Histone H1.2 Represses the Transcription of the p16 Tumor Suppressor Gene.

BACKGROUND/AIM: CDK inhibitor p16 plays a pivotal role in the induction of cellular senescence and functions as a tumor suppressor. Here, we demonstrate that histone H1.2 is involved in p16 repression. MATERIALS AND METHODS: Cells were transfected with siRNAs and subjected to quantitative reverse transcription-polymerase chain reaction, immunoblotting and chromatin immunoprecipitation (ChIP) assay. RESULTS: The decrease in H1.2 by oncogenic RAS was associated with increased levels of p16. Depletion of H1.2 selectively increased p16, but not alternative reading frame (ARF) mRNA. ChIP assay showed that H1.2 directly bound to the p16 promoter. Interestingly, silencing YB-1, a component of H1.2 complex, decreased the expression levels of H1.2, resulting in decreased binding of H1.2 on the p16 promoter. CONCLUSION: These results provide a model in which H1.2 is positively regulated by YB-1 and directly binds to and represses the transcription of p16.

Elimination of Off-Target Effect by Chemical Modification of 5'-End of siRNA.

In this study, the efficiency of RNA interference of small interfering RNAs (siRNAs) bearing 5'-O-methyl-2'-deoxythymidine (X) and 5'-amino-2', 5'-dideoxythymidine (Z) at the 5'-end of the sense strand and the antisense strand of siRNA was investigated in HeLa cells stably expressing enhanced green fluorescent protein. The results indicated that when one strand of siRNA was modified with X or Z and the other was unmodified, the X or Z modification was predominant in the process of strand selection and the unmodified strand was selected as a guide strand. When both strands are modified with X or Z, the modified antisense strand with X or Z will be selected as a guide strand with a certain probability. The resulting mature RNA-induced silencing complex exerted reduced, but still moderate silencing activity remained. These results suggest that the modification of the sense strand with X or Z eliminates the off-target effects caused by the sense strand without affecting the silencing efficiency of the siRNA.

OIP5-AS1 Promotes Proliferation of Non-small-cell Lung Cancer and Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: The long noncoding RNA OIP5 antisense RNA 1 (OIP5-AS1) is overexpressed in various cancer types, such as lung cancer, hepatoblastoma and cervical cancer, and functions to accelerate cell proliferation, invasion and migration. Here, we investigated the roIe of OIP5-AS1 in cell-cycle progression of H1299 and A549 non-small cell lung cancer cells, and FaDu and CAL27 head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: The cells were transfected with small interfering RNA and subjected to cell-cycle analysis and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: Silencing of OIP5-AS1 suppressed the proliferation of H1299, A549, FaDu and CAL27 cells. RT-qPCR and cell-cycle analysis revealed that silencing OIP5-AS1 increased the expression of CDK inhibitors, such as p15, p16, p18 and p19, resulting in G1-phase arrest. CONCLUSION: OIP5-AS1 regulates G1-phase progression by repressing CDK inhibitors and, thus, promotes the proliferation of H1299, A549, FaDu and CAL27 cells.

Long Noncoding RNA ANROC on the INK4 Locus Functions to Suppress Cell Proliferation.

BACKGROUND/AIM: The INK4 locus encodes three important genes p15INK4B, p16INK4A, and ARF, which function to suppress oncogenesis, and a long noncoding RNA, ANRIL, which, in contrast, functions to promote oncogenesis. Herein, we report a fifth genetic element on the INK4 locus, a long noncoding RNA with unknown function named associated negative regulation of cell proliferation (ANROC), which played a role in the suppression of cell proliferation. MATERIALS AND METHODS: Following ANROC silencing in cells by siRNA, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell cycle analysis using flow cytometry were performed. RESULTS: ANROC expression was decreased by oncogenic RAS signalling. ANROC knockdown enhanced HeLa cell proliferation and induced cyclin B1 mRNA, which promotes G2/M progression of the cell cycle. Furthermore, flow cytometric analysis revealed that ANROC knockdown increased the percentage of cells in the S and G2/M phases of the cell cycle. CONCLUSION: ANROC functions to suppress cell cycle progression by suppressing cyclin B1 expression, thus inhibiting cell proliferation.

Telomerase inhibition, telomere attrition and proliferation arrest of cancer cells induced by phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT.

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 µM concentration but less efficiently at 1 µM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells.

