Cyclin-dependent Kinase Inhibitor 1B Research Articles

DNA methylation of antisense noncoding RNA in the INK locus (ANRIL) is associated with coronary artery disease in a Chinese population

To explore the association between methylation of antisense non-coding RNA in the INK4 locus (ANRIL) and coronary artery disease (CAD) development. Methylation levels of ANRIL in 100 subjects with CAD and 100 controls were quantitatively analyzed using Sequenom MassARRAY. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to identify novel pathways. Our analyses indicated that 7 to 8 CpG sites within the 2nd CpG island located upstream of ANRIL, also known as cyclin-dependent kinase inhibitor 2B – antisense 1 (CDKN2B-AS1), are hyper-methylated in CAD subjects compared to controls (p = 0.034). The 40th CpG site within the 2nd CpG island located upstream of CDKN2B-AS1 was methylated to a lesser extent in CAD subjects compared to controls (p = 0.045). Both Pearson and Spearman analyses indicated that methylation levels were significantly associated with total cholesterol (r = 0.204, p = 0.004), fasting high-density lipoprotein cholesterol (r = 0.165, p = 0.020), and fasting low-density lipoprotein cholesterol (r = 0.265, p = 0.000). KEGG pathway analysis revealed a significant enrichment of genes associated with the tumor necrosis factor (TNF) signaling pathway. Among them, CCAAT/enhancer binding protein (C/EBPβ) was identified as a key transcription factor that promotes expression of CDKN2B-AS1 through promotor interaction. DNA methylation of the ANRIL promoter was significantly associated with CAD development in our study. Our analyses suggest that C/EBPβ is a key transcription factor that promotes CDKN2B-AS1 expression by directly interacting with the gene promotor mediated by TNF signaling.

266Loss of pumilio 1 phosphorylation turns off the regulation of miRNA-221 on gene p27kip1 in the heart

Abstract Background MicroRNAs regulate gene expression post-transcriptionally via complementary sequence pairing of mRNA 3'UTR and miRNA seed region. MiR-221 downregulates p27kip1 Cyclin-dependent kinase inhibitor 1B (p27), a cell cycle inhibitor, through direct targeting. The down-regulation of p27 caused by miR-221 upregulation induces cancer cell proliferation and has been reported to be closely associated with human cancer metastasis. This regulation is dependent on the existence of pumilio 1 (PUM1). Meanwhile miR-221 mimics protect cardiac myocytes against ischemic injury. It is a potential therapeutic target for cardioprotection. We investigated whether miR-221 targeted p27 in the heart. Purpose To test the hypothesis that the regulation of miR-221 on p27 is abolished in the heart due to lack of RNA binding protein (RBP) PMU1 phosphorylation but not due to p27 3'UTR shortening. Methods Primary cardiac fibroblasts (cFB) were isolated and cultured under normoxia and hypoxia. After transfections of miR-221 mimics and mimic control (MC), cell proliferation was assessed by cell number, CCk-8 and Ki67 staining. In vivo rats undergoing left coronary artery ligation (MI) and Sham were treated with intravenous miR-221 mimics. Cells or cardiac tissue were collected to measure p27 and PUM1 by RT-qPCR and Western blot (WB). Full length p27 (coding plus 3'UTR sequence) and 3'UTR p27 sequence were quantified using specific primers. The ratio of 3'UTR/Full length was compared to indicate changes between groups. The ratios of 3'UTR/Full length were compared to indicate changes between groups. Results MiR-221 mimics significantly downregulated the expression of p27 mRNA and protein in cFB in both normoxia and hypoxia. P27 downregulation led to cFB proliferation, which could be abrogated by p27 overexpression. miR-221 mimic treatment increased cardiac miR-221 by ∼2–5 fold in Sham and ∼15 fold in MI. Whilst p27 expression was not affected in either Sham or MI hearts. Simultaneously α-SMA+ cells, an indication of cFB number and activation, were reduced by miR-221 mimic in Sham and MI heart. The ratios of p27 3'UTR/full length did not vary significantly between cultured cFB and heart (Sham and MI), indicating the 3'UTR length did not vary between cFB and the whole heart. PUM1 mRNA expression was stable in cFB and the heart. However, phosphorylated PUM1 was highly expressed in cFB but completely diminished in Sham and MI hearts. Conclusion MiR-221 downregulated p27 in primary cFB and induced cell proliferation in vitro. miR-221 could not regulate cardiac p27 expression nor increase cFB proliferation in vivo. MiR-221 induced p27 regulation was correlated with the presence or absence of phosphorylated PUM1. For the first time, we demonstrated that loss of phosphorylated PUM1 turns off the regulation of miRNA-221 on gene p27 in the heart. The mechanism of PUM1 phosphorylation warrant future investigation.

