|Year : 2015 | Volume
| Issue : 1 | Page : 27-31
Evaluating of suppressor of zeste 12 and chromobox homolog 8 genes expression showed two possible origins for gastric cancer development
M Ghalandary, M Behmanesh, M Sadeghizadeh
Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran
|Date of Web Publication||3-Feb-2016|
Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran
Source of Support: The Department of Research Affairs of Tarbiat
Modares University and the Iranian Stem Cell Network provide the
funding of this work., Conflict of Interest: None
Context: Changes in genome, made by multiple genetic and epigenetic alterations result to the cancer initiation and progression. Suppressor of zeste 12 (SUZ12) and chromobox homolog 8 (CBX8) proteins are two components of epigenetic regulators that their function in the initiation and progression of cancers are not well-understood. Aims: The role of SUZ12 and its target CBX8 is examined. Settings And Design: Comparing the expression levels of SUZ12 and CBX8 between 30 gastric tumor and their marginal tissues. Materials And Methods: Quantitative reverse transcription polymerase chain reaction technique was performed. Statistical Analysis: Statistical comparison was carried out using Statistical Program for Social Sciences software 16.0 (Released 2007, SPSS for Windows. SPSS Inc., Chicago, IL, USA) and (GraphPad Prism version 5 for Windows, GraphPad Software, La Jolla, California USA, ww.graphpad.com).Results: Despite the obvious differences in the expression of these genes in each sample for tumor and its marginal tissue, statistical analysis did not show significant differences in the mean of expression for SUZ12 and CBX8 genes in total. Due to the variation in expression levels, the samples could be divided into two groups for each gene; group 1, in which the genes were overexpressed in tumor and group 2, in which the genes were down regulated in tumor samples. Conclusion: We found that in each group, the difference in the SUZ12 and CBX8 genes expression were significantly divergent between tumors and their marginal tissues. It means that the regulatory mechanisms involved in developing and controlling the process of gastric cancer pathogenesis is more complex than it thought. These results also bring new evidence on the possible double origin for gastric cancer development, bone-marrow-derived cells and tissue stem cells.
Keywords: Epigenetics, gastric cancer, polycomb group proteins, quantitative reverse transcription polymerase chain reaction, suppressor of zeste 12
|How to cite this article:|
Ghalandary M, Behmanesh M, Sadeghizadeh M. Evaluating of suppressor of zeste 12 and chromobox homolog 8 genes expression showed two possible origins for gastric cancer development. Indian J Cancer 2015;52:27-31
|How to cite this URL:|
Ghalandary M, Behmanesh M, Sadeghizadeh M. Evaluating of suppressor of zeste 12 and chromobox homolog 8 genes expression showed two possible origins for gastric cancer development. Indian J Cancer [serial online] 2015 [cited 2020 Jan 27];52:27-31. Available from: http://www.indianjcancer.com/text.asp?2015/52/1/27/175566
| » Introduction|| |
Gastric cancer is one of the most common cancers and is the second most frequent cause of death world-wide. Despite of extensive work, still there is no effective therapy for cancer. It might be due to the lack of complete knowledge about certain biological nature of this disease such as cellular heterogeneity and phenotype plasticity.
According to the recent progress, it is becoming increasingly difficult to ignore the importance of epigenetic deregulation in addition to the genetic changes for initiation of cancer. Non-coding ribonucleic acids (RNAs) mainly microRNAs, deoxyribonucleic acid (DNA) hypermethylation of CpG (cytosine-phosphate-guanine) dinucleotide island in the promoter region of tumor suppressor genes and specific post-translational modifications of histones such as phosphorylation, acetylation and methylation on specific amino acid residues are the most potent epigenetic changes, which are involved in tumors formation and progression.,,
Polycomb group proteins (PcG) are the main factors that are known to be associated with cellular memory, epigenetic regulation, cancer progression, development and stem cell self-renewal through histone post-translational modifications and gene silencing.,
The PcG proteins form two biochemically and functionally different classes, called polycomb repressive complexes (PRC1 and PRC2) that were first described in Drosophila, but components of which are conserved from insect to human.
PRC1 complex consist of more than 10 subunits, which specifically binds to the modified H3K27 through human Pc homolog proteins and catalyzes histone H2A ubiquitylation. By the formation of this multi-protein complex the target genes will be repressed through chromatin remodeling.
