SP1-induced circ_0017552 modulates colon cancer cell proliferation and apoptosis via up-regulation of NET1.

Colon cancer (CC) is a common malignancy over the world and its morbidity and mortality significantly went up in China in recent years. Molecular functions in cancers have gradually been the pivot subject in cancer research. Neuroepithelial cell transforming 1 (NET1) was reported to contribute to prostate cancer and gastric cancer. Our study figured out that NET1 was overexpressed in CC cells. Then, loss-of-function assays revealed that NET1 facilitated CC cell proliferation and repressed CC cell apoptosis. Next, miR-338-3p was confirmed to target NET1. After that, we verified that circ_0017552 which originates from NET1 could positively modulate NET1 expression. Besides, circ_0017552 was a sponge of miR-338-3p. Rescue assays' results demonstrated that circ_0017552 could regulate CC cell proliferation and apoptosis through up-regulation of NET1. A transcription factor named Sp1 (SP1) was found to be present in circ_0017552. SP1 induced transcription of circ_0017552 to facilitate CC cell proliferation and inhibit CC cell apoptosis. In a word, SP1-induced circ_0017552 regulated CC cell proliferation and apoptosis through miR-338-3p/NET1 axis.

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

Cell-in-Cell-Mediated Entosis Reveals a Progressive Mechanism in Pancreatic Cancer.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with high intratumoral heterogeneity. There is a lack of effective therapeutics for PDAC. Entosis, a form of nonapoptotic regulated cell death mediated by cell-in-cell structures (CICs), has been reported in multiple cancers. However, the role of entosis in PDAC progression remains unclear. METHODS: CICs were evaluated using immunohistochemistry and immunofluorescence staining. The formation of CICs was induced by suspension culture. Through fluorescence-activated cell sorting and single-cell RNA sequencing, entosis-forming cells were collected and their differential gene expression was analyzed. Cell functional assays and mouse models were used to investigate malignant phenotypes. Clinical correlations between entosis and PDAC were established by retrospective analysis. RESULTS: Entosis was associated with an unfavorable prognosis for patients with PDAC and was more prevalent in liver metastases than in primary tumors. The single-cell RNA sequencing results revealed that several oncogenes were up-regulated in entosis-forming cells compared with parental cells. These highly entotic cells demonstrated higher oncogenic characteristics in vitro and in vivo. NET1, neuroepithelial cell transforming gene 1, is an entosis-related gene that plays a pivotal role in PDAC progression and is correlated with poor outcomes. CONCLUSIONS: Entosis is correlated with PDAC progression, especially in liver metastasis. NET1 is a newly validated entosis-related gene and a molecular marker of poor outcomes. PDAC cells generate a highly aggressive subpopulation marked by up-regulated NET1 via entosis, which may drive PDAC progression.

mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

Src stimulates Abl-dependent phosphorylation of the guanine exchange factor Net1A to promote its cytosolic localization and cell motility.

The neuroepithelial cell transforming gene 1 (Net1) is a guanine nucleotide exchange factor for the small GTPase RhoA that promotes cancer cell motility and metastasis. Two isoforms of Net1 exist, Net1 and Net1A, both of which are sequestered in the nucleus in quiescent cells to prevent aberrant RhoA activation. Many cell motility stimuli drive cytosolic relocalization of Net1A, but mechanisms controlling this event are not fully understood. Here, we demonstrate that epithelial growth factor stimulates protein kinase Src- and Abl1-dependent phosphorylation of Net1A to promote its cytosolic localization. We show that Abl1 efficiently phosphorylates Net1A on Y373, and that phenylalanine substitution of Y373 prevents Net1A cytosolic localization. Furthermore, we found that Abl1-driven cytosolic localization of Net1A does not require S52, which is a phosphorylation site of a different kinase, c-Jun N-terminal kinase, that inhibits nuclear import of Net1A. However, we did find that MKK7-stimulated cytosolic localization of Net1A does require Y373. We also demonstrate that aspartate substitution at Y373 is sufficient to promote Net1A cytosolic accumulation, and expression of Net1A Y373D potentiates epithelial growth factor-stimulated RhoA activation, downstream myosin light chain 2 phosphorylation, and F-actin accumulation. Moreover, we show that expression of Net1A Y373D in breast cancer cells also significantly increases cell motility and Matrigel invasion. Finally, we show that Net1A is required for Abl1-stimulated cell motility, which is rescued by expression of Net1A Y373D, but not Net1A Y373F. Taken together, this work demonstrates a novel mechanism controlling Net1A subcellular localization to regulate RhoA-dependent cell motility and invasion.

