|Year : 2014 | Volume
| Issue : 3 | Page : 636-640
The analysis of deregulated expression and methylation of the PER2 genes in gliomas
Wang Fan1, Xiaowei Chen1, Caiyan Li2, Yongluo1, Lvan Chen1, Pingfei Liu1, Zhijun Chen1
1 Department of Neurosurgery, The First People's Hospital of Jingmen, Jingmen, China
2 Department of Neurosurgery, The second People's Hospital of Jingmen, Jingmen, China
|Date of Web Publication||14-Oct-2014|
Department of Neurosurgery, The First People's Hospital of Jingmen, Jingmen
Source of Support: None, Conflict of Interest: None
Context: Growing evidence shows that disruption of circadian rhythm may be a risk factor in the development of glioma. However, the molecular mechanisms of genes controlling circadian rhythm in glioma cells have not been explored and differential expression of the circadian clock genes in glioma and non-tumor cells may provide a molecular basis for manifesting this mechanism.
Aims: The aim of the following study is to analyze the PER2 expression involved in the pathogenesis of glioma.
Materials and Methods: Using immunohistochemical staining, methylation specific polymerase chain reaction techniques, we examined the expression of the most important clock genes, PER2, in 92 gliomas.
Statistical Analysis Used: The association between tumor grade (high-grade/low-grade gliomas) and expression of the investigated proteins (negative/positive) was assessed using the Spearman, Chi-square test and two-sample t-test, included in the Statistical Package for the Social Science, version 13.0. Using Spearman Correlation to analyse correlation between the expression of PER 2 and PER2 promoter methylation.
Results: Our results reveal disturbances in the expression of the period 2 (PER2) genes in most (52.17%) of the glimoa cells in comparison with the nearby non-cancerous cells and the PER2 gene deregulation is most probably by methylation of the PER2 promoter.
Conclusions: Since, the circadian clock controls expression of cell-cycle related genes, we suggest that disturbances in PER2 gene expression may result in disruption of the control of the normal circadian clock, thus benefiting the survival of cancer cells and promoting carcinogenesis. Differential expression of circadian genes in non-cancerous and cancerous cells may provide a molecular basis for chronotherapy of glioma.
Keywords: Glioma, PER2, tumorigenesis
|How to cite this article:|
Fan W, Chen X, Li C, Yongluo, Chen L, Liu P, Chen Z. The analysis of deregulated expression and methylation of the PER2 genes in gliomas. J Can Res Ther 2014;10:636-40
|How to cite this URL:|
Fan W, Chen X, Li C, Yongluo, Chen L, Liu P, Chen Z. The analysis of deregulated expression and methylation of the PER2 genes in gliomas. J Can Res Ther [serial online] 2014 [cited 2020 Jul 11];10:636-40. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/636/138202
| > Introduction|| |
A circadian clock controls various physiological and behavioral rhythms. In mammals, a master circadian clock exists in the suprachiasmatic nucleus of the hypothalamus, and slave oscillators can be found in most tissues. , The rhythms of the circadian clock are controlled by the interaction between positive and negative feedback loops consisting of key clock regulators (including Clock and Bmal1), two cryptochromes (Cry1 and Cry2), and three period (mPer1, mPer2, and mPer3). , In mammalian cells, circadian oscillations are achieved through feedback loops that are regulated in large part by muscle ARNT-like 1 (Bmal1) protein , and circadian locomotor output cycles kaput (Clock) protein. , Both proteins contain basic helix-loop-helix period, ARNT, SIM domains. These proteins bind to the target proteins Per1, Per2, and Per3, as well as to cryptochrome 1 (Cry1) and Cry2.
Gene knockout studies in mice revealed differential roles for the three mPer proteins in the mammalian circadian rhythm. , The PER genes are key circadian rhythm regulators in mammals, and PER2 is an essential component of the mammalian clock mechanism and robust circadian expression of PER2 is essential for the maintenance of circadian rhythms. Recent studies have demonstrated that PER2 regulates other molecular and biochemical processes beyond their established role in the mammalian circadian clock. More importantly, PER2 appears to act as a tumor suppressor in the mammalian. , The deregulated expression of the PER2 gene can be found in breast cancers, chronic myeloid leukemia, human endometrial carcinoma. , The increased proliferation and decreased apoptosis that are associated with clock disruption in tumors can be subverted through PER2 overexpression in malignant cells. ,, Therefore, PER2 genes may act as tumor suppressors.