The PcG proteins enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12) (Drosophila)) are the core component of PRC2.,, The EZH2 mediates initiation of transcriptional repression through its histone methyl transferase activity, specific for lysine (K) residues of K9 and K27 in histone H3 and its activity needs SUZ12 gene product.
PcG proteins are mandatory to repress genes encoding transcription factors required for differentiation in stem cells. This repression is dynamic and subsequent activation will occur during differentiation; nevertheless, the DNA methylation at the promoter of these genes may lead to the repression of these genes permanently and maintenance of stem cell phenotype and consequently initiation of abnormal clonal expansion.
A considerable amount of literature has been published about the relationship between deregulated activity of the PcG proteins such as BMI 1 (B lymphoma Mo-MLV insertion region 1 homolog), ring finger protein (RING) and EZH2 and development of the different cancers., So far, however, there has been little information about the role of SUZ12 in development of cancer.,,
Gastric cancer is a heterogeneous disease, which is made up of at least two different diseases including intestinal-type and diffuse-type. It has been suggested that cancer cells often have many characteristics of stem cells, but the accurate origin of many cancers such as gastric is unknown yet. It is believed that epithelial cancers are derived from the transformation of tissue stem cells as well as bone-marrow-derived cells (BMDCs) that are often home to the site of chronic injury or inflammation.,
It has been shown that loss of SUZ12 gene expression leads to the amplified activity of progenitor and hematopoietic stem cells (HSCs); besides, PcG proteins are essential for stems cells to maintain their pluripotency by silencing some lineage specific factor genes. According to these facts and the crucial role of epigenetic alterations in gastric carcinoma, in this research the potential role of SUZ12 and its downstream target chromobox homolog 8 (CBX8) in development of gastric tumors is examined by evaluating the expression level of these genes with real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay.
| » Materials and Methods|| |
Patients and samples
Thirty gastric cancer specimens and their marginal tissues surgically resected from gastric cancer patients were obtained from Iran National Tumor Bank (Tehran, Iran). The autopsies consisted of 30 individuals (18 males and 12 females with a mean age of 58 ± 8 years old). The tissues were immediately snap-frozen in liquid nitrogen and then stored at −80°C, until RNA extraction. The histopathological parameters were evaluated by two independent pathologists, according to the grading and tumor, node and metastasis (TNM) system for stage classification of the World Health Organization. The Ethics Committees of Tarbiat Modares University approved the experimental design and the patients' written informed consents were obtained prior to sampling.
RNA extraction and complementary deoxyribonucleic acid synthesis
Total RNA was extracted from gastric tumor and their marginal normal specimens by using the RNX plus solution (Cinnagen, Tehran, Iran) according to the manufacturer's instructions. To remove any genomic DNA contamination, total RNAs were treated by RNase-free DNase I (Fermentas, Lithuania) at 37°C for 30 min. The integrity and quantity of RNAs were estimated by agarose gel electrophoresis and spectrophotometric measurements, respectively.
Three microgram of total RNA from each sample was reverse-transcribed by First-Strand cDNA synthesis kit using MMLV-RevertAid™ Reverse Transcriptase (Fermentas, Lithuania) and Oligo (dT)18-(MWG, Germany) in total volume of 20 µl, according to the manufacture's instruction.
Real time PCR analysis
Study of messenger ribonucleic acids (mRNA) levels was performed to measure the relative levels of SUZ12 and CBX8 genes expression in comparison with the housekeeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) with specific primers. All primers were designed using Primer Express software (Applied Biosystems, Foster City, CA) and their sequences are shown in [Table 1]. The designed primers were for the junction between two adjacent exons or in a different exon.
|Table 1: The sequence of primers that were used for gene expression analysis. All primers were designed using primer express software (Applied Biosystems, Foster City, CA). The primers were designed for the junction between two exons or in a different exon|
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Quantitative real-time PCR performed using an ABI 7500 sequence detection system (Applied Biosystems, USA), with 10 ng cDNA, 200 nM of forward and reverse primers and 10 μL of Syber Green Permix Ex Taq II (TaKaRa, Shiga, Japan) in a final volume of 20 mL, according to the manufacturer's instructions. The PCR reaction was performed as follows: A single cycle of 95°C for 5 min for initial denaturation, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension 60°C for 1 min. Specificity of the PCR products was examined by dissociation curve analysis, restriction endonuclease digestion followed by gel electrophoretic analysis to verify their size. The PCR and digested products were resolved in 12% (W/V) acrylamide gel electrophoresis.
