assessments were then made of the ability of anti-VEGF siRNA to knock down expression of VEGF and the subsequent effect this decreased expression had on breast cancer cell apoptosis. Growth curve construction and nude mice experimentation in vivo were performed to assess the effects of VEGF silencing on tumor growth. Those cells transfected with siRNA targeting VEGF showed a 65% knockdown in VEGF expression and a marked increase in cell apoptosis. The expression of Bcl-2 protein in MCF-7 cells was decreased, the level of Bax protein was kept the same, cytochrome c was released from mitochondria into cytosol, and the cleaved Caspase-3 protein rose after siRNA transfection. The siRNA targeting human VEGF could induce apoptosis in MCF-7 cells and the mechanism of apoptosis is possibly related with changing Bcl-2/Bax expression ratio, releasing cytochrome c from mitochondria into cytosol, and up-regulation of Caspase-3 protein, but also could suppress the growth of breast cancer cells in vivo. VEGF might be a potential therapeutic target for human breast cancer.

Effects of neuregulin on expression of MMP-9 and NSE in brain of ischemia/reperfusion rat.

The aim is to investigate the effects of neuregulin-1beta (NRG-1beta) on expression of matrix metalloproteinase-9 (MMP-9) and neuron-specific enolase (NSE) in brain tissue in rats following cerebral ischemia/reperfusion. One hundred and fifty adult healthy male Wistar rats were used in the present study. Ten of them were randomized into a sham-operation group (n = 10) and the rest suffered surgery operation of middle cerebral artery occlusion/reperfusion with intraluminal monofilament suture from the left external-internal carotid artery. As a result, 100 rats of successful models were randomly divided into a control group (n = 50) and a treatment group (n = 50). Rats in the treatment group were injected 1.5% NRG-1beta at a dosage of 0.3 microg/kg from the stump of the left external carotid artery into the internal carotid artery. The expressions of MMP-9 and NSE proteins were determined by immunohistochemical, immunofluorescent double labeling, and Western blot assay. Ischemia/reperfusion induced morphological changes of brain tissue, including neurocyte shrinkage, chromatin condensation, nuclei fragment, and gliacyte and endothelial cell swelling. NRG-1beta obviously reduced and delayed the cerebral damage. With the duration of ischemia, the expression of MMP-9 gradually increased in the control group. NRG-1beta decreased the level of MMP-9 compared with that in the control group (P < 0.01). NSE immunoreaction transiently elevated at the early stage of cerebral ischemia insult, and then gradually decreased in the control group. The administration of NRG-1beta significantly increased the level of NSE, and thus delayed the time and the degree of neuron damage. There were statistical differences in contrast to the control group (P < 0.01). There was no relationship between the expressions of the two proteins. MMP-9 might aim at various target cells at different stages and contribute to the inflammatory reaction after cerebral ischemia-reperfusion insult. NRG-1beta inhibits the activation of MMP-9 and development of inflammation, enhances the activity of NSE, improves the microenvironment of neuron survivals, and delays the phase of irreversible neuron necrosis. Therefore, NRG-1beta may play a neuroprotective role in cerebral ischemia/reperfusion.

Chemically modified siRNA directed against the KDR gene inhibits the proliferation of breast cancer cells.

Vascular endothelial growth factor receptor-2 or kinase insert domain-containing receptor (VEGFR2/KDR) is secreted by most solid tumors, including breast cancer, and is an important mediator of angiogenesis. To observe the effects of KDR gene expression on cell proliferation and the cell cycle in MCF-7 cells in vitro and in vivo, we used chemically modified siRNA directed against KDR. The results revealed that chemically modified siRNA transfection of the KDR gene effectively inhibited the proliferation of MCF-7 cells, arrested cells in the G1 phase and down-regulated the expression of KDR. In addition, in the progression of cell cycle arrest induced by siRNA, phosphorylated ERK and CDK1 expression was down-regulated (P<0.01). In vivo, the growth of tumors was visibly suppressed. RT-PCR and the results of immunohistochemistry indicated that KDR mRNA and protein expression was reduced in the excised tumors. In contrast, there were no obvious changes in the control groups. This implies that chemically modified KDR siRNA markedly decreases KDR gene expression and inhibits cellular proliferation in vitro, as well as suppressing tumor growth in a xenograft model. KDR may be a new target for breast cancer treatment.