Long Noncoding RNA, ANRIL, Regulates the Proliferation of Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: ANRIL is a long noncoding RNA located on INK4 locus, which encodes p15 and p16 that cause G1 phase arrest in the cell cycle. ANRIL positively regulates proliferation of several kinds of cancer cells such as lung and gastric cancers. This study, examined the effect of ANRIL in head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: Cells were transfected with siRNA oligonucleotides targeting ANRIL. Transfected cells were subjected to cell-cycle and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. RESULTS: Depletion of ANRIL increased p15 mRNA in FaDu cells, and p15 and p16 mRNA in CAL27 cells and inhibited proliferation of these cells. Cell cycle analysis showed that depletion of ANRIL caused arrest at the G1 phase of the cell cycle. CONCLUSION: ANRIL promotes G1 phase progression by repressing p15 and p16, and thus promotes FaDu and CAL27 cell proliferation.

A novel compound, ferulic acid-bound resveratrol, induces the tumor suppressor gene p15 and inhibits the three-dimensional proliferation of colorectal cancer cells.

Resveratrol, a phytoalexin present in grapes and other edible foods, has been reported to have beneficial effects against various diseases including cancer. We previously reported that resveratrol and its derivative, caffeic acid-adducted resveratrol, selectively inhibit the three-dimensional (3D) proliferation of a human colorectal cancer cell line, HCT116 with activating KRAS mutation. Herein, we demonstrated that a novel compound, ferulic acid-bound resveratrol, also represses the 3D proliferation of HCT116 cells. We observed that resveratrol conjugated to two ferulic acids represses the 3D proliferation of HCT116 cells more strongly than resveratrol and resveratrol conjugated to one ferulic acid. Resveratrol conjugated to two ferulic acids also inhibited the 3D proliferation of MCF7 human breast cancer cells. We further uncovered that the resveratrol derivative increases the mRNA level of the tumor suppressor p15, a CDK inhibitor that functions as a brake of cell proliferation in HCT116 cells. These results imply that the resveratrol derivative represses 3D proliferation via increasing p15 expression in HCT116 cells.

A novel resveratrol derivative selectively inhibits the proliferation of colorectal cancer cells with KRAS mutation.

Resveratrol is a polyphenolic compound in many edible foods including grapes, peanuts, and berries. Several studies have revealed the beneficial effects of resveratrol against various diseases such as heart disease, diabetes, obesity, neurological disorders, and cancer. A recent study showed that resveratrol inhibits the proliferation of HCT116 human colorectal cancer cells in three-dimensional culture (3DC) via induction of luminal apoptosis in HCT116 cell spheroids. In this study, we showed that a novel compound, caffeic acid-adducted resveratrol, has a stronger inhibitory effect on the growth of HCT116 cell spheroids in 3DC than resveratrol. It showed almost the same inhibitory efficacy as 5-fluorouracil, a conventional anticancer drug. We further showed that the resveratrol derivative did not affect the growth of HKe3 cell spheroids derived from HCT116 cells by disruption of the activating mutant KRAS gene. These results suggest that the resveratrol derivative inhibits the growth of HCT116 cell spheroids via inhibition of an oncogenic KRAS-mediated signaling pathway.

The Long Noncoding RNA OIP5-AS1 Is Involved in the Regulation of Cell Proliferation.

BACKGROUND/AIM: OPA-interacting protein 5 antisense transcript 1 (OIP5-AS1) is a long noncoding RNA located on human chromosome 15q15.1 and transcribed in the opposite direction to OIP5. Here, we report that OIP5-AS1 is involved in regulating cell proliferation. MATERIALS AND METHODS: HeLa cells were transfected with OIP5-AS1-targeting siRNA oligonucleotides and anti-sense oligonucleotides. The cells were harvested 72 h after transfection and subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and cell-cycle and apoptosis analysis. RESULTS: OIP5-AS1 was expressed at a lower level in cells harbouring an oncogenic kirsten rat sarcoma viral oncogene homolog (K-RAS) mutation than in cells expressing wild-type K-RAS. Silencing OIP5-AS1 with siRNA oligonucleotides or anti-sense oligonucleotides reduced HeLa cell proliferation. Apoptosis and cell-cycle analysis showed that silencing OIP5-AS1 did not cause apoptosis, but did cause G2/M phase cell-cycle arrest. CONCLUSION: These results suggest that OIP5-AS1 positively regulates cell proliferation by promoting G2/M phase progression.

