CPEB2-activated Prdm16 translation promotes brown adipocyte function and prevents obesity.

OBJECTIVE: Brown adipose tissue (BAT) plays an important role in mammalian thermogenesis through the expression of uncoupling protein 1 (UCP1). Our previous study identified cytoplasmic polyadenylation element binding protein 2 (CPEB2) as a key regulator that activates the translation of Ucp1 with a long 3'-untranslated region (Ucp1L) in response to adrenergic signaling. Mice lacking CPEB2 or Ucp1L exhibited reduced UCP1 expression and impaired thermogenesis; however, only CPEB2-null mice displayed obesogenic phenotypes. Hence, this study aims to investigate how CPEB2-controlled translation impacts body weight. METHODS: Body weight measurements were conducted on mice with global knockout (KO) of CPEB2, UCP1 or Ucp1L, as well as those with conditional knockout of CPEB2 in neurons or adipose tissues. RNA sequencing coupled with bioinformatics analysis was used to identify dysregulated gene expression in CPEB2-deficient BAT. The role of CPEB2 in regulating PRD1-BF1-RIZ1 homologous-domain containing 16 (PRDM16) expression was subsequently confirmed by RT-qPCR, Western blotting, polysomal profiling and luciferase reporter assays. Adeno-associated viruses (AAV) expressing CPEB2 or PRDM16 were delivered into BAT to assess their efficacy in mitigating weight gain in CPEB2-KO mice. RESULTS: We validated that defective BAT function contributed to the increased weight gain in CPEB2-KO mice. Transcriptomic profiling revealed upregulated expression of genes associated with muscle development in CPEB2-KO BAT. Given that both brown adipocytes and myocytes stem from myogenic factor 5-expressing precursors, with their cell-fate differentiation regulated by PRDM16, we identified that Prdm16 was translationally upregulated by CPEB2. Ectopic expression of PRDM16 in CPEB2-deprived BAT restored gene expression profiles and decreased weight gain in CPEB2-KO mice. CONCLUSIONS: In addition to Ucp1L, activation of Prdm16 translation by CPEB2 is critical for sustaining brown adipocyte function. These findings unveil a new layer of post-transcriptional regulation governed by CPEB2, fine-tuning thermogenic and metabolic activities of brown adipocytes to control body weight.

CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity.

BACKGROUND: Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. METHODS: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. RESULTS: Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. CONCLUSIONS: We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

CPEB2 inhibits preeclampsia progression by regulating SSTR3 translation through polyadenylation.

AIMS: Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear. METHODS: The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining. RESULTS: CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression. CONCLUSION: CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.

CPEB2 enhances cell growth and angiogenesis by upregulating ARPC5 mRNA stability in multiple myeloma.

BACKGROUND: The process of multiple myeloma (MM) is the result of the combined action of multiple genes. This study aims to explore the role and mechanism of cytoplasmic polyadenylation element binding protein2 (CPEB2) in MM progression. METHODS: The mRNA and protein expression levels of CPEB2 and actin-related protein 2/3 complex subunit 5 (ARPC5) were assessed by quantitative real-time PCR and western blot analysis. Cell function was determined by cell counting kit 8 assay, soft-agar colony formation assay, flow cytometry and tube formation assay. Fluorescent in situ hybridization assay was used to analyze the co-localization of CPEB2 and ARPC5 in MM cells. Actinomycin D treatment and cycloheximide chase assay were performed to assess the stability of ARPC5. The interaction between CPEB2 and ARPC5 was confirmed by RNA immunoprecipitation assay. RESULTS: CPEB2 and ARPC5 mRNA and protein expression levels were upregulated in CD138+ plasma cells from MM patients and cells. CPEB2 downregulation reduced MM cell proliferation, angiogenesis, and increased apoptosis, while its overexpression had an opposite effect. CPEB2 and ARPC5 were co-localized at cell cytoplasm and could positively regulate ARPC5 expression by mediating its mRNA stability. ARPC5 overexpression reversed the suppressive effect of CPEB2 knockdown on MM progression, and it knockdown also abolished CPEB2-promoted MM progression. Besides, CPEB2 silencing also reduced MM tumor growth by decreasing ARPC5 expression. CONCLUSION: Our results indicated that CPEB2 increased ARPC5 expression through promoting its mRNA stability, thereby accelerating MM malignant process.