The relative expression of each gene was carried out using comparative threshold cycle as described by Livak and Schmittgen. Briefly, the mean threshold cycle (mCT) was obtained from triplicated amplification during the exponential phase of amplification. Then mCT value of reference gene of GAPDH was subtracted from mCT value of each SUZ12 and CBX8 genes to obtain ΔCT for each tumor or its normal marginal tissues. The ΔΔCT value of each sample was calculated from corresponding CT values; and finally the relative expression of each gene was estimated by ratio formula (ratio = 2−ΔΔCt). All PCR assays were carried out in triplicate and the mean of triplicates was reported. The standard curve for each transcript was made with five times serially diluted cDNA obtained from human adenocarcinoma of the stomach cell line (AGS).
A paired Student's t-test used to compare the expression of genes between tumor and marginal tissues in mRNA level. The clinico-pathological variables used in this study were as follows: Differentiation tumor grade, lymph node metastasis and invasion depth. Results from correlation analyses are expressed by Pearson's correlation coefficient. A P ≤ 0.05 was considered significant and data were shown as mean ± standard deviation. Statistical comparison was carried out using Statistical Program for Social Sciences (SPSS) software 16.0 (SPSS Imc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).
| » Results|| |
Clinical characteristics of the patients
The range of age of the patients was 58 ± 8 years, eighteen patients were male and 12 were female. Among the 30 patients, seven individuals had cardia stomach cancer and 23 had non-cardia carcinoma. The summery of TNM staging of the samples is shown in [Table 2].
|Table 2: Demographic and clinical characteristics of the patients used in this study. Two independent pathologists evaluated the samples according to the grading and TNM system for stage classification|
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CBX8 and SUZ12 genes expression levels in tumor and marginal samples
To comparing the expression level of SUZ12 and CBX8 the mean of gene expression was used. In this study, GAPDH gene was considered as an internal control and we used a paired t-test to compare the mean of expression for SUZ12 and CBX8 genes between tumor and marginal samples. In almost all samples the expression of interested genes was different between tumors and their corresponding normal margins. Because of the wide variation that was observed in our data, statistical analysis didn't show significant differences in the mean expression of SUZ12 between tumor and marginal tissues (P = 0.463) and CBX8 mean expression between tumor and marginal tissues (P = 0.483) [Figure 1a] and [Figure 1b]. However, for each of these two genes, the samples could be divide into two group according to their expression pattern; group 1 in which the gene expression in tumor tissues were more than marginal one and group 2 in which the gene expression in tumor tissues were less than the marginal samples. The pair t-test was used to analyze the differences of SUZ12 and CBX8 expression between tumor and non-tumor tissues in each group [Figure 2a] and [Figure 2b]. Within each group, we found significant differences in the expression of SUZ 12 and CBX8 in the tumor tissue compared with the marginal tissue (for SUZ 12 in group 1 P = 0.0001 and in group 2 P = 0.0005 and for CBX8 in group 1 P = 0.007 and in group 2 P = 0.0001).
|Figure 1a: Comparison of relative expression of suppressor of zeste 12 between tumor and marginal tissues|
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|Figure 1b: Comparison of relative expression of chromobox homolog 8 between tumor and marginal tissues|
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|Figure 2a: Comparison of relative expression of suppressor of zeste 12 in group 1 and group 2|
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|Figure 2b: Comparison of relative expression of chromobox homolog 8 in group 1 and group 2|
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Correlation between the expression levels of SUZ12 and CBX8 mRNA in tumor tissues
We performed Pearson correlation to evaluate the correlation between the expression level of SUZ12 and CBX8, but with P = 0.876, we couldn't be able to find any significant correlation between the expression of these two genes.