Cyclin-Dependent Kinase Inhibitor 2b Mediates Excitotoxicity-Induced Death of Retinal Ganglion Cells.

Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death. Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs. Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death. Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.

Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on CDKN1B/p27Kip1 3’UTRs in cisplatin treated cells

ABSTRACTPost-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3ʹUTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell’s response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3ʹUTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.

Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential Mechanism.

Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

Downregulated lncRNA-SNHG1 enhances autophagy and prevents cell death through the miR-221/222 /p27/mTOR pathway in Parkinson's disease

Autophagy has been shown to be critically associated with the central mechanisms underlying Parkinson's disease (PD), while the mechanisms contributing to the imbalance of autophagy remain unclear. Small nucleolar RNA host gene 1 (SNHG1), a well-studied long noncoding RNA, has been reported to be significantly increased in PD. The potential biological functions of SNHG1 in the regulation of neuronal autophagy and cell death in PD, however, have not yet been completely elucidated. In this study, we examined the existence of regulatory networks involving SNHG1, the miR-221/222 cluster and the cyclin-dependent kinase inhibitor 1B (CDKN1B/p27)/mammalian target of rapamycin (mTOR) signaling pathway in PD. We observed that SNHG1 expression was gradually upregulated in PD cellular and animal models. Furthermore, silencing SNHG1 promoted autophagy and prevented MPP+-induced cell death, similar to the overexpression of the miR-221/222 cluster. Mechanistically, SNHG1 competitively binds to the miR-221/222 cluster and indirectly regulates the expression of p27/mTOR. In conclusion, these results demonstrated that downregulation of SNHG1 attenuated MPP+-induced decreases in LC3-II (an autophagic marker) levels and cytotoxicity through the miR-221/222/p27/mTOR pathway, suggesting that SNHG1 may be a therapeutic target for neuroprotection and disease treatment in PD.

Gentamicin Targets Acid Sphingomyelinase in Cancer: The Case of the Human Gastric Cancer NCI-N87 Cells.

Emerging literature implicates acid sphingomyelinase in tumor sensitivity/resistance to anticancer treatments. Gentamicin is a drug commonly used as an antimicrobial but its serendipity effects have been shown. Even though many evidences on the role of gentamicin in cancer have been reported, its mechanism of action is poorly understood. Here, we explored acid sphingomyelinase as a possible new target of gentamicin in cancer. Since gastric cancer is one of the most common cancers and represents the second cause of death in the world, we performed the study in NCI-N87 gastric cancer cell line. The effect of the drug resulted in the inhibition of cell proliferation, including a reduction of cell number and viability, in the decrease of MIB-1 proliferative index as well as in the upregulation of cyclin-dependent kinase inhibitor 1A and 1B (CDKN1A and CDKN1B), and growth arrest and DNA-damage 45A (GADD45A) genes. The cytotoxicity was apoptotic as shown by FACS analysis. Additionally, gentamicin reduced HER2 protein, indicating a minor tumor aggressiveness. To further define the involvement of sphingomyelin metabolism in the response to the drug, gene and protein expression of acid and neutral sphingomeylinase was analyzed in comparison with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and vitamin D receptor (VDR), molecules involved in cancer. Gentamicin induced a downregulation of PTEN, VDR, and neutral sphingomyelinase and a strong upregulation of acid sphingomyelinase. Of note, we identified the same upregulation of acid sphingomyelinase upon gentamicin treatment in other cancer cells and not in normal cells. These findings provide new insights into acid sphingomyelinase as therapeutic target, reinforcing studies on the potential role of gentamicin in anticancer therapy.

Fbxw7 and Skp2 Regulate Stem Cell Switch between Quiescence and Mitotic Division in Lung Adenocarcinoma.