Circ_0000745 promotes acute lymphoblastic leukemia progression through mediating miR-494-3p/NET1 axis.

BACKGROUND: Circular RNAs (circRNAs) have shown important regulatory roles in tumorigenesis. However, the role and working mechanism of circ_0000745 in acute lymphoblastic leukemia (ALL) development remain largely unclear. METHODS: The expression of circ_0000745, sperm antigen with calponin homology and coiled-coil domains 1 (SPECC1), microRNA-494-3p (miR-494-3p), and neuroepithelial cell transforming 1 (NET1) messenger RNA (mRNA) and protein was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay. Flow cytometry was performed to assess cell apoptosis and cell cycle progression. Extracellular acidification rate (ECAR) was assessed to analyze cell glycolysis. Cell viability was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Ferroptosis was assessed through measuring the intracellular levels of iron and lipid reactive oxygen species (ROS). Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to validate the interaction between miR-494-3p and circ_0000745 or NET1. RESULTS: Circ_0000745 expression was elevated in ALL patients and cell lines. Circ_0000745 knockdown restrained cell cycle progression and glycolysis and triggered cell apoptosis and ferroptosis. Circ_0000745 acted as a molecular sponge for miR-494-3p in ALL cells. miR-494-3p silencing partly diminished circ_0000745 knockdown-induced anti-tumor effects in ALL cells. NET1 was a target of miR-494-3p, and miR-494-3p overexpression-induced anti-tumor influences were partly counteracted by the accumulation of NET1 in ALL cells. Circ_0000745 can positively regulate NET1 expression by sponging miR-494-3p in ALL cells. CONCLUSION: Circ_0000745 contributed to ALL development partly by binding to miR-494-3p to induce NET1 expression.0020.

Baicalin suppresses lung cancer growth phenotypes via miR-340-5p/NET1 axis.

As a malignant disease, lung cancer has a high morbidity and mortality rate. Baicalin is derived from Radix Scutellariae and has anti-tumor effects, however, its role in lung cancer remains unknown. Here, functional assays suggested baicalin suppressed in vitro lung cancer phenotypes. We used micro (mi)RNA array analysis to explore baicalin effects on miRNA expression. We observed baicalin increased miR-340-5p expression, whereas inhibition of this expression abolished anti-tumor effects of baicalin. Furthermore, neuroepithelial cell transforming 1 (NET1) functioned as a miR-340-5p target, and acted in a baicalin-dependent manner to regulate lung cancer progression. Thus, baicalin elicited antitumor activities by affecting the miR-340-5p/NET1 axis, suggesting a new approach to lung cancer clinical management.

NET1 promotes HCC growth and metastasis in vitro and in vivo via activating the Akt signaling pathway.

Neuroepithelial cell transforming gene 1 (NET1), a member of the guanine nucleotide exchange factor family, is involved in various cancers, including gastric cancer, breast cancer and glioma. However, the role of NET1 in hepatocellular carcinoma (HCC) remains largely uncovered. In this study, we found that NET1 expression was upregulated in HCC, and that upregulated NET1 expression was closely associated with poor prognosis and some clinical characteristics in HCC patients. Whilst forced expression of NET1 in HCC cells was observed to significantly promote cell growth and metastasis in vitro and in vivo; downregulation of NET1 was shown to exhibit an opposite inhibitory effect. RNA-seq analysis and gene set enrichment analysis demonstrated that knockdown of NET1 significantly suppressed the level of Akt phosphorylation level and the expression of Akt downstream genes in HCC cells. Moreover, MK2206, a potent Akt inhibitor was shown to block the NET1-induced effects in HCC. Taken together, this study demonstrated that, through the Akt signaling pathway, NET1 plays an oncogenic role in HCC progression and metastasis. Hence, NET1 may potentially be used as a potential therapeutic target and prognostic marker of HCC.

Role of Small GTPase RhoA in DNA Damage Response.