Reciprocal modulation of long noncoding RNA EMS and p53 regulates tumorigenesis.

p53 plays a central role in tumor suppression. Emerging evidence suggests long noncoding RNA (lncRNA) as an important class of regulatory molecules that control the p53 signaling. Here, we report that the oncogenic lncRNA E2F1 messenger RNA (mRNA) stabilizing factor (EMS) and p53 mutually repress each other's expression. EMS is negatively regulated by p53. As a direct transcriptional repression target of p53, EMS is surprisingly shown to inhibit p53 expression. EMS associates with cytoplasmic polyadenylation element-binding protein 2 (CPEB2) and thus, disrupts the CPEB2-p53 mRNA interaction. This disassociation attenuates CPEB2-mediated p53 mRNA polyadenylation and suppresses p53 translation. Functionally, EMS is able to exert its oncogenic activities, at least partially, via the CPEB2-p53 axis. Together, these findings reveal a double-negative feedback loop between p53 and EMS, through which p53 is finely controlled. Our study also demonstrates a critical role for EMS in promoting tumorigenesis via the negative regulation of p53.

STAT1 mediated long non-coding RNA LINC00504 influences radio-sensitivity of breast cancer via binding to TAF15 and stabilizing CPEB2 expression.

Radiotherapy plays important roles in the treatment of breast cancer (BC), which develops from malignant cells in the breast. Long non-coding RNAs (lncRNAs) have been reported to be implicated in radio-resistance or radio-sensitivity of human cancer, which includes breast cancer. Nevertheless, long intergenic non-protein coding RNA 0504 (LINC00504) has not been investigated in BC. In our study, from RT-qPCR analysis, LINC00504 was found to be up-regulated in BC cells. By conducting in vitro assays, it was confirmed that the knockdown of LINC00504 could enhance the radio-sensitivity of BC cells. The regulatory mechanism of LINC00504 in BC was also verified by chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) and luciferase reporter assays. From the experimental results, we knew that the up-regulation of LINC00504 was mediated by signal transducer and activator of transcription 1 (STAT1). Moreover, LINC00504 stabilized the expression of cytoplasmic polyadenylation element-binding protein 2 (CPEB2) via binding to TATA-box binding protein associated factor 15 (TAF15). Furthermore, rescue assays validated that LINC00504 participated in regulating the radio-sensitivity of BC cells via up-regulating CPEB2. In summary, our study disclosed that STAT1 could mediate LINC00504 and weaken the radio-sensitivity of BC cells via binding to TAF15 and stabilizing CPEB2 expression.

A p53/CPEB2 negative feedback loop regulates renal cancer cell proliferation and migration.

The tumor suppressor p53 transactivates the expression of multiple genes to exert its multifaceted functions and ultimately maintains genome stability. Thus, cancer cells develop various mechanisms to diminish p53 expression and bypass the cell cycle checkpoint. In this study, we identified the gene encoding RNA-binding protein cytoplasmic polyadenylation element-binding protein 2 (CPEB2) as a p53 target. In turn, CPEB2 decreases p53 messenger RNA stability and translation to fine-tune p53 level. Specifically, we showed that CPEB2 binds the cytoplasmic polyadenylation elements in the p53 3'-untranslated region, and the RNA recognition motif and zinc finger (ZF) domains of CPEB2 are required for this binding. Furthermore, we found that CPEB2 was upregulated in renal cancer tissues and promotes the renal cancer cell proliferation and migration. The oncogenic effect of CPEB2 is partially dependent on negative feedback regulation of p53. Overall, we identify a novel regulatory feedback loop between p53 and CPEB2 and demonstrate that CPEB2 promotes tumor progression by inactivating p53, suggesting that CPEB2 is a potential therapeutic target in human renal cancer.