Background/Aims The molecular mechanism of dormancy initiation of cancer stem cells (CSCs) is not clear. This study was to explore the molecular mechanism by which CSCs switch from mitotic division to quiescence. Methods MTT assays, flow cytometry, Western blotting, qRT-PCR, and immunofluorescence staining were used to test cell viability, cell cycle and expression of F-box and WD repeat domain-containing 7 (Fbxw7), c-myc, S phase kinase associated protein-2 (Skp2), cyclin-dependent kinase inhibitor 1B (p27), octamer-binding transcription factor 3/4 (Oct3/4), and β catenin gene in 5-fluorouracil (5-FU)-treated A549 cells. Lung adenocarcinoma xenograft models were employed to detect the effects of Fbxw7 on tumor growth. Results 5-FU inhibited the proliferation of A549 cells, with a median inhibitory concentration (IC50) of 200 μg/ml after 24 h treatment. 5-FU treatment increased the expressions of Oct3/4, Fbxw7, and p27 and increased the number of A549 cells at G0/G1. 5-FU treatment triggered nuclear translocation of β-catenin, decreased the expression levels of c-myc and Skp2, and decreased the number of A549 cells at S phase. Release from 5-FU decreased the expressions of Oct3/4, Fbxw7 and p27; decreased the percentage of cells in the G0/G1 phase; increased the expressions of Skp2 and c-myc; and increased the proportion of cells in S phase. 5-FU treatment led to high expressions of Oct3/4, c-myc, and p27, with low expressions of Fbxw7 and Skp2. Knockdown of Fbxw7 augmented the expression of c-myc and decreased the proportion of A549 cells in Go/G1 phase. Skp2 siRNA increased the expression of p27 and the percentage of G0/G1 phase cells and reduced the proportion of S phase cells. Fbxw7 overexpression inhibited tumor growth in mouse lung adenocarcinoma xenograft models. When Fbxw7 expression was low, Skp2 expression was higher in lung adenocarcinoma tissues and associated with the differentiation of lung adenocarcinoma. Conclusion 5-FU enriches the CSCs in lung adenocarcinoma cells via increasing Fbxw7 and decreasing Skp2 expression, followed by downregulation of c-myc and upregulation of p27, which switches cells to quiescence.

CDKN2B upregulation prevents teratoma formation in multipotent fibromodulin-reprogrammed cells.

Tumorigenicity is a well-documented risk to overcome for pluripotent or multipotent cell applications in regenerative medicine. To address the emerging demand for safe cell sources in tissue regeneration, we established a novel, protein-based reprogramming method that does not require genome integration or oncogene activation to yield multipotent fibromodulin (FMOD)-reprogrammed (FReP) cells from dermal fibroblasts. When compared with induced pluripotent stem cells (iPSCs), FReP cells exhibited a superior capability for bone and skeletal muscle regeneration with markedly less tumorigenic risk. Moreover, we showed that the decreased tumorigenicity of FReP cells was directly related to an upregulation of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression during the FMOD reprogramming process. Indeed, sustained suppression of CDKN2B resulted in tumorigenic, pluripotent FReP cells that formed teratomas in vivo that were indistinguishable from iPSC-derived teratomas. These results highlight the pivotal role of CDKN2B in cell fate determination and tumorigenic regulation and reveal an alternative pluripotent/multipotent cell reprogramming strategy that solely uses FMOD protein.

Overexpression of CDKN2B is involved in poor gastric cancer prognosis.

The objective of this investigation is to elucidate the clinical significance of cyclin-dependent kinase inhibitor 2B (CDKN2B) expression regarding gastric cancer (GC), as well as to detect the involvement of CDKN2B expression in the clinicopathological indexes and prognosis of GC. Immunohistochemical analysis was used for identification of CDKN2B expression in GC specimens. Chi-square (χ2 ) test was applied to detect the association of CDKN2B expression and clinicopathological parameters of GC. The involvement of CDKN2B expression in the prognosis was analyzed via univariate and multivariate analysis. It was indicated that relative to the corresponding para-carcinoma tissues, CDKN2B expression was notably upregulated in GC specimens. Moreover, the expression of CDKN2B was strongly correlated with the differentiation (r = -0.182; P = .015), invasion (r = -0.157; P = .038), distant metastases (r = -0.196; P = .004), and TNM stage (r = -0.204; P = .005). Nevertheless, no remarkable variance was related to age, tumor loci, or sex. Kaplan-Meier survival curve and univariate analysis showed that CDKN2B overexpression predicted poorer disease-free survival (P = .007) and overall survival (P = .005) in those with GC. In addition, Cox proportional hazards regression model revealed that CDKN2B was an isolated biomarker of disease-free survival and overall survival in patients with GC. Taken together, our data demonstrated that the overexpression of CDKN2B could be an isolated factor for GC prognostic in patients. CDKN2B gene may be a useful target and new treatment for improving the prognosis of GC.

