Abstract
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.
INK4 locus (inhibitor of cyclin dependent kinase 4 locus) is located in the human chromosome 9p21 region. This locus encodes p15, p16, and ARF genes, products of which function as tumor suppressors. Both p15 and p16 are CDK inhibitors that cause G1 phase arrest in the cell cycle via inhibition of the CDK4 and CDK6 kinase activities (1). ARF functions to stabilize tumor suppressor p53 at the protein level by inhibiting the ubiquitin ligase activity of MDM2 (2, 3). This locus is frequently either mutated, deleted, or its transcription is repressed in many types of human cancers (4). The transcriptional activation of this locus is therefore thought to be important in tumor suppression.
Long noncoding RNAs (lncRNAs) are more than 200 nucleotides long and lack a functional open reading frame. Recent studies have revealed that many lncRNAs participate in the regulation of differentiation, growth, senescence, and apoptosis (5). An antisense noncoding RNA transcribed from the INK4 locus (ANRIL) is the lncRNA (6). Along with Yap et al., we revealed that ANRIL associates with polycomb repression complex 1 (PRC1) and 2 (PRC2) and recruits them on the INK4 locus to repress the transcription of p15 and p16 genes (7, 8). In the gene silencing model of polycomb protein complexes, PRC2 methylates histone H3 at lysine 27 on the target region, leading to the recruitment of PRC1, which ubiquitinates histone H2A at lysine 119 (9-11). These histone modifications establish and maintain the epigenetic repression of target genes. Silencing ANRIL results in the induction of p15 and p16 transcription, thus inhibiting cell proliferation (7, 8). Other than p15 and p16, ANRIL is involved in the transcriptional regulation of many genes related to cell adhesion, gene expression and apoptosis (12-14), indicating that ANRIL participates in the regulation of diverse cellular functions.
Expression analysis showed that ANRIL is expressed at higher levels in several human cancer cells, including human cervical cancer, osteosarcoma and non-small cell lung cancer than normal lung fibroblasts (15). ANRIL regulates cancer cell proliferation in an INK4 locus-dependent (15) and – independent manner (16). Many studies on clinical cancer specimens have shown that the expression of ANRIL is up-regulated in various human cancers such as lung, gastric, ovarian and cervical cancers (17). It has also been reported very recently that ANRIL expression is up-regulated in oral cancer and that ANRIL participates in the proliferation, migration and invasion of oral squamous cell carcinoma cells (18). However, the role of ANRIL in head and neck squamous cell carcinoma is largely unknown. In this study, we examined the function and mechanism of action of ANRIL in head and neck squamous cell carcinoma cells.
Materials and Methods
Cell culture and RNA interference. A human hypopharyngeal cancer cell line, FaDu and a human oral cancer cell line, CAL27 (American Type Culture Collection, Frederick, MD, USA), were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in an atmosphere containing 5% CO2. For ANRIL silencing, siRNA oligonucleotides targeting ANRIL were transfected by using Lipofectamine RNAiMAX according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). The nucleotide sequence of the ANRIL siRNA oligonucleotide was 5’-GGUCAUCUCAUUGCUCUAU-3’ with 3’ dTdT overhangs.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Seventy-two h after transfection of siRNA oligonucleotides, cells were subjected to qRT-PCR. qRT-PCR was performed as previously described (19). Briefly, the isolation of total RNA was performed using RNeasy Plus kit (Qiagen, Hilden, Germany). cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) and amplified using QuantiTect SYBR Green PCR kit (Qiagen). The specific primer set for ANRIL was 5’-CTATCCGCCAATCAGGAGGC-3’ and 5’-GCGGCAGCGGTTTAGTTTAAT -3’. The specific primer sets for p15, p16, ARF and GAPDH have been previously described (15).
Cell cycle analysis. Cell cycle analysis was performed as previously described (20). Briefly, cells were fixed for at least 3 h at −20°C using 70% ethanol about 72 h after transfecting siRNA oligonucleotides. The fixed cells were processed with Muse™ Cell Cycle Kit (Merck Millipore, Darmstadt, Germany) according to the instructions of the manufacturer and subjected to cell cycle analysis using Muse Cell Analyzer (Merck Millipore).
Statistical analysis. Data are presented as means and standard deviations from triplicate samples. A two-tailed t-test was performed to determine significant differences. A p-values of <0.05 was considered to be statistically significant.
Results
We had previously reported that ANRIL is involved in the repression of cellular senescence in normal lung fibroblasts (8). Recently, several other research groups, including ours, reported that ANRIL functions to promote the proliferation of cancer cells, such as colorectal cancer, lung cancer, cervical cancer, gastric cancer, hepatocellular carcinoma and bladder cancer cells (17). Accordingly, we first examined the involvement of ANRIL in head and neck squamous cell carcinoma cell proliferation. The expression of ANRIL was downregulated using siRNA oligonucleotides that target ANRIL in a human hypopharyngeal cancer cell line, FaDu, and a human oral cancer cell line, CAL27. Depletion of ANRIL significantly repressed the proliferation of both FaDu (Figure 1A and B) and CAL27 (Figure 1C and D) cells, suggesting that ANRIL functions to promote the proliferation of both FaDu and CAL27 cells.