CPEB2-activated PDGFRα mRNA translation contributes to myofibroblast proliferation and pulmonary alveologenesis.

BACKGROUND: Alveologenesis is the final stage of lung development to form air-exchanging units between alveoli and blood vessels. Genetic susceptibility or hyperoxic stress to perturb this complicated process can cause abnormal enlargement of alveoli and lead to bronchopulmonary dysplasia (BPD)-associated emphysema. Platelet-derived growth factor receptor α (PDGFRα) signaling is crucial for alveolar myofibroblast (MYF) proliferation and its deficiency is associated with risk of BPD, but posttranscriptional mechanisms regulating PDGFRα synthesis during lung development remain largely unexplored. Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) is a sequence-specific RNA-binding protein and translational regulator. Because CPEB2-knockout (KO) mice showed emphysematous phenotypes, we investigated how CPEB2-controlled translation affects pulmonary development and function. METHODS: Respiratory and pulmonary functions were measured by whole-body and invasive plethysmography. Histological staining and immunohistochemistry were used to analyze morphology, proliferation, apoptosis and cell densities from postnatal to adult lungs. Western blotting, RNA-immunoprecipitation, reporter assay, primary MYF culture and ectopic expression rescue were performed to demonstrate the role of CPEB2 in PDGFRα mRNA translation and MYF proliferation. RESULTS: Adult CPEB2-KO mice showed emphysema-like dysfunction. The alveolar structure in CPEB2-deficient lungs appeared normal at birth but became simplified through the alveolar stage of lung development. In CPEB2-null mice, we found reduced proliferation of MYF progenitors during alveolarization, abnormal deposition of elastin and failure of alveolar septum formation, thereby leading to enlarged pulmonary alveoli. We identified that CPEB2 promoted PDGFRα mRNA translation in MYF progenitors and this positive regulation could be disrupted by H2O2, a hyperoxia-mimetic treatment. Moreover, decreased proliferating ability in KO MYFs due to insufficient PDGFRα expression was rescued by ectopic expression of CPEB2, suggesting an important role of CPEB2 in upregulating PDGFRα signaling for pulmonary alveologenesis. CONCLUSIONS: CPEB2-controlled translation, in part through promoting PDGFRα expression, is indispensable for lung development and function. Since defective pulmonary PDGFR signaling is a key feature of human BPD, CPEB2 may be a risk factor for BPD.

A positive feedback self-regulatory loop between miR-210 and HIF-1α mediated by CPEB2 is involved in trophoblast syncytialization: implication of trophoblast malfunction in preeclampsia†.

The pregnancy complication preeclampsia is directly associated with hypoxic stress and insufficient trophoblast cell differentiation. The hypoxia-inducible microRNA (miRNA), miR-210, has been identified as a significantly up-regulated miRNA in preeclamptic placenta, and evidence in other cell types has indicated a feedback regulation between miR-210 and hypoxia-inducible factor-1α (HIF-1α) under hypoxic condition. It remains unclear whether and how the feedback loop between miR-210 and HIF-1α may contribute to trophoblast dysfunction in preeclampsia. Here, we proved that cytoplasmic polyadenylation element-binding 2 (CPEB2) was a direct target of miR-210 in human trophoblast. CPEB2 could inhibit the translation of hypoxia-induced HIF-1α via directly binding the cytoplasmic polyadenylation element (CPE) site in the 3'-untranslated region (UTR) of HIF-1α mRNA. The increase in the HIF-1α level upon hypoxia treatment could be efficiently reversed by miR-210 inhibitor. In addition, CPEB2 was primarily expressed in villous syncytiotrophoblasts, and the suppression of trophoblast cell syncytialization by miR-210 could be significantly rescued by CPEB2 overexpression. In preeclamptic placenta, the expression of CPEB2 was evidently lower than normal pregnant control, and the miR-210 level was aberrantly higher and trophoblast syncytialization was limited. The findings revealed a positive feedback loop between miR-210 and HIF-1α that is mediated by CPEB2 in human trophoblasts, and demonstrated a mechanism underlying the insufficient trophoblast syncytialization in preeclampsia under hypoxic stress.