Homeobox Transcription Factor NKX2-1 Promotes Cyclin D1 Transcription in Lung Adenocarcinomas.

The known oncogene cyclin D1 (CCND1) participates in progression of the cell cycle from G1 to S-phase. Expression of cyclin D1 is frequently promoted in multiple human cancers including non-small cell lung cancer (NSCLC). However, a relationship between cyclin D1 expression and the prognosis of NSCLC has not been confirmed. NKX2-1 is a homeobox transcription factor involved in pulmonary development as a differentiation-promoting factor. In NSCLC, it acts as a metastasis suppressor and correlates with a good prognosis. Here, NKX2-1-binding motifs were identified in the cyclin D1 promoter, but it has not been clarified whether NKX2-1 is involved in cyclin D1 expression in NSCLC. To shed light on this issue, endogenous NKX2-1 was depleted in NSCLC cell lines, which resulted in decreased cyclin D1 mRNA and protein. In contrast, forced overexpression of NKX2-1 increased cyclin D1 levels. Moreover, NKX2-1 directly bound to the cyclin D1 promoter and enhanced its activity. Finally, using human NSCLC clinical specimens, it was determined that both NKX2-1 protein and mRNA were significantly correlated with cyclin D1 expression status in adenocarcinomas. These results indicate that NKX2-1 directly and positively regulates transcription of cyclin D1 Finally, expression of NKX2-1, but not cyclin D1, was significantly associated with metastatic incidence as an independent good prognostic factor of adenocarcinoma.Implications: NKX2-1-expressing adenocarcinomas, whereas NKX2-1 promoted cyclin D1 expression, may show good prognosis features by the metastasis inhibition potency of NKX2-1 regardless cyclin D1 expression. Mol Cancer Res; 15(10); 1388-97. ©2017 AACR.

Y-box Binding Protein 1 Is Involved in Regulating the G2/M Phase of the Cell Cycle.

BACKGROUND/AIM: The transcription factor Y-box-binding protein 1 (YB1) is overexpressed in many types of human cancers. YB1 regulates the G1 phase of the cell cycle by controlling transcription of G1 regulators. Here, we report that YB1 is also involved in regulating G2/M phase. MATERIALS AND METHODS: YB1-depleted TKO cells were subjected to quantitative reverse transcription-polymerase chain reaction and cell-cycle analysis. RNA immunoprecipitation (RIP)-chip assay was performed using anti-YB1 antibodies. Precipitated RNAs were subjected to microarray analysis. RESULTS: Silencing YB1 inhibited the proliferation of TKO cells, which lost the machinery required for G1 phase arrest. Cell-cycle analysis showed that silencing YB1 caused G2/M phase cell-cycle arrest. RIP-chip assay showed that YB1 associated with mRNA of multiple cell-cycle-related genes, including G2/M phase regulators. CONCLUSION: YB1 positively regulates not only the G1 phase but also G2/M phase by regulating multiple cell-cycle-related genes.

Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideß7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideß7 is a promising candidate for in vivo use and therapeutic applications.

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

BACKGROUND: A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. MATERIALS AND METHODS: U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. RESULTS: PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G1 phase arrest. CONCLUSION: These results suggest that PANDA promotes G1-S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation.

Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

BACKGROUND: P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. MATERIALS AND METHODS: U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. RESULTS: Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. CONCLUSION: These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53.

Oncogenic Ras influences the expression of multiple lncRNAs.

Recent ultrahigh-density tiling array and large-scale transcriptome analysis have revealed that large numbers of long non-coding RNAs (lncRNAs) are transcribed in mammals. Several lncRNAs have been implicated in transcriptional regulation, organization of nuclear structure, and post-transcriptional processing. However, the regulation of expression of lncRNAs is less well understood. Here, we show that the exogenous and endogenous expression of an oncogenic form of small GTPase Ras (called oncogenic Ras) decrease the expression of lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), which is involved in the regulation of cellular senescence. We also show that forced expression of oncogenic Ras increases the expression of lncRNA PANDA (p21 associated ncRNA DNA damage activated), which is involved in the regulation of apoptosis. Microarray analysis demonstrated that expression of multiple lncRNAs fluctuated by forced expression of oncogenic Ras. These findings indicate that oncogenic Ras regulates the expression of a large number of lncRNAs including functional lncRNAs, such as ANRIL and PANDA.

