Banjarnahor P. R. S, Rahardjo S. P, Savitri E, Hatta M, Suhadi F. X. B, Bukhari A. The Correlation Between Tnm and Yy1 and P53 Mrna Expression in Nasopharyngeal Cancer. Biomed Pharmacol J 2021;14(1).

---

Manuscript received on :19-01-2021
Manuscript accepted on :19-03-2021
Published online on: 31-03-2021

---

Plagiarism Check: Yes
Reviewed by: Dr. Osama Mosa  
Second Review by: Dr. Ankur Singh Bist
Final Approval by: Dr Mohammed Rachidi

---

How to Citeclose    |   Publication History close

 Views: (Visited 444 times, 1 visits today)    PDF Downloads: 381

Pulo RS Banjarnahor¹, Sutji Pratiwi Rahardjo², Eka Savitri², Mochammad Hatta³, FX Budhianto Suhadi⁴ and Agussalim Bukhari⁵

¹Hasanuddin University, Faculty of Medicine, Makassar, Indonesia.

²Department of Ear, Nose and Throat, Faculty of Medicine, Hasanuddin University, Faculty of Medicine, Makassar,  Indonesia

³Molecular Biology and Immunology Laboratory,Faculty of Medicine, Hasanuddin University, Makassar, Indonesia.

⁴Medical Education Unit, Faculty of Medicine, Pelita Harapan University, Lippo Karawaci, Tangerang, Indonesia.

⁵Department of Nutrition, Hasanudin University,Faculty of Medicine, Makassar, Indonesia.

Corresponding Author E-mail: hattaram@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/2104

Abstract

Nasopharyngeal cancer is the fifth most severe malignant disease in the head and neck in the human body. The main treatment is chemoradio therapy. The protein gene 53 (p53) is a tumor suppressor gene. YinYang1 (YY1) is a transcription factor having an important role in cell cycle control. YY1 can function as an activator, suppressor or initiator of the gene transcription process. This research aims to see the relationship between p53 mRNA gene expression and YY1 mRNA gene expression on the NPC TNM Stage. Materials andmethods cross-sectional research at 20 WHO type 3 NPC in which 3 samples after chemoradiotherapy, 17 non-chemoradio therapy samples consisted of 8 stage two samples, 7 stage three samples and 2 stage four samples. With RT-PCR, YY1 mRNA gene expression and p53 mRNA gene expression were measured, then the T-test was independent of the average chemoradio therapy group. It was concluded, at a higher NPC stage, the level of YY1 mRNA gene expression was relatively higher while p53 expression was lower. Post-chemoradio therapy levels of p53 mRNA gene expression were higher and YY1 expression was lower than in the non-chemoradio therapy group.

Keywords

Nasopharyngeal cancer TNM stage; p53; YY1

Introduction

Nasopharyngeal carcinoma (NPC) is a malignancy which accounts for about 2% of squamous cell carcinoma of the head and neck. The NPC case is the fifth most frequent case after cervical cancer, breast cancer, lymphoma and skin cancer. In this journal it was said that NPC was associated with the EBV virus 100%.^1-2

WHO divides NPC into 3 types namely type 1 horn-coated flat cell carcinoma, type 2 and 3 are flat cell carcinoma without horny layers. In fact, the most often found is undifferentiated type III NPC according to WHO classification.^3-8

The p53 gene is a tumor suppressor gene or tumor suppressor. Initially, p53 was thought to be an oncogene because it was found in excessive amounts or overexpression in malignant cells, the p53 gene was able to inhibit cell growth caused by oncogenes and could inhibit the tumorigenic potential of cells in animals. This proves that p53 is a tumor suppressor gene.^8-11

Yin Yang1 (YY1) is a multifunctional protein that can act as a transcriptional repressor, activator, or initiator element binding to a protein that directs and initiates transcription in vitro.¹² YY1 is a ubiquitous & multifunctional zinc-finger transcription factor, which has an important role in the control of the cell cycle. YY1 has a regulatory role in cell growth, development, and differentiation by influencing the levels of Cyclin D1, c-Myc, Rb, MDM2 and p53. YY1 is able to do negative regulation on p53 by increasing Murine Double Minute 2(MDM2)-p53 interaction which will lead to ubiquitination and degradation of p53. It is estimated that YY1 may regulate approximately 10% of the total genes in humans. In addition, Zaravinos who examined the expression of YY1 in tumors in humans explained that YY1 has a role as an inhibitory signal in p300 which is a co-activator of p53.^13-17

The management of NPC varies based on the staging of the NPC itself. The staging system used for NPC was created by the American Joint Committeeon Cancer, based on the TNM system.⁸

This study was conducted with the aim of analyzing the relationship between YY1 mRNA gene expression and p53 mRNA gene expression in NPC patients to be assessed qualitatively using RT PCR analysis and its relationship to TNM staging. With this new exploration, it is hoped that it can be the basis for assessing the severity and further research in efforts to treat nasopharyngeal cancer.