Abstract 1860: Muscadine grape extract prevents proliferation of trastuzumab-sensitive and -resistant HER2 positive breast cancer cells and has synergistic effects when combined with trastuzumab

Abstract Human epidermal growth factor receptor 2 (HER2) positive breast cancer, characterized by overexpression of the HER2 protein, is an aggressive type of breast cancer comprising ~20% of diagnosed cases. Although targeted HER2 drugs, such as trastuzumab (TRZ), have reduced mortality by about one third, many patients still experience primary or acquired resistance. Grape seed/skin extracts are popular dietary supplements due to their potent antioxidant and anti-inflammatory properties. However, extracts from muscadine grapes are not extensively studied, and information regarding their therapeutic effects on cancer is limited. The objective of this study was to determine whether a muscadine grape skin and seed extract (MGE) inhibits the proliferation of TRZ-sensitive and -resistant HER2 positive breast cancer cells, and determine if combination treatment with MGE and TRZ has synergistic effects. Treatment with increasing concentrations of MGE for 48h significantly inhibited proliferation of TRZ-sensitive SKBR3 and -resistant HCC1954 human HER2 overexpressing breast cancer cells in a time and dose-dependent manner. HER2 heterodimerization and phosphorylation results in activation of the protein kinase B (AKT) pathway, which regulates breast cancer proliferation and cell survival. Treatment with 20 μg/mL MGE for 24h reduced HER2 protein expression by 52% (p≤0.01) in SKBR3 and 60% (p≤0.01) in HCC1954 cells. AKT activation was also reduced by 63% (p≤0.001) and 65% (p≤0.0001) in SKBR3 and HCC1954 cell lines, respectively. Active AKT stabilizes s-phase kinase-associated protein 2 (SKP2), ultimately leading to increased ubiquitination and degradation of cell-cycle inhibitor cyclin-dependent kinase inhibitor 1B (p27) and its transcription factor forkhead box protein 1 (FOXO1). Treatment with 20 μg/mL MGE for 24h increased total FOXO1 and p27 levels by 4-fold (p≤0.01) and 2.5-fold (p≤0.05), respectively, in SKBR3 cells. In addition, FOXO1 was increased 5-fold (p≤0.0001) and p27 4-fold (p≤0.05) in HCC1954 cells. Furthermore, SKP2 protein expression was reduced by 66% (p≤0.01) in SKBR3 and 73% (p≤0.01) in HCC1954 cells. MGE, in combination with TRZ, was synergistic at five different dose combinations (determined using Chou and Talalay method of synergy), and significantly inhibited proliferation to a greater extent than either agent alone in TRZ-sensitive SKBR3 cells. In summary, treatment with MGE reduced proliferation, inhibited components of the AKT pro-proliferation/survival pathway, and had synergistic effects when combined with TRZ in HER2 positive breast cancer cells. Thus, MGE may serve as an effective therapeutic either administered singly or in combination with targeted therapies for the treatment of HER2 positive breast cancer. Citation Format: Jessica Mackert, Patricia E. Gallagher, E. Ann Tallant. Muscadine grape extract prevents proliferation of trastuzumab-sensitive and -resistant HER2 positive breast cancer cells and has synergistic effects when combined with trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1860.

