MicroRNA-143 Targets Syndecan-1 to Repress Cell Growth in Melanoma

Melanoma is the most aggressive type of skin cancer with a rapid progression and a limited efficiency of therapeutics. Recently, studies have identified some microRNAs playing important roles in the development of melanoma. Syndecan-1 (Syn-1), dysregulated in many cancers, plays important roles in tumor progression by controlling cell proliferation. In this study, we investigated whether microRNA-143 (miR-143) is involved in the regulation of Syn-1 and thus plays a functional role in melanoma. We found that miR-143 expression was significantly lower in melanoma tissues than in normal tissues and its low expression was closely related to clinical stages of melanoma. Further experiments showed that consistent with the inhibitory effects induced by knockdown of Syn-1, overexpression of miR-143 suppressed cell proliferation, promoted G1 phase arrest and induced apoptosis in melanoma. Downregulation of miR-143 apparently produced opposite effects. Combined treatment of miR-143 overexpression and Syn-1 knockdown induced remarkable synergistic effects, while reconstitution of miR-143-resistant Syn-1 reversed the inhibitory activity of miR-143. Moreover, miR-143 level was inversely correlated with Syn-1 expression in melanoma cells. miR-143 directly targeted the 3′-untranslated regions of Syn-1 mRNA and they were in the same Argonaute2 complex. Taken together, this study revealed a link between miRNA-143 and Syn-1 in the pathogenesis of melanoma. MiR-143 plays an important role in the regulation of cell growth in melanoma. Restoration of miR-143 expression may represent a promising and efficient therapeutic approach for targeting malignant melanoma.     

Myocilin Regulates Cell Proliferation and Survival

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.     

MiR-371-5p Facilitates Pancreatic Cancer Cell Proliferation and Decreases Patient Survival

Background

microRNAs (miRNAs) play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.

Methods

The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC) and their adjacent normal pancreatic tissues (ANPT) or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). Protein expression was analyzed by Western blot.

Results

The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1) downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.

Conclusions

our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.     

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis

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MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA

Background

Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis.

Methodology/Principal Findings

The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA).

Conclusions

Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.     

OCT4 Positively Regulates Survivin Expression to Promote Cancer Cell Proliferation and Leads to Poor Prognosis in Esophageal Squamous Cell Carcinoma

Background

OCT4 and Survivin are important factors for cancer cell proliferation, renewal and dedifferentiation, and correlate with resistance to radiotherapy and chemotherapy in most human cancers, but their regulatory mechanisms are not well known.

Methodology/Principal Findings

In this study, 50 patients with esophageal squamous cell carcinoma (ESCC) were retrospectively analyzed. OCT4 was expressed in 13 cases (26%), and survivin was positively expressed in 31 cases (62%), examined by immunochemistry. OCT4 was found to be an independent predictive factor for median survival time, and the patients from the subgroup with both high expression of OCT4 and Survivin had the worst prognosis investigated by log-rank test. To further explore the molecular regulatory mechanism between OCT4 and Survivin, we constructed the specific small hairpin RNA (shRNA)-expressing vectors targeting OCT4 or/and Survivin and manipulated the expression of OCT4 and Survivin. By Western blotting and RT-PCR, we found that OCT4 could up-regulate Survivin expression in the esophageal cancer cell lines Eca109 and TE1. Simultaneously knockdown of OCT4 and Survivin expression induced cell apoptosis and G2-phase decrease of cell cycle by flow cytometry, and finally exerted an enhanced anti-proliferation potency in Eca109 and TE1 cell lines by MTT assay.

Conclusions

This study shows that OCT4 and Survivin expression were correlated with poor survival in patients with ESCC. OCT4 and Survivin may be regarded as targets in ESCC biotherapy.     

Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5′ untranslated region (5′UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5′UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.Targeting the translation initiation pathway is emerging as a potential therapy for inhibiting cancer cell growth (35, 38). Ribosome recruitment to the 5′ ends of eukaryotic mRNAs proceeds via translation initiation mechanisms that are dependent either on the 5′ cap structure (m⁷GpppN, where N is any nucleotide) or an internal ribosome entry site (IRES). The majority of translation initiation events in eukaryotes are mediated through cap-dependent translation whereby the 40S ribosomal subunit is recruited to the vicinity of the mRNA 5′ cap structure by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of eIF4E (the cap-binding subunit), eIF4A (an RNA helicase), and eIF4G (a large scaffolding protein for eIF4E, eIF4A, and other initiation factors). Once assembled at the 5′ cap, the 40S ribosomal subunit in association with several initiation factors scans the 5′ untranslated region (5′UTR) of the mRNA until it encounters a start codon in a favorable context, followed by polypeptide synthesis (37).Early in vitro studies have shown that the initiation factor eIF4B acts to potentiate ribosome recruitment to the mRNA (3, 45). eIF4B stimulates translation of both capped and uncapped mRNAs in vitro (1, 36). This function is exerted through stimulation of the helicase activity of eIF4A (43), possibly through direct interactions with eIF4A (44) or with mRNA, the ribosome-associated eIF3, and 18S rRNA (28, 29, 44). Thus, eIF4B is thought to form auxiliary bridges between the mRNA and the 40S ribosomal subunit. Toeprinting studies using mammalian eIF4B underscored its importance in the assembly of the 48S initiation complex, especially on mRNAs harboring secondary structures in the 5′UTRs (11).In vivo studies of eIF4B are limited. Ectopic expression of eIF4B in cultured Drosophila melanogaster cells and in developing eye imaginal discs stimulated cell proliferation (16). Enhanced cell proliferation is most likely mediated by increased translation of a subset of mRNAs, since knockdown of Drosophila eIF4B by RNA interference (RNAi) caused a modest reduction in global translation but compromised the survival of insect cells grown under low serum conditions (16). Studies of eIF4B in mammalian cells yielded contradictory results. Transient overexpression of eIF4B stimulated translation initiation in a phosphorylation-dependent manner in some cells (18, 49) while inhibiting translation in others (30, 31, 41). These differences might be attributed to disparate levels of eIF4B overexpression.To address the physiological role of eIF4B in mRNA translation in the cell, RNAi knockdown of eIF4B was used here. We demonstrate that eIF4B is required for optimal translation. Importantly, the translation of mRNAs bearing structured 5′UTRs, such as the cell cycle regulators Cdc25C, c-myc, and ODC (ornithine decarboxylase), and the antiapoptotic factors Bcl-2 and XIAP (X-linked inhibitor of apoptosis), was reduced as a result of eIF4B silencing by RNAi. Furthermore, eIF4B silencing promoted caspase-dependent apoptosis. Thus, we show that mammalian eIF4B is required for cell proliferation and survival, whereby it acts by regulating the translation of a functionally related subset of mRNAs.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO₂) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl₄ or Zirconium (IV) oxynitrate (ZrO(NO₃)₂) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.     

Oxysterol-binding Protein (OSBP)-related Protein 4 (ORP4) Is Essential for Cell Proliferation and Survival

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.     

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3′-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)—a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway—by directly binding their 3′-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1β were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.     

REDD1 Is Essential for Optimal T Cell Proliferation and Survival

REDD1 is a highly conserved stress response protein that is upregulated following many types of cellular stress, including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. Recently, REDD1 was shown to be involved in dexamethasone induced autophagy in murine thymocytes. However, we know little of REDD1’s function in mature T cells. Here we show for the first time that REDD1 is upregulated following T cell stimulation with PHA or CD3/CD28 beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although markers of activation appear normal. These findings demonstrate a previously unappreciated role for REDD1 in T cell function.