Method

This research uses cross sectional study. The study was conducted on patients who came before or after the action of chemoradiotherapy at Siloam Hospital, Dharmais Hospital, Presidential Hospital atJakarta, Indonesia diagnosed with nasopharyngeal cancer and the Molecular Biology and Immunology Laboratory of the Faculty of Medicine, Hasanudin University, Makassar, Indonesia. Data collection will be conducted in the period of December 2018 to April 2020. The total number of subjects included in this study are 20 patients with diagnosed as nasopharyngeal cancer.

Using the quantitative real time PCR, samples was measured the levels of p53 mRNA gene expression and YY1 mRNA gene expression for each samplewith triplicate according to previous several studies.^18-23

Statistic Analysis

Statistical analysis was performed comparing the mean expression of p53 mRNA gene between groups of stages as well as the mean expression of mRNA YY1 genes between stages groups using independent T Test.²⁴

Result

The youngest respondents were 27 years old and the oldest was 64 years with an average (mean) and median age of respondents 47 years. The number of samples is 12 men 8 women.Histopathology all subjects are WHO type III.

The sample studied consisted of 20 consisting of 17 who had never had chemoradiotherapy and 3 samples after chemoradiotherapy. Of the three samples, one had cycplatin 3 cycles and 6600cGy radiotherapy, one cisplatin 2 cycles  and 6600cGy radiotherapy, one cisplatin 2 cycles and 4000 cG radiotherapy. Three samples that had been able to undergo chemoradiotherapy and their tumors regressed to be minimal tumoron TNM stages.⁸

In table 1 showed that grouped eight stadium II samples, seven stadium III samples, two stadium IV samples and three minimal stage samples or first stage (post-chemoradiotherapy).

Table 1: Distribution of Age, Sex, Chemo radiotherapy, and TNM stadium of Nasopharyngeal Cancer.

Measurement of YY1 mRNA gene expression at stage after chemo radiotherapy showed a mean value of 7.910 (6.389-8.817). Stage II shows the mean 10.921 (9.649-11.71). Stage III has an average of 11.816 (9.269-14.44) and Stage IV has an average of 13.976 (13.143 – 14.809) (table 2)

Table 2: The Differences of YY1 mRNA gene expression in NPC stages.

Measurement of the p53 gene showed a mean in stage post-chemoradiotherapy of 12.924 (11.421-13.975), Stage II had an average of 9.146 (8.105-10.492). Stage III has an average of 7.881 (5.166-10.256) and the last stage IV has an average of 6.736 (5.838-7.634) (table 3).

Table 3: The Differences of p53 mRNA gene expression in NPC stages.

The first group (we call Group A) consists of respondents who have a post-chemo radiotherapy stage and the second group (we call Group B) is stage two, then the second stage compared to the stage three, the second stage compared with stage four, stage three compared to stage four. The post-chemo radiotherapy stage was compared with the stage three. The post-chemo radiotherapy stage was compared with the stage four. The post-chemo radiotherapy stage was compared with the combined two, three and four stage groups that were not yet chemo radiotherapy.

Because group A and group B are not samples from the same population, we will do the unpaired T test at α = 0.05. By conducting the Fisher Variance test we can find out the type of the Independent Two-Sample T Test used whether using the assumption of variance between samples is the same or different.

YY1 mRNA gene expression post chemoradiotherapy compared with YY1 stage II, with stage III, with stage IV and with combined stage II and III and IV, stage II with stage IV statistically respectively each proven to be lower.

Whereas Stage II YY1 mRNA gene expression was compared with YY1 stage III mRNA gene expression, stage III with stage IV was not statistically proven to be lower.

Expression of p53 mRNA gene post chemoradiotherapy compared with p53 stage II, with stage III, with stage IV, and with combined stage II and III and IV, stage II with stage IV statistically respectively each proved to be higher.

Whereas Stage II p53 mRNA gene expression was compared with p53 stage III mRNA gene expression, stage III with stage IV was not statistically proven to be higher.