Accumulating evidence has suggested a role of the small GTPase Ras homolog gene family member A (RhoA) in DNA damage response (DDR) in addition to its traditional function of regulating cell morphology. In DDR, 2 key components of DNA repair, ataxia telangiectasia-mutated (ATM) and flap structure-specific endonuclease 1 (FEN1), along with intracellular reactive oxygen species (ROS) have been shown to regulate RhoA activation. In addition, Rho-specific guanine exchange factors (GEFs), neuroepithelial transforming gene 1 (Net1) and epithelial cell transforming sequence 2 (Ect2), have specific functions in DDR, and they also participate in Ras-related C3 botulinum toxin substrate 1 (Rac1)/RhoA interaction, a process which is largely unappreciated yet possibly of significance in DDR. Downstream of RhoA, current evidence has highlighted its role in mediating cell cycle arrest, which is an important step in DNA repair. Unraveling the mechanism by which RhoA modulates DDR may provide more insight into DDR itself and may aid in the future development of cancer therapies.

Cdk1 phosphorylation negatively regulates the activity of Net1 towards RhoA during mitosis.

The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.

MiRNA505/NET1 Axis Acts as a CD8+ T-TIL Regulator in Non-Small Cell Lung Cancer.

INTRODUCTION: Lung adenocarcinoma (LUAD), which is the most important and common subtype of non-small cell lung cancer (NSCLC), is highly heterogeneous with a poor prognosis and poses great challenges to health worldwide. MicroRNAs (miRNAs) are regulators of gene expression with recognized roles in physiology and diseases, such as cancers, but little is known about their functional relevance to CD8+ T cell infiltration regulation in the tumor microenvironment (TME) of NSCLC patients, especially LUAD patients. METHODS: Bioinformatic analysis was used to analyze TCGA data. RT-PCT, Western blot, luciferase assay and immunohistochemistry were used to detect the expression levels and bindings of genes and miRNA. ELISA and cytotoxic assay were used to evaluate CD8+ T cell function. RESULTS: In this study, bioinformatic analysis unveiled the miR-505-3p/NET1 pair as a CD8+ T-tumor-infiltrating lymphocyte (TIL) regulator. Then, we confirmed the bioinformatic results with LUAD patient samples, and NET1 was shown to be a direct target of miR-505-3p in a luciferase assay. Functional experiments demonstrated that miR-505-3p enhanced CD8+ T-TIL function, while NET1 impaired CD8+ T-TIL function and partly reversed the effects of miR-505-3p. The observed effects might be exerted via the regulation of immunosuppressive receptors in T cells. DISCUSSION: Our study may provide novel insights into LUAD progression related to the TME mechanism and new possibilities for improving adoptive immunotherapy.

Collective cancer cell invasion requires RNA accumulation at the invasive front.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

MicroRNAs and Xenobiotic Toxicity: An Overview.

The advent of new technologies has paved the rise of various chemicals that are being employed in industrial as well as consumer products. This leads to the accumulation of these xenobiotic compounds in the environment where they pose a serious threat to both target and non-target species. miRNAs are one of the key epigenetic mechanisms that have been associated with toxicity by modulating the gene expression post-transcriptionally. Here, we provide a comprehensive view on miRNA biogenesis, their mechanism of action and, their possible role in xenobiotic toxicity. Further, we review the recent in vitro and in vivo studies involved in xenobiotic exposure induced miRNA alterations and the mRNA-miRNA interactions. Finally, we address the challenges associated with the miRNAs in toxicological studies.

LncRNA CTC-497E21.4 promotes the progression of gastric cancer via modulating miR-22/NET1 axis through RhoA signaling pathway.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as important roles in gastric cancer (GC). However, the role of the dysregulated lncRNAs in GC remained large unknown. We investigated the clinical significance, biological function and mechanism of CTC-497E21.4 in GC. METHODS: Firstly, RTFQ-PCR was used to detect the expression of CTC-497E21.4 in GC. Furthermore, knockdown of CTC-497E21.4 was conducted to assess the effect of CTC-497E21.4 in vitro and vivo. Subcellular localization of CTC-497E21.4 was determined by nuclear plasmolysis PCR and FISH. We also predicted CTC-497E21.4 binding miRNAs and downstream target genes and evaluated its regulation of miR-22 by acting as a ceRNA. RESULT: CTC-497E21.4 was upregulated in GC tissues and GC cell lines (P < 0.05), and the expression was associated with depth of invasion, lymph node metastasis, and neurological invasion. Besides, knockdown of CTC-497E21.4 inhibited cell proliferation, invasion and promoted cell cycle arrest in vitro and inhibited tumorigenesis in vivo. Mechanistic investigations indicated that CTC-497E21.4 acted as a ceRNA for miR-22 and regulated NET1 expression. CTC-497E21.4/miR-22-3p/NET1 participated in the RhoA signaling pathway in the GC progression. CONCLUSION: CTC-497E21.4 competed with miR-22 to regulate the expression of NET1 and regulated the malignant progression of GC through RhoA signaling pathway.