Knockdown of long non-coding RNA PVT1 inhibits the proliferation of Raji cells through cell cycle regulation.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be associated with oncogenesis. However, the functional role of PVT1 in Burkitt lymphoma has not yet been addressed. The purpose of the present study was to investigate the effect of PVT1 knockdown by small interfering RNA (siRNA) on the proliferation of Burkitt lymphoma Raji cells and to explore its possible mechanism of action. An effective siRNA targeting PVT1 was screened and the corresponding short hairpin RNA (shRNA) was reconstructed into a lentiviral vector. Cell proliferation and cell cycle distribution were assessed by Cell Counting kit-8 assay and flow cytometry, respectively. Protein expression levels of c-Myc, cyclin-dependent kinase inhibitor1A (CDKN1A, P21) and cyclin E1 (CCNE1) were detected by western blotting. A polymerase chain reaction (PCR) array was used to analyse the expression of genes associated with the cell cycle. PVT1 knockdown markedly suppressed proliferation, and induced cell cycle arrest at the G0/G1 phase in Raji cells. Protein expression levels of c-Myc and CCNE1 were reduced, whereas P21 protein expression was markedly increased following downregulation of PVT1 in Raji cells. The cell cycle PCR array revealed that 54 genes were upregulated and 26 genes were downregulated in Raji cells following PVT1 knockdown. Reverse transcription-quantitative PCR demonstrated that cyclin G2 (CCNG2), CDKN1A, Retinoblastoma-like 2 (RBL2, p130), HUS1 checkpoint homolog, cyclin dependent kinase inhibitor 3 (CDKN3) and cyclin dependent kinase inhibitor 1B (CDKN1B) expression were upregulated, whereas the expression levels of CCNE1, cyclin D1 (CCND1) and cell division cycle 20 (CDC20) were downregulated in Raji cells with PVT1 knockdown. In conclusion, PVT1 knockdown may inhibit the proliferation of Raji cells by arresting cells in G0/G1 phase. Furthermore, inhibition of cell proliferation may be associated with a reduction inc-Myc expression and alterations in the expression levels of cell cycle-associated genes.

Cyclin-dependent kinase inhibitor 2B gene is associated with the sensitivity of hepatoma cells to Sorafenib.

Purpose: The sensitivity of advanced hepatocellular carcinoma (HCC) to Sorafenib is low. The purpose of this study was to investigate the effects of cyclin-dependent kinase inhibitor 2B (CDKN2B) gene on the prognosis of HCC and the sensitivity of HCC cells to Sorafenib.Patients and methods: Streptavidin-perosidase (SP) staining was performed to determine the expression of CDKN2B in HCC tissues and adjacent tissues. The cell counting kit-8 (CCK-8) assay was carried out to determine cell viability. CDKN2B mRNA and protein were tested by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. CDKN2B gene was silenced or over-expressed in the cells by plasmid transfection technique. Flow cytometry was carried out to detect cell cycle and apoptosis.Results: SP staining results showed that CDKN2B was positive in adjacent tissues and in HCC tissues from partial response (PR) patients, CDKN2B was slightly positive in stable disease (SD) patients, but negative in progression disease (PD) patients. The survival rate of patients with low expression of CDKN2B was low. Up-regulation of CDKN2B expression could promote the pro-apoptotic effect of Sorafenib and cell arrest in G1 phase. When the CDKN2B gene expression was down-regulated, the cell apoptosis rate and the proportion of cells treated with Sorafenib in G1 phase decreased. Silencing CDKN2B reversed CDKN2B overexpression caused by Sorafenib.Conclusion: CDKN2B genes were lowly expressed in tumor tissues from HCC patients who were treated with Sorafenib and had a poor prognosis. Up-regulation of CDKN2B promoted sensitivity of HCC to Sorafenib, and similarly down-regulation of CDKN2B reduced the sensitivity.

Exosomes derived from M1 macrophages aggravate neointimal hyperplasia following carotid artery injuries in mice through miR-222/CDKN1B/CDKN1C pathway

The role of M1 macrophages (M1M)-derived exosomes in the progression of neointimal hyperplasia remains unclear now. Using a transwell co-culture system, we demonstrated that M1M contributed to functional change of vascular smooth muscle cell (VSMC). We further stimulated VSMCs with exosomes isolated from M1M. Our results demonstrated that these exosomes could be taken up by VSMCs through macropinocytosis. Using a microRNA array assay, we identified that miR-222 originated from M1M-derived exosomes triggered the functional changes of VSMCs. In addition, we confirmed that miR-222 played a key role in promoting VSMCs proliferation and migration by targeting Cyclin Dependent Kinase Inhibitor 1B (CDKN1B) and Cyclin Dependent Kinase Inhibitor 1C (CDKN1C) in vitro. In vivo, M1M-derived exosomes significantly aggravated neointima formation following carotid artery ligation injury and wire injury and these effects were partly abolished by miR-222 inhibitor 2′OMe-miR-222. Our findings thus suggest that exosomes derived from M1M could aggravate neointimal hyperplasia through delivering miR-222 into VSMCs. Future studies are warranted to validate if the post-injury vascular neointimal hyperplasia and restenosis could be attenuated by inhibiting miR-222.

Knockdown of NANOG Reduces Cell Proliferation and Induces G0/G1 Cell Cycle Arrest in Human Adipose Stem Cells.

The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG, SOX2 and OCT4 represent the core regulatory network that suppresses differentiation-associated genes, maintaining the pluripotency of mesenchymal stem cells. The roles of NANOG in maintaining self-renewal and undifferentiated status of adult stem cells are still not perfectly established. In this study we define the effects of downregulation of NANOG in maintaining self-renewal and undifferentiated state in mesenchymal stem cells (MSCs) derived from subcutaneous adipose tissue (hASCs). hASCs were expanded and transfected in vitro with short hairpin Lentivirus targeting NANOG. Gene suppressions were achieved at both transcript and proteome levels. The effect of NANOG knockdown on proliferation after 10 passages and on the cell cycle was evaluated by proliferation assay, colony forming unit (CFU), qRT-PCR and cell cycle analysis by flow-cytometry. Moreover, NANOG involvement in differentiation ability was evaluated. We report that downregulation of NANOG revealed a decrease in the proliferation and differentiation rate, inducing cell cycle arrest by increasing p27/CDKN1B (Cyclin-dependent kinase inhibitor 1B) and p21/CDKN1A (Cyclin-dependent kinase inhibitor 1A) through p53 and regulate DLK1/PREF1. Furthermore, NANOG induced downregulation of DNMT1, a major DNA methyltransferase responsible for maintaining methylation status during DNA replication probably involved in cell cycle regulation. Our study confirms that NANOG regulates the complex transcription network of plasticity of the cells, inducing cell cycle arrest and reducing differentiation potential.

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis.

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis.

MAPKAPK2 plays a crucial role in the progression of head and neck squamous cell carcinoma by regulating transcript stability

BackgroundHead and neck squamous-cell carcinoma (HNSCC) ranks sixth among cancers worldwide. Though several molecular mechanisms of tumor initiation and progression of HNSCC are known, others remain unclear. Significance of p38/MAPKAPK2 (Mitogen-activated protein kinase-activated protein kinase-2) pathway in cell stress and inflammation is well established and its role in tumor development is being widely studied.MethodsWe have elucidated the role of MAPKAPK2 (MK2) in HNSCC pathogenesis using clinical tissue samples, MK2-knockdown (MK2KD) cells and heterotropic xenograft mice model.ResultsIn patient-derived tissue samples, we observed that MK2 is reproducibly overexpressed. Increased stability of cyclin-dependent kinase inhibitor 1B (p27), mitogen-activated protein kinase phosphatase-1 (MKP-1) transcripts and decreased half-life of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) transcripts in MK2KD cells suggests that MK2 regulates their transcript stability. In vivo xenograft experiments established that knockdown of MK2 attenuates course of tumor progression in immunocompromised mice.ConclusionAltogether, MK2 is responsible for regulating the transcript stability and is functionally important to modulate HNSCC pathogenesis.

MON-335 Phenocopy of Multiple Endocrine Neoplasia Type 1 (MEN1) Due to a Germline Cell Division Cycle 73 (CDC73) Variant