Discussion

Based on general carcinogenesis, the process of change into cancer is caused by mutations in the gene controlling the cell cycle. The controlling genes are protooncogen, tumor suppressor gene, and repair genes.Generally patients come to health facilities after stadium 2 and above.

Treatment for NPC is chemo radiotherapy. Chemotherapy makes the cell situation in a state of rapid division which will facilitate the effects of radiation. The radiation process will reduce the expression of the YY1 gene mRNA in nasopharyngeal tumors via p300 which will trigger an increase in p53, known as protooncogen.^9–11,16Changes in the mean expression of the YY1 mRNA gene in stage two increased in stage three and more increased in stage four but the samples that had received chemo radiotherapy decreased are seen in table 2.But statistically the increased expression of the YY1 mRNA gene was seen to be in a higher stage only evident from stage two compared to stage four. This is because the change between stage two to stage three and from stage three to stage four is not very noticeable. So it can be stated there is a change in the process of increasing levels of YY1 mRNA gene expression in accordance with an increase in a higher stage. So there is an increased correlation between staging with high levels of YY1 mRNA gene expression.

Statistically T-test samples that received mean chemo radiotherapy YY1 mRNA gene expression levels were lower than each mean YY1 mRNA gene expression in stage two, or stage three or stage four, as well as post-chemo radiotherapy samples compared to the combined groups of stage two, three and four or a sample group that has not received chemo radiotherapy. So it can be stated that the expression of the YY1 mRNA gene will be seen to be decreased if chemo radiotherapy is performed.¹⁵

The change in mean expression of p53 mRNA gene in stage two decreases in stage three and more decreases in stage four but the samples that have received chemo radiotherapy increased expression. But statistically decreased expression of p53 mRNA genes is seen according to a higher stage and is only evident from the stadium two compared to stage four. This is because the change between stage two to stage three and from stage three to stage four is not very noticeable. So it can be stated that there is a process of changing the level of p53 mRNA gene expression in accordance with an increase in a higher stage. So there is an increased correlation between staging and the low expression of the p53 mRNA gene(table3)

Samples that received chemo radiotherapy mean p53 mRNA gene expression appeared statistically higher T Test compared to each p53 mRNA gene expression levels in stage two, or stage three or stage four, as well as post-chemo radiotherapy samples compared to the combined groups of stage two, three and four. So it can be stated that the expression of the p53 mRNA gene will be seen to be increased if the chemo radiotherapy is performed because it will trigger an increase in p53 function (table 3).

Protein 53 or p53 is a polypeptide expressed or coded by the p53 gene that plays a role in maintaining cell integrity or genome integrity through transcription and translation processes. The p53 gene is a tumor suppressor gene or tumor suppressor. In cells that have mutations, the cell will express p53 to try to repair damaged cells in accordance with the composition of the previous normal amino acids because of its protoonchogen nature (table3).

Chemotherapy and radiation will affect ATR which is a serine / threonine specific protein kinase that is involved in sensing DNA damage and activates a site of DNA damage assessment, which leads to the timing of cell cycle capture.^25–27

The mean expression of the YY1 mRNA gene in stages two, three and four appeared to be more increased whereas the expression of the p53 mRNA gene was more decreased in NPC before this chemo radiotherapy showed the process of destruction of the polypeptide chain which in the advanced stage was more severe.^28-30 The mean YY1 mRNA gene expression in the stage before chemo radiotherapy was higher than the mean YY1 mRNA gene expression post-chemo radiotherapy reverse the mean expression of p53 mRNA gene before chemo radiotherapy was lower than that after chemo radiotherapy showed that the process of chemo radiotherapy went well to improve cells with increased expression of p53 mRNA genes, which in turn were proto oncogenic mRNA genes.²⁵

The role of p53 in the cell cycle greatly contributes to preventing cancer growth. Ablation of YY1 results in the accumulation of the amount of p53, so conversely if an overexpression of YY1 stimulates the reduction and degradation of the p53 gene so that endogenous levels of p53 decrease. YY1 fights p53 with various mechanisms including increasing p53 ubiquination and degradation of p53, blocking p53 acetylation, weakening the stabilization of p14ARF, and inhibiting the mediating transcription of p53.^9–10,18