ANRIL regulates the proliferation of human colorectal cancer cells in both two- and three-dimensional culture.

ANRIL is a long noncoding RNA transcribed from the INK4 locus that encodes three tumor suppressor genes, p15, p16, and ARF. Previous studies demonstrated that ANRIL represses p15 and p16, which positively regulate the pRB pathway, leading to repression of cellular senescence of human normal fibroblasts. However, the role of ANRIL in cancer cell proliferation is less well understood. Here we report that ANRIL is involved in the proliferation of colorectal cancer HCT116 cells in two- and three-dimensional culture. Silencing ANRIL by both transfection with small interfering RNA and retrovirally produced small hairpin RNA reduced HCT116 cell proliferation in both two- and three-dimensional culture. HCT116 cells depleted for ANRIL were arrested in the S phase of cell cycle. Notably, silencing ANRIL did not result in the activation of expression of the INK4 locus. These results suggest that ANRIL positively regulates the proliferation of HCT116 cells in two- and three-dimensional culture in a p15/p16-pRB pathway-independent manner.

Long Noncoding RNA ANRIL Regulates Proliferation of Non-small Cell Lung Cancer and Cervical Cancer Cells.

BACKGROUND: Long noncoding RNA ANRIL (antisense non-coding RNA in the INK4 locus) represses p15 and p16, which induce cell-cycle arrest at G1 phase, leading to enhanced cell proliferation of normal fibroblasts. Herein we report that ANRIL is also involved in the regulation of cancer-cell proliferation. MATERIALS AND METHODS: HeLa and H1299 cells were transfected with ANRIL siRNAs. At 72 h post-transfection, cells were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell-cycle analysis. RESULTS: qRT-PCR showed that ANRIL is highly expressed in these cancer cells compared to normal fibroblasts. Depletion of ANRIL increased p15 expression, with no impact on p16 or ARF (alternative reading frame) expression, and caused cell-cycle arrest at the G2/M phase, leading to inhibition of proliferation of H1299 and HeLa cells. CONCLUSION: ANRIL positively regulates the proliferation of cancer cells, such as H1299 and HeLa cells, via regulating p15 and other genes related to G2/M phase control.

Transcriptional Regulation of the p16 Tumor Suppressor Gene.

The p16 tumor suppressor gene encodes a specific inhibitor of cyclin-dependent kinase (CDK) 4 and 6 and is found altered in a wide range of human cancers. p16 plays a pivotal role in tumor suppressor networks through inducing cellular senescence that acts as a barrier to cellular transformation by oncogenic signals. p16 protein is relatively stable and its expression is primary regulated by transcriptional control. Polycomb group (PcG) proteins associate with the p16 locus in a long non-coding RNA, ANRIL-dependent manner, leading to repression of p16 transcription. YB1, a transcription factor, also represses the p16 transcription through direct association with its promoter region. Conversely, the transcription factors Ets1/2 and histone H3K4 methyltransferase MLL1 directly bind to the p16 locus and mediate p16 induction during replicative and premature senescence. In the present review, we discuss the molecular mechanisms by which these factors regulate p16 transcription.

YB-1 promotes transcription of cyclin D1 in human non-small-cell lung cancers.

Cyclin D1, an oncogenic G1 cyclin, and YB-1, a transcription factor involved in cell growth, are both over-expressed in several human cancers. In human lung cancer, the functional association between YB-1 and cyclin D1 has never been elucidated. In this study, we show YB-1 is involved in the transcription of cyclin D1 in human lung cancer. Depletion of endogenous YB-1 by siRNA inhibited progression of G1 phase and down-regulated both the protein and mRNA levels of cyclin D1 in human lung cancer cells. Forced over-expression of YB-1 with a cyclin D1 reporter plasmid increased luciferase activity, and ChIP assay results showed YB-1 bound to the cyclin D1 promoter. Moreover, the amount of YB-1 mRNA positively correlated with cyclin D1 mRNA levels in clinical non-small-cell lung cancer (NSCLC) specimens. Immunohistochemical analysis also indicated YB-1 expression correlated with cyclin D1 expression in NSCLC specimens. In addition, most of the cases expressing both cyclin D1 and CDC6, another molecule controlled by YB-1, had co-existing YB-1 over-expression. Together, our results suggest that aberrant expression of both cyclin D1 and CDC6 by YB-1 over-expression may collaboratively participate in lung carcinogenesis.