Genetic deletion of the Rho GEF Net1 impairs mouse macrophage motility and actin cytoskeletal organization.

Macrophages are innate immune cells that constantly patrol an organism to fulfill protective and homeostatic roles. Previous studies have shown that Rho GTPase activity is required for macrophage mobility, yet the roles of upstream regulatory proteins controlling Rho GTPase function in these cells are not well defined. Previously we have shown that the RhoA GEF Net1 is required for human breast cancer cell motility and extracellular matrix invasion. To assess the role of Net1 in macrophage motility, we isolated bone marrow macrophage (BMM) precursors from wild type and Net1 knockout mice. Loss of Net1 did not affect the ability of BMM precursors to differentiate into mature macrophages in vitro, as measured by CD68 and F4/80 staining. However, Net1 deletion significantly reduced RhoA activation, F-actin accumulation, adhesion, and motility in these cells. Nevertheless, similar to RhoA/RhoB double knockout macrophages, Net1 deletion did not impair macrophage recruitment to the peritoneum in a mouse model of sterile inflammation. These data demonstrate that Net1 is an important regulator of RhoA signaling and motility in mouse macrophages in vitro, but that its function may be dispensable for macrophage recruitment to inflammatory sites in vivo.

Regulation of RhoA activation and cell motility by c-Jun N-terminal kinases and Net1.

Jnks are mitogen activated protein kinases that are best known for regulating transcription and apoptotic signaling. However, they also play important roles in controlling cell motility and invasion by phosphorylating many actin and microtubule regulatory proteins. These mechanisms have important implications for normal cell motility as well as cancer metastasis. Jnks are activated by growth factors and cytokines that stimulate cell motility, and this often requires upstream activation of Rho GTPases. Our recent work indicates that Jnks may also regulate Rho GTPase activation. Specifically, we found that Jnk-dependent phosphorylation of the RhoA guanine nucleotide exchange factor (RhoGEF) Net1A promotes its cytosolic accumulation to drive RhoA activation and actin cytoskeletal reorganization. Net1A is unusual among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation. Importantly, Jnk-stimulated cytosolic localization of Net1A is sufficient to stimulate cell motility and extracellular matrix invasion in non-invasive breast cancer cells. Since Net1A expression is critical for cancer cell motility and invasion in vitro, and breast cancer metastasis in vivo, these data uncover a previously unappreciated regulatory mechanism that may contribute to metastasis in multiple types of cancer.

Preparation of novel targeting nanobubbles conjugated with small interfering RNA for concurrent molecular imaging and gene therapy in vivo.

The aim of this study was to establish a novel targeting nanobubble (TNB) conjugated with small interfering RNA (siRNA)-cyanine 5 (Cy5) and to validate its theranostic ability in vivo. The TNB conjugated with neuroepithelial transforming gene 1 (NET-1) siRNA-Cy5 was prepared by thin-film hydration and mechanical sonication method. A hepatocellular carcinoma (HCC) xenograft model was established by subcutaneously injecting SMMC-7721 cells in BALB/c nude mice. The NET-1 siRNA-conjugated TNB was utilized for accurate contrast-enhanced ultrasound in vivo imaging, which was enabled by the target ligand GPC-3 antibody and specific gene transfection with the aid of low-frequency ultrasound (LFUS) irradiation. BALB/c nude mice bearing tumors were randomized into 5 groups and irradiated with LFUS for 5 min after TNB administration; mice were treated twice a week for a total of 60 d. The mean particle size of TNB was <500 nm. Mice treated with NET-1 siRNA-conjugated TNB showed a significant decrease in tumor growth and the highest survival rate. Our findings offer an effective and safe gene vehicle and probe for molecular imaging in vivo. It may improve the early diagnosis and treatment effects of HCC.-Wu, B., Shang, H., Liang, X., Sun, Y., Jing, H., Han, X., Cheng, W. Preparation of novel targeting nanobubbles conjugated with small interfering RNA for concurrent molecular imaging and gene therapy in vivo.