YY1 is a nuclear protein expressed in all tissues. The name “Yin Yang” represents two opposing functions, namely as a TF to act as a repressor or activator. YY1 is a TF belonging to the GLI-Krüppel class of zinc finger protein. This has a fundamental role in embryogenesis, cell proliferation, and differentiation. YY1 exerts its biological effects through its ability to transactivate or suppress gene expression, depending on the gene that binds it. Recent studies have shown that YY1 indirectly activates or suppresses gene expression without DNA binding, through interactions with histone modifiers and chromatin remodeling proteins. Besides binding directly to p53, YY1 also binds to Arf and Mdm2 and increases ubiquitination and p53 degradation, so YY1 is a negative regulator of p53.^12,18

It has been explained that YY1 and p53 are inversely related. Statistically, the relationship between YY1 and p53 gene expression has a large correlation. An increase in YY1 was followed by a significant decrease in p53.^9-10,12,18

Conclusion

High levels of YY1 mRNA gene expression are associated with high stages of nasopharyngeal cancer. The low level of expression of the p53 mRNA gene is associated with TNM stage of high nasopharyngeal cancer. After chemo radiotherapy, low expression of YY1 mRNA gene and high expression of p53 mRNA gene were found compared to before chemo radiotherapy.

Acknowledgement

Authors would like to thank Mr. Romi Usman, Mus Helminus, and Marwani for their excellent technical laboratory work in Molecular Biology and Immunology Laboratory for Infection Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