Circadian rhythms, and PER2 proteins in particular, may even influence cancer. , Overexpression of PER2 sensitizes human cancer cells to deoxyribonucleic acid (DNA) damage-induced apoptosis, significantly increasing apoptosis in tumor cells. ,, In addition, PER2 have been reported to be down regulated in several human cancers  and overexpression of either gene inhibits the growth of cancer cells. , These studies show that high expression of PER2 can promote tumor cell apoptosis.
Gliomas are the most common form of primary brain tumors and its morbidity is increasing year by year and no treatment is yet available against them. However, the precise mechanism is not elucidated until now. Differential expression of the circadian clock genes in glioma and non-tumor cells may provide a molecular basis for manifesting this mechanism.  In the present study, we used immunohistochemical staining, methylation-specific polymerase chain reaction (PCR) to explore the expression level changes in the most important circadian genes (PER2) in human glioma.
| > Materials and methods|| |
During the period of the months from May 2006 to June 2012, 92 patients with diffuse subcortical gliomas were operated on in Department of Neurosurgery, and the glioma tissue samples of these patients were used for this study. A total of 92 resected glioma tissue samples with the surrounding non-cancerous tissues were collected. The paired non-cancerous tissue confirmed by histopathological analysis was collected from the normal part of glioma tissue without contamination from glioma cells. The age of the patient ranged from 12 to 69 with a mean of 40.5 years, with 57 men and 35 women. The tissues were frozen or formalin-fixed immediately after surgical resection and stored in liquid nitrogen as Hui et al. previously described. The glioma tissue and tumor free specimens were surgically obtained between 11:00 and 15:00. The glioma tissues in our study include 37 astrocytomas, 29 oligodendrogliomas and 26 glioblastoma. According to WHO pathology grading, 22 cases were Stage I, 26 were Stage II, 25 were Stage III, and 19 were Stage IV.
Paraffin-embedded tissue sections (4 μm) on poly-1-lysine coated slides were deparaffinized. The sections were treated with ethylenediaminetetraacetic acid in a pressure cooker, heated at boiling temperature for 2.5 min, cooled and incubated with 3% H 2 O 2 for 10 min to block endogenous peroxidase, and then incubated with gradient alcohol and washed in phosphate buffered saline (PBS) 3 times for 2 min each time. After being bathed in PBS, sections were incubated with antibodies for PER1 (1:400, Santa Cruz Biotechnology, CA) for 2 h at 37°C.
The slides were washed 3 times with PBS and incubated with secondary anti-body, anti-rabbit antibody (PV-6001, Zhongshan Golden bridge Biotechnology Co., China) and antimouse antibody (PV-6002, Zhongshan Golden bridge Biotechnology Co., China) at 37°C for 30 min, then thoroughly washed 3 times with PBS. Detection of immunostaining was performed using diaminobenzidine (Zhongshan Golden bridge Biotechnology Co. China.) as chromogene, and then a counterstain was performed using hematoxylin.
Staining was evaluated by a pathologist and an investigator blind to diagnosis and sections were classified as positive or negative. Cell nucleus can be observed through staining.
Positive cells had yellow staining in the cell nucleus. Cells were quantified by cell numbers per one high power field, with no positive cell graded as 0, 1-25% of the cells as 1, 26-50% as 2, 51-75% as 3, 75-100% as 4. The staining intensity was graded, with no coloration graded as 0, light yellow as 1, yellow as 2, and brown as 3. The two scales were multiplied, the cells with a value greater than or equal to 2.0 were counted as positive. 
Methylation-specific PCR analysis of PER2
Genomic DNA was modified with sodium bisulfite and methylation-specific PCRs were performed as described  with some modifications. Primer pairs for detection of the methylated and unmethylated sequences in the promoter of PER2 are shown in [Table 1]. Briefly, 4 ug of genomic DNA in 40 ml H 2 O was denatured by incubation with 10 ul 1 N NaOH at 37°C for 10 min followed by modification with 30 ul of 10 mM hydroquinone and 520 ul 1.5 M sodium bisulfite (pH 5.0) at 50°C for 16 h. The DNA samples were eluted with 100 ul prewarmed H 2 O (65-70°C) in a wizard DNA purification kit (Promega). A volume of 50 μl of 1 N NaOH was added to the eluent and the mixture was incubated at room temperature for 5 min. After the pellet was precipitated with 150 ul 100% isopropanol and washed with 70% ethanol, it was resuspended in 45 ul H 2 O. Modified DNA was amplified in a total volume of 20 ul solution containing 1 × PCR buffer, 1.0 mM MgCl 2 , 100 ng of each primer, 0.2 mM of each dNTP and 2.5 U Taq polymerase. The PCR was performed in a thermal cycler for 40 cycles; each cycle consisted of denaturation at 94°C for 1 min, annealing at 60°C for both methylated and unmethylated primers for 1 min extension at 72°C for 1 min and a final 5 min extension at 72°C. PCR products were then loaded and electrophoresed on 3.5% agarose gels, stained with ethidium bromide and visualized under ultraviolet illumination.