Canine mammary tumour cells exposure to sevoflurane: effects on cell proliferation and neuroepithelial transforming gene 1 expression.

OBJECTIVE: The influence of perioperative factors, such as anaesthetic and analgesic techniques, on metastatic spread following surgery for primary cancer removal is of growing interest. The present study investigated the effects of sevoflurane on canine mammary tumour cell proliferation (MTT colorimetric assay) and on the expression of neuroepithelial transforming gene 1 (NET1). STUDY DESIGN: Prospective controlled in vitro trial. STUDY MATERIAL: Primary (CIPp) and metastatic canine tubular adenocarcinoma (CIPm) cells. METHODS: To perform MTT tests, cell lines were seeded at a density of 3000 cells per well and incubated with sevoflurane (1, 2.5 or 4 mM) or only with the culture medium (control). Sevoflurane was added to the cell cultures every hour to avoid changes in drug concentration. MTT assays were performed after 6 hours of exposure obtaining absolute values of absorbance. The RNA isolated from the lysates of the same cell lines underwent quantitative polymerase chain reaction to evaluate NET1 gene expression changes compared with controls. One- or two-way analysis of variance was used as appropriate (p < 0.05). RESULTS: A significant increase in cell proliferation compared with controls was observed in CIPp treated with lower sevoflurane concentrations, whereas a significant decrease in cell proliferation was found in CIPm treated with all the sevoflurane concentrations. All CIPp treatments did not induce changes in gene expression compared with controls, whereas a significant increase in gene expression was observed in CIPm between controls and the higher sevoflurane concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Sevoflurane treatments modified the cell proliferation rate in both cell lines showing an increase or decrease when applied to CIPp or CIPm, respectively. Expression of the NET1 gene increased after treatment with sevoflurane 4 mM in metastatic cells. The role of sevoflurane on cancer recurrence should be further investigated.

NET-1 promotes invasion, adhesion and growth of hepatocellular carcinoma by regulating the expression of BAX, caspase 3, caspase 8 and BCL2.

Dysregulation of neuroepithelial transforming gene-1 (NET-1) has been shown in hepatocellular carcinoma (HCC) patients. We aimed to evaluate the influence of NET-1 on HCC invasion, adhesion and growth. In vitro cellular functional assays including invasion and adhesion were performed to evaluate the effects of knockdown and overexpression of NET-1. HCC cells were transplanted into nude mice, and tumor growth was assessed. BAX, caspase 3, caspase 8 and BCL2 protein levels were detected by western blot. After transfection with NET-1 siRNA, NET-1 positive ratio in HCC cells significantly decreased. Cell invasion and adhesion assay showed that knockdown of NET-1 reduced the invasion and adhesion ability of HCC cells, whereas overexpression of NET-1 increased the ability. The evaluation of tumor growth revealed that NET-1 knockdown significantly decreased tumor volume and weight, while NET-1 overexpression promoted tumor growth in nude mice. Western blot showed that NET-1 knockdown increased BAX, caspase 3 and caspase 8 expression but decreased BCL2 expression, whereas NET-1 overexpression significantly down-regulated BAX, caspase 3 and caspase 8 expression but increased BCL2 expression. Our data suggest that NET-1 promotes HCC invasion, adhesion and growth by regulating BAX, caspase 3, caspase 8 and BCL2 expression.

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease.

Ulp1 and Ulp2, in the yeast Saccharomyces cerevisiae, are the founding members of deSUMOylating enzymes. These enzymes remove small ubiquitin-like modifier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme. However, the mechanism for Ulp2's substrate specificity has been insufficiently understood. Here we show that the C-terminal regulatory domain of Ulp2 contains three distinct, yet conserved, motifs that control its in vivo substrate specificity and cell growth. Among them, a SUMO-interacting motif (SIM) was found to coordinate with the domain of Ulp2 that binds to the nucleolar protein Csm1 to ensure maximal deSUMOylation of Ulp2's nucleolar substrates. We found that whereas the Csm1-binding domain of Ulp2 recruits this enzyme to the nucleolus, Ulp2's C-terminal SIM promotes its SUMO protease activity and plays a key role in mediating the in vivo specificity of Ulp2. Thus, the substrate specificity of Ulp2 is controlled by both its subcellular localization and the SUMOylation status of its substrates. These findings illustrate the highly coordinated and dynamic nature of the SUMO pathways in maintaining homeostasis of intracellular SUMOylation.