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells.

BACKGROUND: Over-expression of cyclooxygenase (COX)-2 promotes breast cancer progression by multiple mechanisms, including induction of stem-like cells (SLC). Combined gene expression and microRNA microarray analyses of empty vector vs COX-2- transfected COX-2 low MCF7 breast cancer cell line identified two COX-2-upregulated microRNAs, miR-526b and miR-655, both found to be oncogenic and SLC-promoting. Cytoplasmic Polyadenylation Element-Binding Protein 2 (CPEB2) was the single common target of both microRNAs, the functions of which remain controversial. CPEB2 has multiple isoforms (A-F), and paradoxically, a high B/A ratio was reported to impart anoikis-resistance and metastatic phenotype in triple- negative breast cancer cells. We tested whether CPEB2 is a tumor suppressor in mammary epithelial cells. METHODS: We knocked-out CPEB2 in the non-tumorigenic mammary epithelial cell line MCF10A by CRISPR/Cas9-double nickase approach, and knocked-down CPEB2 with siRNAs in the poorly malignant MCF7 cell line, both lines being high CPEB2-expressing. The resultant phenotypes for oncogenity were tested in vitro for both lines and in vivo for CPEB2KO cells. Finally, CPEB2 expression was compared between human breast cancer and non-tumor breast tissues. RESULTS: CPEB2 (isoform A) expression was inversely correlated with COX-2 or the above microRNAs in COX-2-divergent breast cancer cell lines. CPEB2KO MCF10A cells exhibited oncogenic properties including increased proliferation, migration, invasion, EMT (decreased E-Cadherin, increased Vimentin, N-Cadherin, SNAI1, and ZEB1) and SLC phenotype (increased tumorsphere formation and SLC marker-expression). Tumor-suppressor p53 protein was shown to be a novel translationally-regulated target of CPEB2, validated with polysome profiling. CPEB2KO, but not wild-type cells produced lung colonies upon intravenous injection and subcutaneous tumors and spontaneous lung metastases upon implantation at mammary sites in NOD/SCID/IL2Rϒ-null mice, identified with HLA immunostaining. Similarly, siRNA-mediated CPEB2 knockdown in MCF7 cells promoted oncogenic properties in vitro. Human breast cancer tissues (n = 105) revealed a lower mRNA expression for CPEB2 isoform A and also a lower A/B isoform ratio than in non-tumour breast tissues (n = 20), suggesting that CPEB2A accounts for the tumor-suppressor functions of CPEB2. CONCLUSIONS: CPEB2, presumably the isoform A, plays a key role in suppressing tumorigenesis in mammary epithelial cells by repressing EMT, migration, invasion, proliferation and SLC phenotype, via multiple targets, including a newly-identified translational target p53.

CPEB2 Is Necessary for Proper Porcine Meiotic Maturation and Embryonic Development.

Oocyte meiotic maturation and embryogenesis are some of the most important physiological processes that occur in organisms, playing crucial roles in the preservation of life in all species. The post-transcriptional regulation of maternal messenger ribonucleic acids (mRNAs) and the post-translational regulation of proteins are critical in the control of oocyte maturation and early embryogenesis. Translational control affects the basic mechanism of protein synthesis, thus, knowledge of the key components included in this machinery is required in order to understand its regulation. Cytoplasmic polyadenylation element binding proteins (CPEBs) bind to the 3'-end of mRNAs to regulate their localization and translation and are necessary for proper development. In this study we examined the expression pattern of cytoplasmic polyadenylation element binding protein 2 (CPEB2) both on the mRNA (by real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR) and protein (by Western blotting, WB) level, as well as its localization during the meiotic maturation of porcine oocytes and early embryonic development by immunocytochemistry (ICC). For the elucidation of its functions, CPEB2 knockdown by double-strand RNA (dsRNA) was used. We discovered that CPEB2 is expressed during all stages of porcine meiotic maturation and embryonic development. Moreover, we found that it is necessary to enable a high percentage of oocytes to reach the metaphase II (MII) stage, as well as for the production of good-quality parthenogenetic blastocysts.