|Table 1: Primers used for the detection of CpG methylation and unmethylation in the promoter of the PER2 genes|
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CpG methylase (SssI)-treated genomic DNA was used as the positive control for methylation-specific primers. DNA samples extracted from blood samples of healthy individuals tested negative in methylation and positive in unmethylation in the PER2 gene was used as positive controls for unmethylation. To ensure specificity in methylation in the PER2 gene, unmodified genomic DNA samples from non-cancerous and cancerous parts of the glioma patients were also carried out as negative controls.
The association between tumor grade (high-grade/low-grade gliomas) and expression of the investigated proteins (negative/positive) was assessed using the Spearman, Chi-square test and two-sample t-test, included in the Statistical Package for the Social Science, version 13.0.
| > Results|| |
The level of PER2 in glioma and non-tumor tissue
The expression of PER2 can be observed in gliomas and normal tissues (non-tumor brain sample) at different levels, all the staining was in the cell nucleus [Figure 1]. The expression of PER2 in glioma tissues is 52.17% (48/92), while out of 92 non-tumor brain tissues, 75 were PER2-positive (81.52%). The expression levels of PER2 in the glioma cells were significantly different from those in the non-tumor brain tissue cells (P < 0.01, two-sample t-test).There was no significant difference in the expression rates of PER2 between low-grade gliomas (I, II) and non-tumor tissues around gliomas [P > 0.05, Chi-square test, [Table 2]], whereas the expression of PER2 in high-grade gliomas (III, IV) is significantly lower than that of low-grade gliomas (r = −0.431, P = 0.014 < 0.05) non-tumor tissues around high-grade gliomas (r = −0.382, P = 0.026 < 0.05).
|Figure 1: Immunohistochemical analysis of PER2 for representative cases and positive staining in the nucleus are found (×20). (a and c) were the tissues of Grade I or II glioma and the non-tumor brain tissues around Grade I or II glioma tissue; (b and d) were the tissues Grade III or IV glioma and the non-tumor brain tissues around the Grade of III or IV glioma|
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Methylation PCR analysis of promoter sequences of the PER2 genes
To investigate whether aberrant CpG methylation of the promoter region was the cause for differential PER2 gene expression in gliomas, we analyzed the methylation status of the promoter sequences of the PER2 genes. We designed two primer pairs to discriminate between methylated and unmethylated alleles and between bisulfite-modified and unmodified DNA. In [Figure 2], we show the methylation status of the PER2 promoter region for two representative cases. They represent similar expression patterns in both cancerous and non-cancerous tissues (case 1) and elevated PER2 expression in the non-cancerous tissue (case 2), respectively. CpG methylation of the PER2 promoter was observed in 8/92 (8.70%) cancerous tissues and in 1/92 (1.08%) non-cancerous tissues.
|Figure 2: Analysis of the PER2 promoter in two human glioma cases with different PER2 expressions. Case 1 had similar expression patterns in both cancerous and non-cancerous tissues; case 2 had elevated PER2 expression in the noncancerous tissue. m: 100 bp ladder marker, M and U: mPCR using methylation or unmethylation specific primers, N: Non-cancerous tissue, T: Cancerous tissue, SssI: SssI methylate treated deoxyribonucleic acid (DNA) as positive control, G: unmodified genomic DNA|
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Correlation between the expression of PER2 proteins and PER2 promoter methylation
The correlation between the expression profiles of the PER2 proteins and the results of methylation PCR analysis is shown in [Table 3]. There were 39 cases in the Type I protein profile that showed 2 cases methylation in the promoters of PER2 genes; 1 case showed promoter methylation in non-cancerous tissues, the remaining case was methylated in the tumor tissue. A total of 53 cases belonged to the Type II protein profile, 7 cases showed promoter methylation in PER2 gene in tumor tissues.
