NBR2 promotes the proliferation of glioma cells via inhibiting p15 expression.

PURPOSE: The purpose of this study was to investigate whether NBR2 can affect the proliferation of glioma cells by inhibiting the expression of p15, so as to promote the occurrence and development of glioma. METHODS: The expression of NBR2 in 44 glioma tissue specimens was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of NBR2 on cell viability, cell colony formation as well as cell migration and invasion abilities were examined by cell counting kit-8 (CCK-8) assay, plate cloning assay and Transwell assay. p15 protein was detected using Western blot. After simultaneous knockdown of NBR2 and p15, qRT-PCR, CCK-8, and plate cloning experiments were adopted to analyze p15 gene level, cell viability and proliferation ability, respectively. RESULTS: NBR2 was highly expressed in glioma tissues, and the level in stage III/IV glioma tissues was conspicuously higher than that in stage I/II. The overall survival rate of glioma patients with high NBR2 level was conspicuously lower than those with low NBR2 expression. Clinical data analysis revealed that NBR2 expression was correlated with the WHO stage of clinical patients. After knockdown of NBR2, it was found that NBR2 level, cell viability, cell proliferation ability as well as migration and invasion abilities were all conspicuously reduced. In addition, the protein level of p15 was significantly increased after NBR2 was inhibited. Meanwhile, knockout of p15 could reverse the inhibitory effect of NBR2 on glioma cell proliferation. CONCLUSIONS: The highly-expressed NBR2 inhibits the expression of p15, thus promoting the proliferation of glioma cells.

Long noncoding RNA HOTAIR promotes the self-renewal of leukemia stem cells through epigenetic silencing of p15.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder initiated from a small subset of leukemia stem cell (LSC), which presents unrestricted self-renewal and proliferation. Long non-coding RNA HOTAIR is abundantly expressed and plays oncogenic roles in solid cancer and AML. However, whether HOTAIR regulates the self-renewal of LSC is largely unknown. Here, we reported that the expression of HOTAIR was increased in LSC than in normal hematological stem and progenitor cells (HSPCs). HOTAIR inhibition by short hairpin RNAs (shRNAs) decreased colony formation in leukemia cell lines and primary AML blasts. We then investigated the role of HOTAIR in leukemia in vivo. HOTAIR knockdown extends the survival time in U937-transplanted NSG mice. Furthermore, HOTAIR knockdown reduced infiltration of leukemic blasts, decreased frequency of LSC, and prolonged overall survival in MLL-AF9-induced murine leukemia, suggesting that HOTAIR is required for the maintenance of AML. Mechanistically, HOTAIR inhibited p15 expression through zeste homolog 2 (EZH2)-enrolled tri-methylation of Lys 27 of histone H3 (H3K27me3) in p15 promoter. In addition, p15 partially reversed the decrease of colony and proliferation induced by HOTAIR knockdown, suggesting that p15 plays an important role in the leukemogenesis by HOTAIR. In conclusion, our study suggests that HOTAIR facilitates leukemogenesis by enhancing self-renewal of LSC. HOTAIR might be a potential target for anti-LSC therapy.

Prognostic impact of p15 gene aberrations in acute leukemia.

The p15 gene (also known as CDKN2B, INK4B, p15INK4B), located in band 9p21, encodes a protein that induces a G1-phase cell cycle arrest through inhibition of CDK4/6 (cyclin-dependent kinase 4/6). It also plays an important role in the regulation of cellular commitment of hematopoietic progenitor cells and myeloid cell differentiation. p15 can be silenced by several mechanisms, including deletion and hypermethylation of its promoter. Homozygous p15 deletion is rare in acute myeloblastic leukemia (AML) and myelodysplastic syndromes (MDS) but frequent in acute lymphoblastic leukemia (ALL). On the contrary, methylation of the p15 promoter is identified in some 50% of the patients with AML and MDS, but is less frequent in ALL. The analysis of the 28 studies available in the literature revealed conflicting results (unfavorable, favorable or no impact) that can be due, at least in part, to methodological and/or biological pitfalls. Among those, are the heterogeneity of the methylation patterns of the p15 gene and the lack of a comprehensive analysis including transcriptional and translational inactivation that have major impact on its expression. Therefore, detection of the p15 mRNA expression (quantitative or not) may represent a more appropriate method to determine the prognostic impact of the p15 gene.

Long intergenic non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer.

Long noncoding RNAs (lncRNAs) play important regulatory roles in several human cancers. Integrated analysis revealed that expression of long intergenic non-coding RNA 152 (LINC00152) was significantly upregulated in gastric cancer (GC). Further analysis in a cohort of 97 GC patients revealed that LINC00152 expression was positively correlated with tumor invasion depth, lymph node metastasis, higher TNM stage, and poor survival. Gene set enrichment analysis revealed that cell proliferation and cell cycle progression were increased in patients with high LINC00152 expression. In both GC cell lines and xenograft systems, LINC00152 overexpression facilitated GC cell proliferation by accelerating the cell cycle, whereas LINC00152 knockdown had the opposite effect. Moreover, by binding to enhancer of zeste homolog 2 (EZH2), LINC00152 promotes GC tumor cell cycle progression by silencing the expression of p15 and p21. These findings suggest that LINC00152 may play contribute to the progression of GC and may be an effective therapeutic target.