CPEB2-dependent translation of long 3'-UTR Ucp1 mRNA promotes thermogenesis in brown adipose tissue.

Expression of mitochondrial proton transporter uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is essential for mammalian thermogenesis. While human UCP1 mRNA exists in a long form only, alternative polyadenylation creates two different isoforms in mice with 10% of UCP1 mRNA found in the long form (Ucp1L) and ~90% in the short form (Ucp1S). We generated a mouse model expressing only Ucp1S and found that it showed impaired thermogenesis due to a 60% drop in UCP1 protein levels, suggesting that Ucp1L is more efficiently translated than Ucp1S. In addition, we found that ß3 adrenergic receptor signaling promoted the translation of mouse Ucp1L and human Ucp1 in a manner dependent on cytoplasmic polyadenylation element binding protein 2 (CPEB2). CPEB2-knockout mice showed reduced UCP1 levels and impaired thermogenesis in BAT, which was rescued by ectopic expression of CPEB2. Hence, long 3'-UTR Ucp1 mRNA translation activated by CPEB2 is likely conserved and important in humans to produce UCP1 for thermogenesis.

CPEB2 Activates GRASP1 mRNA Translation and Promotes AMPA Receptor Surface Expression, Long-Term Potentiation, and Memory.

Activity-dependent synthesis of plasticity-related proteins is necessary to sustain long-lasting synaptic modifications and consolidate memory. We investigated the role of the translational regulator cytoplasmic polyadenylation element binding protein 2 (CPEB2) in learning and memory because regulated mRNA translation contributes to synaptic plasticity. Forebrain-restricted CPEB2 conditional knockout (cKO) mice exhibited impaired hippocampus-dependent memory in contextual fear conditioning and Morris water maze tests. CPEB2 cKO hippocampi showed impaired long-term potentiation in the Schaffer collateral-CA1 pathway. Reduced surface, but not total, expression of AMPA receptors (AMPARs) in CPEB2 KO neurons led us to identify that CPEB2 enhanced the translation of GRASP1 mRNA to facilitate recycling and maintain the surface level of AMPARs. Ectopic expression of CPEB2 or GRASP1 in CA1 areas of CPEB2 cKO mouse hippocampi rescued long-term potentiation and spatial memory in a water maze test. Thus, CPEB2-regulated GRASP1 mRNA translation is pivotal for AMPAR recycling, long-term plasticity, and memory.

Splice variants of cytosolic polyadenylation element-binding protein 2 (CPEB2) differentially regulate pathways linked to cancer metastasis.

The translational regulator cytosolic polyadenylation element-binding protein 2 (CPEB2) has two isoforms, CPEB2A and CPEB2B, derived by alternative splicing of RNA into a mature form that either includes or excludes exon 4. Previously, we reported that this splicing event is highly dysregulated in aggressive forms of breast cancers, which overexpress CPEB2B. The loss of CPEB2A with a concomitant increase in CPEB2B was also required for breast cancer cells to resist cell death because of detachment (anoikis resistance) and metastasize in vivo To examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes, we used next generation sequencing of triple negative breast cancer cells in which the isoforms were specifically down-regulated. Down-regulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition and hypoxic response, whereas down-regulation of the CPEB2A isoform did not have this effect. Examining key nodes of these pathways showed that CPEB2B induced the expression of regulatory DNA trans-factors (e.g. HIF1α and TWIST1). Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1α. Functional studies showed that specific down-regulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce the acquisition of anoikis resistance and drive metastasis. Overall, this study demonstrates that CPEB2 alternative splicing is a major regulator of key cellular pathways linked to anoikis resistance and metastasis.

Identification of microRNA 885-5p as a novel regulator of tumor metastasis by targeting CPEB2 in colorectal cancer.