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid tumors, and neuroendocrine tumors (NETs) of the pituitary and pancreas. MEN1 is caused by germline mutations of the tumor suppressor gene MEN1, which are found in >75% of MEN1 patients. The remaining patients may have mutations involving: as yet unidentified genes; or other genes with known involvement in endocrine tumors, e.g. cyclin dependent kinase inhibitor 1B (CDKN1B) which is associated with MEN4. Here, we report a patient who had primary hyperparathyroidism, acromegaly, and a pancreatic NET that showed immuno-staining for glucagon and chromogranin A, which are consistent with MEN1. However, mutational analysis did not identify a MEN1 mutation, and analysis of other genes was undertaken, after informed consent was obtained from the patient. These genes include: CDKN1B; CDKN1A; CDKN2B; CDKN2C;cell cycle division 73 (CDC73), causative for hyperparathyroidism-jaw tumour (HPT-JT) syndrome, an autosomal dominant disorder characterised by occurrence of parathyroid tumors, ossifying fibromas of the jaw, renal tumours and uterine tumors; calcium sensing receptor (CASR), causative for familial hypocalciuric hypercalcemia type 1 (FHH1); and aryl-hydrocarbon receptor-interacting protein (AIP), causative for familial pituitary tumors. This revealed a heterozygous c.1138C>T (p.Leu380Phe) missense mutation of CDC73. This is likely a disease-causing mutation as: 1) the p.Leu380Phe substitution is not present in >120,000 alleles of The Exome Aggregation Consortium (ExAc) database; 2) involves an evolutionary conserved Leu380 residue; and 3) is situated within the parafibromin domain predicted to interact with the polymerase associated factor 1 (PAF1) and RNA polymerase II, (POLR2A) complex. In addition, RNA-Scope analysis, to detect specific CDC73 mRNA transcripts in paraffin embedded sections, of the patient’s pancreatic NET revealed that CDC73 mRNA expression was significantly decreased by >13% (p<0.005) in the tumor cells, compared to normal adjacent islets. Thus, our results suggest the spectrum of tumours associated with CDC73 mutations also includes pancreatic NETs, and demonstrate that CDC73 mutations may result in a phenocopy of MEN1.

A transcriptomic signature of tertiary Gleason 5 predicts worse clinicopathological outcome.

To investigate the genomic features of tertiary pattern 5 (TP5) on radical prostatectomy specimens in an effort to explain the poor clinical outcomes associated with this disease subtype. Data from 159 men with Gleason Grade Group (GGG) 3 or 4 were considered. All patients had Decipher diagnostic testing with transcript profiles and single-channel array normalisation (SCAN)-normalised expression of coding genes. The relationship between Decipher and TP5 was investigated by linear and binary logistic regressions. A differential transcriptomic analysis between patients with and without TP5 was performed. The prognostic role of these genes on progression-free survival (PFS) and overall survival (OS) was evaluated using The Cancer Genome Atlas. In all, 52/159 (33%) patients had GGG 3-4 with TP5 disease. TP5 was associated with a higher Decipher score (β 0.07, 95% confidence interval [CI] 0.02-0.13; P=0.04) and higher likelihood of falling within the intermediate- or high-risk categories (odds ratio 3.34, 95% CI 1.34-8.35; P=0.01). Analysis of microarray data revealed an 18-gene signature that was differentially expressed in patients with TP5; 13 genes were over- and five under-expressed in the TP5 cohort. The overexpression of cyclin dependent kinase inhibitor 2B (CDKN2B), polo-like kinase 1 (PLK1), or cell division cycle 20 (CDC20) was associated with worse PFS. The group harbouring overexpression of at least one gene had a 5-year PFS rate of 50% vs 74% in the group without overexpression (P<0.001). Our studies have elucidated unique genomic features of TP5, whilst confirming previous clinical findings that patients harbouring TP5 tend to have worse prognosis. This is the first RNA-based study to investigate the molecular diversity of TP5 and the first correlating CDKN2B to poorer prognosis in patients with prostate cancer.

CDCA8 is a key mediator of estrogen-stimulated cell proliferation in breast cancer cells

Endocrine therapy is effective in the early stage of breast cancer treatment, and most tumor cells will gain the ability to proliferate under residual amounts of estrogen, which will cause the recurrence of the disease. The role of cell division cycle associated 8 (CDCA8) in Estradiol (E2)-stimulated breast cancer cells growth is investigated in this research. CDCA8 showed higher mRNA expression in E2-stimulated MCF7 and T47D cells, and such an increase could also be observed in tumor samples. CDCA8 shRNA inhibited the survival and growth detected by cell number and colony formation, while promoted cell cycle G1 phase arrest determined with flow cytometry, which coordinated with a decrease in E2-induced molecules, namely Cyclin D1 (CCND1), B-Cell CLL/Lymphoma 2 (BCL2), and an increase in apoptosis-related molecules, such as cyclin-dependent kinase inhibitor 1a (P21) and cyclin-dependent kinase inhibitor 1b (P27). Kaplan-Meier plot analysis indicated that higher CDCA8 expression was positively associated with poor prognosis with a probability lower than 0.4 at the five-year interval (p = 0.035). All of these suggest that CDCA8 is a key mediator of estrogen-stimulated breast cancer cell growth and survival, which can be utilized as a novel target in breast cancer treatment.