[Effects of isatin on cell cycle arrest and apoptosis of neuroblastoma cell line SH-SY5Y].

BACKGROUND & OBJECTIVE: Isatin (ISA) is a natural material that exists in mammalian body fluids and tissues. ISA could inhibit the growth of tumors, but the mechanism remains unclear. This study aimed to explore the effects of ISA on the cell cycle and apoptosis of neuroblastoma cell line SH-SY5Y. METHODS: Fluorescent staining, flow cytometry, and Western blot were performed to analyze the cell cycle arrest and apoptosis of SH-SY5Y cells after treatment of ISA at different concentrations (0, 100, 200, 400 micromol/L). RESULTS: When treated with 400 micromol/L ISA for 48 h, SH-SY5Y cells showed typical apoptotic morphologic changes including chromatin condensation and DNA fragment. Along with the increase of ISA concentration, Bcl-2 expression was decreased, the ratio of Bcl-2 to Bax was significantly decreased (P<0.05). When treated with ISA (100, 200, 400 micromol/L) for 48 h, the positive rates of activated Caspase-3 in SH-SY5Y cells were significantly higher than that in control SH-SY5Y cells (19.28%, 25.88%, and 33.43% vs. 10.58%, P<0.05). Moreover, inhibitor of caspase-activated DNase (ICAD), the substrate of Caspase-3, was degraded. In addition, the proportion of SH-SY5Y cells at G1 phase was significantly increased with an apparent G1 phase arrest when treated with ISA (100, 200, 400 micromol/L) for 48 h. In the progress of cell cycle arrest induced by ISA, phosphorylated ERK and CDK1 expression were down-regulated (P<0.05). CONCLUSION: ISA can induce apoptosis and G1 phase arrest in SH-SY5Y cells, possibly by decreasing Bcl-2/Bax, activating Caspase-3 and down-regulating the expression of phosphorylated ERK and CDK1.

[Small interference RNAs directed against KDR gene inhibit the proliferation of breast cancer cells in vitro and in vivo].

AIM: To inhibit kinase insert domain-containing receptor (KDR) expression chemically modified siRNA in vitro and in vivo and to investigate the feasibility and specificity of gene therapy for breast cancer. METHODS: In vitro, siRNA was transfected into MCF-7 cells to induce RNAi by using cationic liposome Lipofectamine2000(TM). The changes of KDR mRNA and protein expression in both siRNA treatment group and control group were measured by MTT assay and RT-PCR, In vivo, the siRNA was transfected into transplanted tumor in nude mice by using cationic polymer nanoparticle In vivo jetPEI(TM). Tumor growth was observed. The mRNA and protein expression of KDR was measured by RT-PCR and immunohistochemical staining. RESULTS: Experiments in vitro showed that siRNA directed against KDR effectively inhibited the proliferation of MCF-7 cells and downregulated KDR mRNA expression. In vivo, the growth of tumor was visibly suppressed. Furthermore, RT-PCR and immunohistochemical results indicated that KDR mRNA and protein expression was reduced in excised tumors. CONCLUSION: RNAi mediated by chemically modified siRNA markedly decreased KDR gene expression and inhibited cellular proliferation. It may have the potential as a therapeutic method to treat human cancer.

Inhibitory effect of vascular endothelial growth factors-targeted small interfering RNA on proliferation of gastric cancer cells.

AIM: To examine the effects of vascular endothelial growth factor (VEGF)-targeted small interfering RNA (siRNA) on proliferation of gastric cancer cells in vitro. METHODS: Several siRNAs were transfected into human gastric cancer cell line SGC-7901 with Lipofectamine 2000. Cells not transfected with Lipofectamine 2000 or scrambled (SCR) siRNA served as controls. The inhibitory effect of siRNA on the expression of VEGF mRNA and protein was detected by RT-PCR and ELISA. MTT assay was used to examine the inhibition rate of cell growth. The change in cell cycling of siRNA-treated cells was detected by flow cytometry. RESULTS: siRNA targeting human VEGF effectively inhibited the proliferation of gastric cancer cell line SGC-7901 and the distribution of cell cycle. The percentage of G(0)/G(1) phase was significantly higher in siRNA(1)- and siRNA(2)-transfected cells than in control cells. The expression of VEGF mRNA was significantly inhibited in siRNA(1)- and siRNA(2)-transfected cells compared with that in control cells. VEGF protein notably decreased in siRNA-transfected cells, but had no effect on SCR siRNA. CONCLUSION: VEGF siRNA inhibits proliferation of gastric cancer cells in vitro.