Colorectal cancer is the third most common cancer in the world and liver is the most frequent site of distant metastasis with poor prognosis. The aim of this study is to investigate microRNAs leading to liver metastasis. We applied microarray analysis and quantitative PCR to identify and validate dysregulated miRNAs in liver metastases when compared to primary CRCs. Functional significance and the underlying molecular mechanism of selected miRNA was demonstrated by a series of in vitro and in vivo assays. Our microarray analysis and subsequent quantitative PCR validation revealed that miR-885-5p was strongly up-regulated in liver metastases and in CRC cell-lines derived from distant metastases. Overexpression of miR-885-5p significantly induced cell migration, cell invasion, formation of stress fibre in vitro and development of liver and lung metastases in vivo. MiR-885-5p induced metastatic potential of CRC by repressing cytoplasmic polyadenylation element binding protein 2 transcription through directly binding to two binding sites on its 3' untranslated region, and consequently led to up-regulation of TWIST1 and hence epithelial-mesenchymal transition. Our findings demonstrated the overexpression of miR-885-5p in liver metastasis and its roles in inducing CRC metastasis, potentiating development of miR-885-5p inhibitor to treat advanced CRC in the future.

TUG1 mediates methotrexate resistance in colorectal cancer via miR-186/CPEB2 axis.

Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. Methotrexate (MTX) is one of the earliest cytotoxic drugs and serves as an anti-metabolite and anti-folate chemotherapy for various types of cancer. However, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the efficacy of MTX therapies in clinics. Long non-coding RNAs (lncRNAs) have gained widespread attention in recent years. More and more evidences have shown that lncRNAs play regulatory roles in various biological activities and disease progression including drug resistance in cancer cells. Here, we observed lncRNA TUG1 was associated to the MTX resistant in colorectal cancer cells. Firstly, quantitative analysis indicated that TUG1 was significantly increased in tumors which were resistant to MTX treatment. TUG1 knockdown re-sensitized the MTX resistance in colorectal cancer cells, which were MTX-resistant colorectal cell line. Furthermore, bioinformatics analysis showed that miR-186 could directly bind to TUG1, suggesting TUG1 might worked as a ceRNA to sponge miR-186. Extensively, our study also showed that CPEB2 was the direct target of miR-186 in colorectal cancer cells. Taken together, our study suggests that lncRNA TUG1 mediates MTX resistance in colorectal cancer via miR-186/CPEB2 axis.

LncRNA CCAT1 modulates the sensitivity of paclitaxel in nasopharynx cancers cells via miR-181a/CPEB2 axis.

Recent studies reported that long non-coding RNA (lncRNA) might play critical roles in regulating chemo-resistant of multiple types of cancer. This study aimed to investigate whether long non-coding RNA CCAT1 was involved in Paclitaxel resistance in nasopharyngeal carcinoma (NPC). qRT-PCR was used for testing the expression of CCAT1, miR-181a and CPEB2 in tumor tissues and NPC cancers. NPC cells were transfected with siRNAs to suppress the mRNA level of CCAT1 in NPC cells. MTT assays and flow cytometry analysis were used to assess the sensitivity of paclitaxel in NPC cells. Luciferase reporter assays were used to examine the interaction of CCAT1 or CPEB2 to miR-181a. Our findings revealed that the upregulated CCAT1 results in significantly enhancing paclitaxel resistance in nasopharyngeal cancer cells. Bioinformatics analysis and luciferase reporter assay indicated that the upregulated CCAT1 sponges miR-181a in NPC cells. Furthermore, RNA immuno-precipitation assays showed that miR-181a could directly bind to CCAT1 mRNA in NPC cells. We restored miR-181a in NPC cells, and found restoration of miR-181a re-sensitized the NPC cells to paclitaxel in vitro. In addition, our results also showed that miR-181a was a modulator of paclitaxel sensitivity due to its regulative effect on cell apoptosis via targeting CPEB2 in NPC cells. Taken together, lncRNA CCAT1 regulates the sensitivity of paclitaxel in NPC cells via miR-181a/CPEB2 axis.

Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.