|Table 3: Relationship between promoter methylation and PER2 protein patterns in cancerous and non-cancerous tissues of glioma|
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| > Discussion|| |
Generally speaking, the circadian clock and cell cycle are two global regulatory systems in most eukaryotic organisms.  Recently, research has shown that the circadian system is not only essential for proper growth control, but it also regulates cell proliferation and apoptosis. ,,, As a set of core circadian genes, PERs have a role in a wide variety of physiological processes, including the circadian rhythm of cells and sustaining the normal cell cycle. It has been reported that 2-10% of all mammalian genes are clock-controlled, ,,, and recent studies reported that approximately 7% of all clock-controlled genes identified in rodents regulate either cell proliferation or apoptosis. , Therefore, the development of glioma may be related to the disruption of PER2.
The molecular changes in circadian rhythm controlled genes in glioma cells are still unexplored. In this study, we analyzed and compared the expression status of the PER2 proteins in glioma and non-cancer brain tissues obtained at the same time in each case so that tissue pairs were synchronized with respect to their circadian clocks, and we find differential expression patterns in the PER2 genes in glioma cells in most of the glioma cases (48/92) when analyzed in comparison with their paired nearby non-cancer brain tissues. The deregulation of the circadian clock may be one of the most important factors in the proliferation of glioma. Since expression of PER2 play a central role in circadian rhythm, our results suggest that the circadian clock in the tumor cells of most glioma cases behaves differently than in nearby non-cancer brain tissues.
Furthermore, we also observed PER2 expression patterns in different cell populations of the different grade glioma tissue to be 76.74% for low-grade and for 30.61% high-grade. In nearby non-tumor brain tissue around high-grade and low-grade, the expression of PER2 are 79.59% and 83.72%, suggesting that several asynchronized circadian clocks may be in operation in the same glioma tissue. This can show that the heterogeneity has been in glioma cell population in glioma tissue. Our results that supported the hypothesis that the heterogeneity has been in glioma cell population in glioma tissue.
In our study, we found that the PER2 protein profiles was disturbed in human glioma tissues. Chen et al. found 50% of the differential expression of the PER proteins in the breast cancerous tissues can be explained by promoter methylation of the PER genes and it has been shown that CpG methylation of promoter sequences, an epigenetic alteration, can inactivate promoter functions leading to downregulation and inhibition of gene expression.  To elucidate a possible mechanism to explain the PER2 expression patterns, we further explored whether CpG methylation had occurred in the PER2. Our results show that 8.70% of the differential expression of the PER2 proteins in the glioma tissues can be explained by promoter methylation of the PER2 genes, and because the cancerous cells were also defective in the cell cycle or signal transduction pathway resulting in disturbance of circadian gene expression. We propose that the differential expression of the PER2 proteins between cancerous and non-cancerous tissues in each glioam patient is partly due to promoter methylation of the PER2, or as a result of promoter methylation of other circadian genes resulting in deregulation of the PER2, or disruption of the signal transduction pathway or cell cycle influencing the PER protein expression.
In another separate study, it was observed that the loss of PER1 protein was commonly observed in human endometrial carcinoma but not in the adjacent normal cells.  Xia et al.  propose that inactivation of the PER1 and PER2 in glioma cells may result in deregulation of the cell cycles thus favoring the proliferation of glioma cells. Recently, Chen et al.  have shown that the circadian clock controls the expression of cell cycle related genes and the intracellular circadian clockwork is able to control the cell-division cycle directly and in a unidirectional mode in proliferating cells. And the temporal expression of genes involved in cell cycle regulation and tumor suppression was also deregulated in mPer2 mutant mice.  Based on these results, we propose that deregulated of the PER2 genes in glioma cells in deregulation of the cell cycle favoring proliferation of tumor cells.
| > Conclusion|| |
Deregulated expression of the PER2 genes is common in glioma, and inactivation of the PER2 in glioma cells may result in deregulation of the cell cycles thus favoring the proliferation of glioma cells.
| > References|| |
Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature 2002;418:935-41.
Morse D, Sassone-Corsi P. Time after time: Inputs to and outputs from the mammalian circadian oscillators. Trends Neurosci 2002;25:632-7.
Czeisler CA, Duffy JF, Shanahan TL, Brown EN, Mitchell JF, Rimmer DW, et al
. Stability, precision, and near-24-hour period of the human circadian pacemaker. Science 1999;284:2177-81.