[Effect of siRNA transfection targeting VEGF gene on proliferation and apoptosis of human breast cancer cells].

AIM: To explore the effect of RNA interference-mediated vascular endothelial growth factor (VEGF) gene silencing on proliferation and apoptosis of human breast cancer MCF-7 cells. METHODS: Small interfering RNA (siRNA) targeting VEGF gene was designed and DNA template was synthesized, then siRNA was obtained by in vitro transcription. After ds-siRNA was transfected into MCF-7 cells with Lipofectamine, the proliferation of MCF-7 cells was detected by MTT colorimetry, and the apoptosis was measured by Hoechst33258 staining. The change of cell cycle was analyzed by flow cytometry (FCM). The expression of VEGF on mRNA and protein level was analyzed by RT-PCR and immunohistochemical technology respectively. RESULTS: The siRNA targeting human VEGF gene effectively inhibited the proliferation of MCF-7 cells, induced cell apoptosis, arrested the cell cycle in G(0)/G(1) phase, and decreased the expression of VEGF on both mRNA and protein level. But these effects did not appear in scrambled siRNA (siRNA(SCR)) control group. CONCLUSION: The siRNA targeting human VEGF gene could reduce VEGF expression, inhibit cellular proliferation and induce apoptosis in MCF-7 cells in vitro.

Effects of ghrelin on hypothalamic glucose responding neurons in rats.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) with potent stimulatory effects on food intake. The aim of the present study was to investigate the effects of ghrelin on neuronal activity of hypothalamic glucose responding neurons. Single unit discharges in the lateral hypothalamic area (LHA), the ventromedial hypothalamic nucleus (VMH), and the parvocellular part of the paraventricular nucleus(pPVN) were recorded extracellularly by means of four-barrel glass micropipettes in anesthetized rats. The activity of glucose-sensitive neurons (GSNs) in the LHA, pPVN, and of glucoreceptor neurons (GRNs) in the VMH modulated by administration of ghrelin was analyzed. In the LHA, the majority of GSNs (17/25) increased in frequency due to ghrelin. Whereas the majority of VMH-GRNs (27/33) and pPVN-GSNs (9/13) was inhibited. The responses to ghrelin were abolished by pretreatment of [D-Lys-3]-GHRP-6, ghrelin receptor antagonist. These data indicate that the glucose responding neurons in the LHA, VMH, and pPVN are also involved in the orexigenic actions of ghrelin in the hypothalamic circuits, although AgRP/NPY neurons in the arcuate nucleus (ARC) are the primary targets of ghrelin.

Anorexigenic melanocortin signaling in the hypothalamus is augmented in association with failure-to-thrive in a transgenic mouse model for Prader-Willi syndrome.

As in Prader-Willi syndrome (PWS) infants, mouse models of PWS display failure-to-thrive during the neonatal period. In rodents, the hypothalamic neuropeptide, Neuropeptide Y (NPY) and Agouti-related peptide (AgrP) stimulate while alpha-melanocyte stimulating hormone (alpha-MSH) inhibits appetite. We hypothesized that altered expression of these neuropeptides in the hypothalamus may underlie the failure-to-thrive in PWS neonatal mice. To test this hypothesis we evaluated mRNA expression of Npy, Agrp, and Pomc by in situ hybridization in the hypothalamic arcuate nucleus (ARC) of 3-day-old female and male PWS neonates. The results showed that Agrp mRNA expression was decreased relative to wild-type (WT) controls in neonates of both sexes, while mRNA expression of Pomc was upregulated in PWS neonates. Since AgrP and the Pomc-derived peptide, alpha-MSH, are functional antagonists at melanocortin 4 receptors in the hypothalamic regulation of appetitive behavior, these results show that robust anorexigenic melanocortin signaling, may contribute to the failure-to-thrive in PWS neonatal mice.