Conflict of Interest

Nil 

Funding Source

Self Funded

References

1. Xu, Y F, Liu W L, Dong, et al. Sequencing of DC-SIGN promoter indicates an association between promoter variation and risk of nasopharyngeal carcinoma in cantonese. BMC Med Genet.11(1): 161 (2010).https://doi: 10.1186/1471-2350-11-161
   CrossRef
2. Adham M, Antonius KN, Ika A. Nasopharyngeal carcinoma in Indonesia: epidemiology, incidence, signs, and symptoms at presentation. Chin J Cancer. 31(4):185-96 (2012).https://doi: 10.5732/cjc.011.10328
   CrossRef
3. Friborg JT, Yuan JM, Wang R, et al. A prospective study of tobacco and alcohol use as risk factors for pharyngeal carcinomas in Singapore Chinese. Cancer.;109(6): 1183-91 (2007). https://doi.org/10.1002/cncr.22501
   CrossRef
4. Saad S, Wang TJC. Nasopharyngeal carcinoma: Current treatment options and future directions. J NasoPharyngeal Carcinoma.1:16 (2014).
5. Yao M, Epstein JB, Modi BJ, et al. Current surgical treatment of squamous cell carcinoma of the head and neck. Oral Oncol.43(3): 213-23 (2007).https://doi: 10.1016/j.oraloncology.2006.04.013
   CrossRef
6. Savvides PP. The role of chemotherapy in the management of patients with head and neck cancer. Semin Plast Surg. 24(2):137–47 (2010). https://doi.org/10.1055/s-0030-1255331
   CrossRef
7. Jund R, Feldmann HJ, Wollenberg B, et al. Changes in head and neck tumor hypoxic fraction during split course radio chemo therapy.  Ann Otol Rhinol Laryngol.108(1):73-8 (1999).https://doi: 10.1177/000348949910800111
   CrossRef
8. AJCC Cancer Staging Supplement to the AJCC Cancer Staging Manual, Eighth Edition. Springer. 18-9 (2017)
9. Vogelstein B, Lane D, Levine A. Surfing the p53 network. Nature.408:307–10 (2000). https://doi.org/10.1038/35042675
   CrossRef
10. Fridman JS,Lowe SW. Control of apoptosis by p53. Oncogene.22(56):9030-40 (2003). https://doi.org/10.1038/sj.onc.1207116
    CrossRef
11. Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci. 102(23):8204-9 (2005). doi:10.1073/pnas.0502857102
    CrossRef
12. Shi Y, Seto E, Chang LS,Shenk T. Transcriptional repression by YY1, a human GLI-Krüppel-related protein, and relief of repression by adenovirus E1A protein.Cell.67(2):377-88 (1991).https://doi: 10.1016/0092-8674(91)90189-6
    CrossRef
13. Bauknecht T, Jundt F, Herr I, et al. A switch region determines the cell type-specific positive or negative action of YY1 on the activity of the human papillomavirus type 18 promoter. J Virol.69(1):1-12 (1995). https://doi:10.1128/JVI.69.1.1-12.1995
    CrossRef
14. Flanagan JR. Autologous stimulation of YYI transcription factor expression: role of an insulin-like growth factor. Cell Growth Differ.6:185-90 (1995).
15. Dong XP, Pfister H. Overlapping YY1-and aberrant SP1-binding sites proximal to the early promoter of human papillomavirus type 16. J Gen Virol.80(8):2097-101 (1999). https://doi: 10.1099/0022-1317-80-8-2097
    CrossRef
16. Lee JS, Galvin KM, See RH, et al. Relief of YY1 transcriptional repression by adenovirus E1A is mediated by E1A-associated protein p300.Genes Dev.9(10):1188-98 (1995). https://doi:10.1101/gad.9.10.1188
    CrossRef
17. Meliala ITS, Hosea R, Kasim V, Wu S. The biological implications of Yin Yang 1 in the hallmarks of cancer. Theranostics.10(9):4183-4200 (2020).https://doi:10.7150/thno.43481
    CrossRef
18. Jacobus J. W, Rauf S, Hatta M, Natsir R, Massi M. N, Cangara M. H, Masadah R, Pattelongi I. J, Salim J. YinYang 1 (YY1) and P53 Gene Expression Analysis in Cervical Cancer and Its Relationship with Cancer Staging. Biomed Pharmacol J. 13(3):1095-1101 (2020). https://dx.doi.org/10.13005/bpj/1977.
    CrossRef
19. Syamsuri F, Hatta M, Natzir R, et al. Expression of TLR-4 in Salmonella typhi-Induced Balb/c Mice Treated by Miana Leaves (Coleus scutellaroides (L) Benth). Indian J Pub Health Res Dev.9(12):1449-54 (2018)
    CrossRef
20. Hatta M, Surachmanto EE, Islam AA, Wahid S. Expression of mRNA IL-17F and sIL-17F in atopic asthma patients. BMC Res Notes. 10(1):202 (2017). https://doi:10.1186/s13104-017-2517-9
    CrossRef
21. Mariana N, Islam AA, Hatta M,et al. IL23 mRNA expression in hirschsprung-associatedenterocolitis. J Med Sci.20:39-43(2020). https://doi: 10.3923/jms.2020.39.43
    CrossRef
22. Gunawan I, Hatta M, Benyamin AF, et al. The hypoxic response expression as a survival biomarkers in treatment-naive advanced breast cancer. Asian Pac J Cancer Prev.21(3):629-37 (2020). https://doi: 10.31557/APJCP.2020.21.3.629.
    CrossRef
23. Purnamanita, Budu, Hatta M, et al.The Effectiveness of Triamcinolone Injection on Risk of Postoperative Operations with the Conjunctiva Autograft Technique and Its Association with Change of VEGF mRNA Expression. Biomed Pharmacol J. 13(2):543-49 (2020). http://doi.org/10.13005/bpj/1916
    CrossRef
24. Walpole R. One and Two-Sample Tests of Hypotheses. In: Myers R, editor. Probability and Statistics for Engineers and Scientist. 8th ed. Pearson Enducation International.Macmillan New York.288–93 (2007).
25. Summerer I, Hess J, Pitea A, et al. Integrative analysis of the microRNA-mRNA response to radiochemotherapy in primary head and neck squamous cell carcinoma cells. BMC Genomics. 16(1):654 (2015). https://doi:10.1186/s12864-015-1865-x
    CrossRef
26. Li M, Liu Y, Wei Y, et al. Zinc-finger protein YY1 suppresses tumor growth of human nasopharyngeal carcinoma by inactivating c-Myc-mediated microRNA-141 transcription. J Biol Chem. 294(15):6172-6187 (2019). https:// doi:10.1074/jbc.RA118.006281
    CrossRef
27. Domoto T, Uehara M, Bolidong D, et al. Glycogen Synthase Kinase 3β in Cancer Biology and Treatment. Cells. 9(6):1388 (2020).https://doi.org/10.3390/cells9061388
    CrossRef
28. Zou H, Wu LX, Tan L, et al. Significance of Single-Nucleotide Variants in Long Intergenic Non-protein Coding RNAs. Front Cell Dev Biol. 8:347 (2020). https://doi:10.3389/fcell.2020.00347
    CrossRef
29. Nie H,Wang Y, Liao Z, Zhou J, Ou C. The function and mechanism of circular RNAs in gastrointestinal tumours. Cell Proliferation. 43:e12815 (2020).https://doi.org/10.1111/cpr.12815
    CrossRef
30. Nandi S, Liang G, Sindhava V, et al.YY1 control of mitochondrial‐related genes does not account for regulation of immunoglobulin class switch recombination in mice. Eur JImmunol. 50(6):822-38 (2020). https://doi: 10.1002/eji.201948385
    CrossRef