We (8), along with Yap et al. (7) have previously shown that ANRIL inhibits p15 and p16 expression, thus resulting in the promotion of cell proliferation. Accordingly, we examined the involvement of ANRIL in p15 and p16 expression in FaDu and CAL27 cells. Data obtained from qRT-PCR demonstrated that transfection with siRNA oligonucleotides significantly reduced ANRIL expression (Figure 2A and B) in both FaDu and CAL27 cells. Depletion of ANRIL resulted in an increase in p15 mRNA expression in FaDu cells (p16 was not detected) (Figure 2A). Interestingly, silencing ANRIL also increased the expression levels of ARF mRNA that is transcribed from the INK4 locus and functions to stabilize the p53 tumor suppressor protein. Depletion of ANRIL resulted in an increase in p15, p16 and ARF mRNA expression levels in CAL27 cells (Figure 2B). These results suggest that ANRIL is also involved in the repression of p15 and ARF transcription in FaDu cells, and p15, p16 and ARF in CAL27 cells.
Given that depletion of ANRIL increased the mRNA levels of p15 and p16, both of which cause arrest at the G1 phase of the cell cycle, we next examined whether depletion of ANRIL affects the cell cycle in FaDu and CAL27 cells. Cell cycle analysis showed that depletion of ANRIL significantly reduced the number of cells in S and G2/M phase and increased the number of cells in the G1 phase in both FaDu (Figure 3A and B) and CAL27 (Figure 3C and D) cells. This result suggests that ANRIL is involved in the regulation of G1 phase progression in FaDu and CAL27 cells.
Discussion
Recent studies have shown that ANRIL induces proliferation of several cancer cells by regulating gene expression (15-17). In this study, we also showed that silencing ANRIL inhibits the proliferation of head and neck squamous cell carcinoma cells such as the human hypopharyngeal cancer cell line, FaDu and the human oral cancer cell line, CAL27, indicating that ANRIL functions as a positive regulator of proliferation of these cells. Furthermore, silencing ANRIL increased the expression of CDK inhibitors p15 and p16 on the INK4 locus, which lead to G1 phase arrest in the cell cycle in FaDu and CAL27 cells, suggesting that ANRIL functions to promote G1-S transition via repression of p15 and p16. ANRIL is known to repress p15 and p16 transcription through the recruitment of polycomb complexes PRC1 and PRC2 on INK4 locus (7, 8). ANRIL also participates in the regulation of cell adhesion, apoptosis and metabolic activity via regulation of the transcription of many genes, in a trans-acting manner (14). The roles of ANRIL in processes other than cell cycle regulation and in target genes other than those of the INK4 locus are yet to be determined in head and neck squamous cell carcinoma and are important topics that warrant further study.
Many studies have shown that ANRIL expression is up-regulated in various human cancers such as lung, gastric, ovarian and cervical cancers (17). It has also been reported very recently that ANRIL is highly expressed in head and neck squamous cell carcinoma and correlates with tumor progression (21). Accordingly, we propose that ANRIL up-regulation causes aberrant progression of the cell cycle via repressing p15 and p16 transcription, resulting in head and neck squamous cell carcinoma progression.
Depletion of ANRIL inhibits proliferation of FaDu and CAL27 cells. A: FaDu cells were seeded at 5×105 cells/100mm dish. One day later, the cells were transfected with either control siRNA (Ctr-i) or siRNA silencing ANRIL (ANRIL-i) and 72 h later, cells were photographed. B: The number of viable cells was measured by trypan blue staining. **p<0.01. C: CAL27 cells were transfected with siRNA and observed as in (A). D: Viable CAL27 cells were counted as in (B). ***p<0.001.
Depletion of ANRIL increases the expression of INK4 locus genes. A: FaDu cells were seeded at 5×105 cells/100mm dish and cultured for one day, transfected with either control siRNA (Ctr-i) or siRNA silencing ANRIL (ANRIL-i) and 72 hours later, were harvested to determine the mRNA levels of ANRIL, p15 and ARF by qRT-PCR. The results were expressed relative to the corresponding values for FaDu cells/Ctr-i. **p<0.01. B: qRT-PCR using CAL27 cells transfected with siRNA was performed and the data was analyzed as in (A). *p<0.05, **p<0.01.
Depletion of ANRIL causes arrest in the G1 phase of the cell cycle in both FaDu and CAL27 cells. A: At 72 h after transfection with either control siRNA (Ctr-i) or siRNA silencing ANRIL (ANRIL-i), FaDu cells were subjected to cell-cycle analysis by flow cytometry. The x-axis of histogram indicates propidium iodide staining intensity which is reflective of DNA content. The y-axis indicates cell count. B: The graph indicates percentages of FaDu cells in G1, S and G2/M phases. ***p<0.001. C: CAL27 cells transfected with siRNA were subjected to cell-cycle analysis as in (A). D: The graph indicates percentages of CAL27 cells in G1, S and G2/M phases. **p<0.01, ***p<0.001.
Acknowledgements
The Authors would like to thank Hiroki Tsujioka, Takeshi Tsuruda, Kousei Nagayasu, Seiki U, Takazumi Uenishi and Ryotaro Okada for their helpful discussions and technical assistance. This work was supported by JSPS KAKENHI Grant Number 17K07184 (to YK) and the Naito Foundation (to YK).
Footnotes
Authors' Contributions
Conceptualization and design, N.M., T.W., R.Y., and Y.K.; Supervision, Y.K.; Materials, N.M., T.W., R.Y., and Y.K.; Data collection, N.M., and Y.K.; Analysis, N.M., and Y.K.; Writer; Y.K.
Conflicts of Interest
The Authors have no conflicts of interest directly relevant to the contents of this article.
- Received May 14, 2019.
- Revision received June 17, 2019.
- Accepted June 18, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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