Reppert SM, Weaver DR. Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 2001;63:647-76.
Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, et al
. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 2002;109:307-20.
Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH, et al
. Extensive and divergent circadian gene expression in liver and heart. Nature 2002;417:78-83.
Delaunay F, Laudet V. Circadian clock and microarrays: Mammalian genome gets rhythm. Trends Genet 2002;18:595-7.
Miki T, Xu Z, Chen-Goodspeed M, Liu M, Van Oort-Jansen A, Rea MA, et al
. PML regulates PER2 nuclear localization and circadian function. EMBO J 2012;31:1427-39.
Yang X, Wood PA, Ansell C, Hrushesky WJ. Circadian time-dependent tumor suppressor function of period genes. Integr Cancer Ther 2009; 8:309-16.
Zheng B, Albrecht U, Kaasik K, Sage M, Lu W, Vaishnav S, et al
. Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 2001;105:683-94.
Fu L, Pelicano H, Liu J, Huang P, Lee C. The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo
. Cell 2002;111:41-50.
Gery S, Komatsu N, Baldjyan L, Yu A, Koo D, Koeffler HP. The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. Mol Cell 2006;22:375-82.
Chen ST, Choo KB, Hou MF, Yeh KT, Kuo SJ, Chang JG. Deregulated expression of the PER1, PER2 and PER3 genes in breast cancers. Carcinogenesis 2005;26:1241-6.
Yang MY, Yang WC, Lin PM, Hsu JF, Hsiao HH, Liu YC, et al
. Altered expression of circadian clock genes in human chronic myeloid leukemia. J Biol Rhythms 2011;26:136-48.
Gery S, Virk RK, Chumakov K, Yu A, Koeffler HP. The clock gene Per2 links the circadian system to the estrogen receptor. Oncogene 2007;26:7916-20.
Hua H, Wang Y, Wan C, Liu Y, Zhu B, Yang C, et al
. Circadian gene mPer2 overexpression induces cancer cell apoptosis. Cancer Sci 2006;97:589-96.
Yang MY, Chang JG, Lin PM, Tang KP, Chen YH, Lin HY, et al
. Downregulation of circadian clock genes in chronic myeloid leukemia: Alternative methylation pattern of hPER3. Cancer Sci 2006;97:1298-307.
Xia HC, Niu ZF, Ma H, Cao SZ, Hao SC, Liu ZT, et al
. Deregulated expression of the Per1 and Per2 in human gliomas. Can J Neurol Sci 2010;37:365-70.
Wood PA, Yang X, Taber A, Oh EY, Ansell C, Ayers SE, et al
. Period 2 mutation accelerates ApcMin/+ tumorigenesis. Mol Cancer Res 2008;6:1786-93.
Sun CM, Huang SF, Zeng JM, Liu DB, Xiao Q, Tian WJ, et al
. Per2 inhibits k562 leukemia cell growth in vitro
and in vivo
through cell cycle arrest and apoptosis induction. Pathol Oncol Res 2010;16:403-11.
Sato F, Nagata C, Liu Y, Suzuki T, Kondo J, Morohashi S, et al
. PERIOD1 is an anti-apoptotic factor in human pancreatic and hepatic cancer cells. J Biochem 2009;146:833-8.
Duffield GE, Best JD, Meurers BH, Bittner A, Loros JJ, Dunlap JC. Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells. Curr Biol 2002;12:551-7.
Kornmann B, Preitner N, Rifat D, Fleury-Olela F, Schibler U. Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs. Nucleic Acids Res 2001;29:E51-1.
Le Minh N, Damiola F, Tronche F, Schütz G, Schibler U. Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators. EMBO J 2001;20:7128-36.
Hrushesky WJ, Bjarnason GA. Circadian cancer therapy. J Clin Oncol 1993;11:1403-17.
Lévi F. Circadian chronotherapy for human cancers. Lancet Oncol 2001;2:307-15.
Clark SJ, Melki J. DNA methylation and gene silencing in cancer: Which is the guilty party? Oncogene 2002;21:5380-7.
Chen Z, Liu P, Li C, Luo Y, Chen I, Liang W, et al
. Deregulated expression of the clock genes in gliomas. Technol Cancer Res Treat 2013;12:91-7.
Luo Y, Wang F, Chen LA, Chen XW, Chen ZJ, Liu PF, et al
. Deregulated expression of cry1 and cry2 in human gliomas. Asian Pac J Cancer Prev 2012;13:5725-8.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]