| » Discussion|| |
In recent years, many studies have shown that PcG proteins are involved in maintaining pluripotency of embryonic stem cells and tissue stem cells through repression of cell-specific transcription factors.SUZ12 is one of the core components of PRC2 which is required for its integrity and activity. Components of PcG proteins are normally expressed at a very low level in normal adult tissue, even though; in embryonic tissues, their expression is high.,, Recently, researchers have shown that SUZ12 protein expression is restricted to the proliferating cells in normal human tissues such as germinal cells in the testis, reactive lymphoid tissue and the epithelium of various organs. So far, however, there has been little discussion about the role of SUZ12 in the development of cancer. Previous studies have demonstrated that SUZ12 is up-regulated in colon, liver, breast tumors as well as mantle cell lymphoma. ,SUZ12 gene is located at the 17q11.2 locus, which is commonly found to be translocated in endometrial stromal tumors by the use of fluorescence In situ hybridization method. It has been shown that SUZ12 overexpression in a subset of primary human tumors is related to gene locus amplification., Loss of SUZ12 function in HSCs, associated with increased proliferative activity while disruption of PRC1, leads to HSC/progenitor cell defects. More recently, finding loss-of-function mutations and deletions of the EZH2 and SUZ12 genes in T-cell acute lymphoblastic leukemia, suggests a tumor suppressor role for PRC2 in human leukemia. We quantified SUZ12 gene expression at the RNA level by means of real-time quantitative RT-PCR in gastric cancer tumors in comparison with normal marginal tissues. Despite large differences between each tumor and its marginal tissue in gene expression, no obvious significant differences between tumor and marginal tissues was observed in total (P = 0.46). The expression pattern of SUZ12 in our samples showed dichotomy, in a subset of samples there was a large increase in expression of SUZ12 between tumor and marginal samples while the others showed a reduction in the gene expression in tumor samples. In order to understand why our data sets in two separate groups, we investigated the correlation between our data and tumor grade, stage and patient's gender, but we could not find any significant relationship and our data.
Two types of cells including BMDCs and tissue stem cells are considered as the origin of gastric cancer. It has been shown that chronic infection with Helicobacter stimulates repopulation of the stomach with BMDCs. In addition, another study have shown that a bone marrow population enriched for HSC can differentiate into epithelial cells of the liver, lung, GI tract and skin. According to the different role of SUZ12 between HSCs and tissue stem cells and discrepancy between the role of this gene as oncogene, which is overexpressed in a group of human tumors- and tumor suppressor gene, it can be concluded that the difference that is seen between samples may be due to different origin of tumors. For further research, CBX8 Gene expression between tumoral and marginal samples was evaluated. CBX8 is one of the members of PcG protein family that its inhibition in human and mouse fibroblasts leads to cell growth cessation and is considered as a positive regulator of cell proliferation. However, far too little attention has been paid to the function of CBX8 in cancer, but it has been shown that this chromodomain containing polycomb protein, binds to BMI1 in human cells and connects to the locus is cyclin-dependent kinase inhibitor 2A (INK4A-ARF) locus in human and mouse cells. it has been proved that BMI1 is extensively expressed in various human tumors; besides, CBX8 and BMI1 are being tied together for binding to INK4A-ARF locus. Furthermore, SUZ12 binds to the CBX8 locus on ES cells and in SUZ12 deficient mouse, CBX8 gene expression is up regulated. ChIP-on-chip analysis on embryonic diploid fibroblasts revealed CBX8 as a potential SUZ12 target genes that its expression is significantly changed upon down-regulation of it. We evaluated the expression of CBX8 on our samples and the result was similar to our result for SUZ12, despite the differences between each tumor and marginal samples statistical analysis did not disclose any significant differences in the expression of CBX8 between tumor and marginal samples. Here, the dichotomy was also seen among samples, but the pattern of expression was not similar to SUZ12 gene expression. Since CBX8 was reported as a potential target for SUZ12, we analyzed the correlation between the expression of SUZ12 and CBX8, but we could not find any association between them; this can be due to the different targets of SUZ12 in epithelial cells since the tumors were adenocarcinomas.
Until date, there is no evidence on possible involvement of SUZ12 and CBX8 in gastric cancer. This study provides some novel information that further elucidates the complexity of gastric cancer and the causative role of epigenetic regulation in this cancer. Because of the heterogeneous nature of gastric cancer and dual role of SUZ12 on HSC and tissue stem cells, a more accurate conclusion requires further studies with larger samples to identify the specific factor that may explain the observed dichotomy.
| » Acknowledgments|| |
The authors gratefully acknowledge the contribution of the patients and institutions in this study. The Department of Research Affairs of Tarbiat Modares University and the Iranian Stem Cell Network provide the funding of this work.
| » References|| |
Baylin SB, Ohm JE. Epigenetic gene silencing in cancer-A mechanism for early oncogenic pathway addiction? Nat Rev Cancer 2006;6:107-16.
Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999;21:163-7.
Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349:2042-54.
Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415-28.
Martinez AM, Cavalli G. The role of polycomb group proteins in cell cycle regulation during development. Cell Cycle 2006;5:1189-97.
Gil J, Bernard D, Peters G. Role of polycomb group proteins in stem cell self-renewal and cancer. DNA Cell Biol 2005;24:117-25.
Rajasekhar VK, Begemann M. Concise review: Roles of polycomb group proteins in development and disease: A stem cell perspective. Stem Cells 2007;25:2498-510.
Cao R, Zhang Y. SUZ12
is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 2004;15:57-6.
Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, et al
. Epigenetic stem cell signature in cancer. Nat Genet 2007;39:157-8.
Hyland PL, McDade SS, McCloskey R, Dickson GJ, Arthur K, McCance DJ, et al
. Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes. Journal of Virology2011;85:10999-1006.
van Leenders GJLH, Dukers D, Hessels D, van den Kieboom SWM, Hulsbergen CA, Witjes JA, et al
. Polycomb-group oncogenes EZH2, BMI1, and RING1 are overexpressed in prostate cancer with adverse pathologic and clinical features. European urology2007;52:455-63.
Wang WH, Studach LL, Andrisani OM. Proteins ZNF198 and SUZ12
are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication. Hepatology 2011;53:1137-47.
Kirmizis A, Bartley SM, Farnham PJ. Identification of the polycomb group protein SU(Z) 12 as a potential molecular target for human cancer therapy. Mol Cancer Ther 2003;2:113-21.
Majewski IJ, Blewitt ME, de Graaf CA, McManus EJ, Bahlo M, Hilton AA, et al
. Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity. PLoS Biol 2008;6:E93.
Cervantes A, Rodríguez Braun E, Pérez Fidalgo A, Chirivella González I. Molecular biology of gastric cancer. Clin Transl Oncol 2007;9:208-15.
Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, et al
. Gastric cancer originating from bone marrow-derived cells. Science 2004;306:1568-71.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) Method. Methods 2001;25:402-8.
Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, et al
. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006;441:349-53.
Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green R, et al
. Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes Dev 2004;18:1592-605.
Kuzmichev A, Margueron R, Vaquero A, Preissner TS, Scher M, Kirmizis A, et al
. Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation. Proc Natl Acad Sci U S A 2005;102:1859-64.
Squazzo SL, O'Geen H, Komashko VM, Krig SR, Jin VX, Jang SW, et al
binds to silenced regions of the genome in a cell-type-specific manner. Genome Res 2006;16:890-90.
Martín-Pérez D, Sánchez E, Maestre L, Suela J, Vargiu P, Di Lisio L, et al
. Deregulated expression of the polycomb-group protein SUZ12
target genes characterizes mantle cell lymphoma. Am J Pathol 2010;177:930-42.
Nucci MR, Harburger D, Koontz J, Dal Cin P, Sklar J. Molecular analysis of the JAZF1-JJAZ1 gene fusion by RT-PCR and fluorescence in situ
hybridization in endometrial stromal neoplasms. Am J Surg Pathol 2007;31:65-70.
Koontz JI, Soreng AL, Nucci M, Kuo FC, Pauwels P, van Den Berghe H, et al
. Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors. Proc Natl Acad Sci U S A 2001;98:6348-53.
Ntziachristos P, Tsirigos A, Van Vlierberghe P, Nedjic J, Trimarchi T, Flaherty MS, et al
. Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia. Nat Med 2012;18:298-301.
Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, et al
. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369-77.
Dietrich N, Bracken AP, Trinh E, Schjerling CK, Koseki H, Rappsilber J, et al
. Bypass of senescence by the polycomb group protein CBX
8 through direct binding to the INK4A-ARF locus. EMBO J 2007;26:1637-48.
Cao L, Bombard J, Cintron K, Sheedy J, Weetall ML, Davis TW. BMI1 as a novel target for drug discovery in cancer. J Cell Biochem 2011;112:2729-41.
Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, et al
. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006;125:301-13.
Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 2006;20:1123-36.
[Figure 1a], [Figure 1b], [Figure 2a], [Figure 2b]
[Table 1], [Table 2]