Yin Yang 1 (YY1) And P53 Gene Expression Analysis in Cervical Cancer and Its Relationship with Cancer Staging

1Post Graduate Program of Medical Science, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. 2Department of Obstetrics and Gynecology, Faculty of Medicine, Pelita Harapan University & Siloam Hospitals Lippo Village, Tangerang, Indonesia. 3Department of Obstetrics and Gynecology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia. 4Molecular Biology and Immunology Laboratory, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. 5Department of Biochemistry, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. 6Department of Pathological Anatomy, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. 7Department of Biostatistics, Faculty of Medicine, University of Hasanuddin, Makassar, Indonesia. *Corresponding author Email: hattaram@yahoo.com

HPV (Human Papilloma Virus) is the main etiology of cervical cancer,with intertwined risk factors including multiple sexual partners and age of sexual activity. 3 HPV infection stimulates carcinogenesis in cervical epithelial cells through inhibition of tumor suppressor gene by E6 and E7 HPV-Encoded viral oncoproteins. 4 HPV-Encoded E6 oncoprotein has the ability to bind directly to p53 and cause degeneration via the E6-APmediated ubiquitination pathway. 5 Yin Yang 1 (YY1) is a ubiquitous zincfinger transcription factor, which has an important role in the controlling the cell cycle. 6 YY1 has a regulatory role in cell growth, development, and differentiation by influencing MDM2 and p53 levels withits rolein increasing the MDM2-p53 interaction, leading to the ubiquitination and degradation of p53. 7,8 Based on several studies over the past 2 decades, YY1 is gene transcription activator, repressor, and initiator that regulate the human genes. 9 While examining the tumor's YY1 expression, Zaravinos explained that YY1 has a role in inhibiting p300, a co-activator of p53. 9 Moreover, Wang portrayed that YY1 overexpression was associated with low-grade to high-grade cervical intraepithelial neoplasia (CIN) progression. 10 In turn, YY1 also able to bind to the HPV-18 upstream regulatory region (URR) to regulate viral oncogenes E6 and E7 transcription. 11,12 In HPV induced cervix cancer, p53 has an essential role as a tumor suppressor. 13 Wild type p53, which is unstable and only can last for 20-30 minutes, can act as cell cycle negative control and genome guardian; in which will then be degraded to p53-E6 complex or mutated p53. 13 Thereafter, p53 also utilized as a molecular prognostic indicator for pre-cancer lesion development or cancer treatment viability. 14 According to the author's knowledge, there are many comprehensive works dedicated to the research of cervical cancer. However, there is little to none published analysis of the intergenic factors affecting cervical cancer carcinogenicity of YY1 and p53. Therefore, it is sensible to modify the material and methods in order to improve and fully realize the potential of YY1 and p53 in cervical cancer.

MATERIALS And METHOdS
The study encompasses 20 cervical cancer patients in Siloam Teaching Hospital, Tangerang in July-September 2016 respondents who have signed the written informed consent. Ethical consideration was obtained from Hasanuddin University medical ethics committee with the references of 901/ H4.8.4.5.31/PP36-KOMETIK/2017.
The interventional study observes age, occupation, education, income, religion, contraception method, family history, smoking, cancer operability, medical history, chemotherapy, and cancer stage relations to cervical cancer gene expression, where blood samples are used for laboratory parameter analysis. YY1 and p53 mRNA gene expression were measured by real-time PCR assay and analyzed using Bio Rad CFX Manager 3.1.
In detecting mRNA YY1 and p53 gene expression quantitative real-time PCR was used. The study utilized a protocol which is optimized for CFX Connect system instruments, Biorad Laboratories, Real Time PCR 96 well 0.1 ml, USA; including the adaptation of diluting agents according to recommended factory manuals for the RT-PCR program. [15][16][17][18][19] The protocol involves initial DNA denaturation with 94 o C for 60 seconds, then 32 times cycles loop of 40 seconds 94 o C and 30 seconds 54 o C. In addition, qRT-PCR employs the one step Green QRT-PCR master kit.
The study sample was taken by random sampling and calculated by 49.65% prevalence, 0.883 proportion difference, 5% alpha and 80% power through ; while the results analyzed through t-test and correlation.

RESULTS And dISCUSSIOn
Within the research period, there are 20 eligible cervical cancer patients comprising 80% housewives, 40% elementary school students, and  19 52 -CGCCCAGCACGATGAAA-32 52 -CCGCCGATCCACACAGA-32 p53 23 5  21 Similarly, many studies portray protumor qualities of YY1 overexpression in breast cancer and primary tumors due to AP2 inducing the Erbb2 oncogene. 22 Baritaki et al explained that YY1 overexpression was also found in cervical cancer patients rooted from HPV-18 or HPV-16 infections. 22 Numerous research demonstrated that the majority of p53 mutations in cancers is missense mutation on the nucleus DNA binding domain. 23 In its application, p53 gene infusion into cancer cells, which beforehand has to lose its endogenic p53, display tumorigenesis reduction while vice versa for mutated p53 infusion. 24,25 The p53 levels also determined to be null or decremented on 71.05% blood samples and 72.73% of cervical cancer patients. Based on the cancer staging, 20% overexpression happens on stage I, 20% on stage II, 25% on stage III, and 66% on stage IV. 26 Significant difference of YY1 and p53 gene concentrations to cancer operability are observed (p: < 0.0001). Inoperable patients have 7.917 ± 1.110 and 10.492 ± 1.050 for YY1 and p53 concentration respectively, while operable patients have 12.891 ± 1.509 and 7.735 ± 1.153. On the other hand, age and marriage do not contribute to the operability of cervical cancer patients (p: 0.0971 and 0.9550) with an 8.00 years difference of age and 0.1 years old gap of marriage age between groups.
The correlation of gene expression and cancer stage as well as between the gene itself produces significant results (p: 0.023, 0.035,  and < 0.0001). Although, p53 expression has a proportional relationship with a coefficient of 0.473, YY1 expression has an inverse correlation with coefficient of -0.505. Likewise, the expression of YY1 against p53 shows a strong correlation, yet inversely proportional (r: -0.905). Viral load measurements were done and showed negative results on all respondents. Figure 1 demonstrates the correlation of YY1 gene expression on the cervical cancer stage showed that each increase of the cancer stage is followed by a decrease in YY1 gene expression. Although mildly related, this trend signifies the action of YY1 as an inhibitor of cell growth as well as YY1 incapability to keep up with the cell growth as cancer progresses.
Nevertheless, the correlation coefficient between p53 and cervical cancer stage display that each increase in staging is followed by an increase in p53 expression due to the gene mutations or dysregulation. YY1 and p53 gene expression relationshiphas the largest statistical correlation where p53 increase was followed by a significant YY1 decrease (p < 0.0001).
The study limitations include small quantities of respondents as compared to similar studies, study design, and study timing; in which cervical cancer can develop until 10-20 years after HPV infection.
Study describes the correlation between YY1 and p53 gene expressions in cervical cancer stages. It was found that the downregulated YY1 expression in cervical cancer as the stage progress is directly opposed to the p53 gene trend. The higher YY1 and lower p53 gene expressions dictate

Further prospects
The study serves as a basis for further investigation to analyze YY1 as well as p53 gene inhibitor and dysregulation nature respectively in neoplasm.