ANRIL inhibits p15(INK4b) through the TGFß1 signaling pathway in human esophageal squamous cell carcinoma.

The INK4b-ARF-INK4a gene cluster encodes three tumor suppressors: p15(INK4b), p14(ARF), and p16(INK4a). Antisense non-coding RNA in the INK4 locus (ANRIL) is transcribed in the opposite direction from this gene cluster. Recent studies suggest that ANRIL represses the expression of p15(INK4b), p14(ARF), and p16(INK4a); however, the underlying mechanism is unclear. In this study, the expressions of ANRIL in human esophageal squamous cell carcinoma (ESCC) tissues and matched adjacent non-tumor tissues were examined by quantitative real-time polymerase chain reaction. Compared with matched adjacent non-tumor tissues, the expression levels of ANRIL in ESCC tissues were significantly increased. Furthermore, inhibition of ANRIL was found to increase the expression of p15(INK4b) and transforming growth factor ß1 (TGFß1) and depletion of ANRIL in ESCC cell lines may inhibit cellular proliferation. Thus, our findings suggest a significant role of ANRIL in the occurrence and development of ESCC through TGFß1 signaling pathways.

Silence of p15 expression by RNAi enhances cisplatin resistance in hepatocellular carcinoma cells.

The insensitivity of hepatocellular carcinoma to chemotherapy is associated with alternation in tumor cell cycling. This current study was designed to investigate the impact of p15 silencing on the sensitivity of Human hepatocellular carcinoma HepG2 cells to cisplatin. HepG2/CDDP/1.6 and HepG2/CDDP/2.0 cells were induced by culture with increased doses of cisplatin and their sensitivities to cis-Diamine dichloroplatinum (CDDP) were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The impacts of p15 silencing on the cell cycling and P-gp expression were characterized by flow cytometry, RT-PCR and Western blot assays, respectively. Knockdown of p15 expression dramatically reduced the relative levels of p15 expression and the frequency of phase G1, promoting cell cycling. On the other hand, knockdown of p15 expression significantly up-regulated the expression of P-glycoprotein (P-gp) in HepG2/CDDP/2.0 cells, associated with the increased resistance of HepG2 cells to CDDP in vitro. In conclusion, the p15 may be a critical regulator of the development of CDDP resistance in HepG2 cells.

Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1.

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (DeltaNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which DeltaNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of DeltaNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, DeltaNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G(0)/G(1) arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in DeltaNLS-PTH-related protein VSMC. More importantly, p15(INK4) and p27(kip1), two critical inhibitors of the G(1/S) progression, were markedly increased. Normalization of both p15(INK4b) and p27(kip1) by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15(INK4b) and p27(kip1) fully account for the marked cell cycle slowing induced by DeltaNLS-PTH-related protein in VSMCs. Finally, DeltaNLS-PTH-related protein is able to induce p15(INK4) and p27(kip1) expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of DeltaNLS-PTH-related protein actions, and suggest that DeltaNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis.

Methylation-independent silencing of the tumor suppressor INK4b (p15) by CBFbeta-SMMHC in acute myelogenous leukemia with inv(16).

The tumor suppressor gene INK4b (p15) is silenced by CpG island hypermethylation in most acute myelogenous leukemias (AML), and this epigenetic phenomenon can be reversed by treatment with hypomethylating agents. Thus far, it was not investigated whether INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21), and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared with the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable with that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded core binding factor beta-smooth muscle myosin heavy chain (CBFbeta-SMMHC). The silencing was manifested in an inability to activate the normal expression of INK4b RNA as shown in vitamin D3-treated U937 cells expressing CBFbeta-SMMHC. CBFbeta-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of INK4b. Importantly, this study (a) establishes that the gene encoding the tumor suppressor p15(INK4b) is a target of CBFbeta-SMMHC, a finding relevant to the leukemogenesis process, and (b) indicates that, in patients with inv(16)-containing AML, reexpression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drugs.

Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

PURPOSE: To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA). METHODS: Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay. RESULTS: TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus. CONCLUSIONS: These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

Histone deacetylase 3 represses p15(INK4b) and p21(WAF1/cip1) transcription by interacting with Sp1.

Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the overexpression of HDAC3 repressed transcription of p15(INK4b) and p21(WAF1/cip1) genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunoprecipitated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15(INK4b) and p21(WAF1/cip1) transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15(INK4b), but not that of p21(WAF1/cip1), implicating the different roles of HDAC3 in repression of p15(INK4b) and p21(WAF1/cip1) transcription. Data from this study indicate that the inhibition of p15(INK4b) and p21(WAF1/cip1) may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis.