Investigation of RASSF4 gene in head and neck cancers Ba ş Boyun Kanserlerinde RASSF4 Geninin

Objectives: RASSF gene family can inhibit the growth of RAS oncogene. This gene family is suggested to have a role in cell cycle control, apoptosis, cell migration, and mitosis control. This study evaluated RASSF4 gene expression levels, SNPs and serum levels in tissues dissected from both healthy individuals and patients diagnosed with head, and neck cancer. Methods: RASSF4 gene expression levels were determined using the RT-PCR. Serum levels of RASSF4 were tested using the Enzyme-Linked Immuno Sorbent Assay technique in study groups. RASSF4 rs7896801 and rs884879 genotypes were identified using by the RT-PCR. Results: No statistical difference was observed between study groups according to RASSF4 gene expression levels. According to SNP results, rs7896801 revealed a 2.4 fold increase of G-allele presence in patients (p=0.015). The increase in the presence of AA genotype was statistically signi ﬁ cant for the control group (p=0.015). Distribution of genotypes and alleles for rs884879 showed a 2.2 fold increase in CC genotype for healthy group (p=0.031) how-ever, the presence of T allele showed a signi ﬁ cant increase in the patients (p=0.048). Conclusions: We suggest that this study will play a pio-neering role for the next studies on RASSF4 gene, especially on SNPs.


Introduction
Head and neck cancers are of complex nature that require extensive diagnosis, and treatment. These cancers involve the oral cavity, larynx, pharynx, salivary glands, and nasal cavity [1]. Alcohol and tobacco use are the two most important factors of head and neck cancers. HPV is also one of risk factors for head and neck cancer [2,3].
RAS has been identified to have the ability to activate both growth-enhancing and growth-inhibiting pathways [4][5][6]. These adverse activities suggest that activation of potent oncogenes such as RAS can control biological processes in a potential tumor cell. The discovery of the RASSF family allows for the disclosure of some of the growth inhibitory effects of RAS [7].
RASSF4 is expressed in various human tissues. The C-terminal has the RASSF4 SARAH area in the RASSF group [13]. The expression of RASSF4 inhibits the growth of human tumor cells. This effect is enhanced by the addition of a RAS CAAX membrane localization signal to the -COOH terminus of RASSF4. RASSF4 were shown to play a role in apoptosis, and cell cycle arrest, and often reduced in many cancers [7,14].
The aim of this study was to investigate the effect of RASSF4 gene on head and neck cancers at expression, SNPs and serum levels.

Materials and methods
The samples used in the study consist of tumor tissues, peripheral blood tissues, and tumor tissues of 68 patients who were diagnosed with head and neck cancer in the Department of Otorhinolaryngology, Istanbul Faculty of Medicine, Istanbul University. The system prescribed by the "American Joint Committee on Cancer (AJCC)" and "The International Union for Cancer Control (UICC)" was used for tumor staging.
The samples in control group, was obtained from 70 healthy individuals with no diagnosis of head and neck cancer. Informed consent forms were obtained from all participants according to the Helsinki Declaration. Ethics committee approval was obtained from the medical ethics committee of Istanbul Faculty of Medicine (1138/2017).
The clinical evaluations were performed, and the tissues, and blood samples were collected in the Department of Otorhinolaryngology, Istanbul Faculty of Medicine, Istanbul University. The blood, and tissue samples were preserved under proper conditions until the study was performed.
Total RNA, and DNA were isolated from tissue samples taken for expression, and polymorphism. Serum was obtained from the blood samples, and RASSF4 levels were determined.

DNA isolation
INVITROGEN PureLink ® Genomic DNA Kit was used for DNA isolation from tissues. The contents of the kit were prepared in accordance with the instructions, and the isolation was performed.

RNA isolation
Easy-BLUE ™ Total RNA Extraction Kit (iNtRON Biotechnology, Inc., Korea) was used for RNA isolation from tissues. The contents of the kit were prepared in accordance with the instructions, and the isolation was performed.

Reverse transcription
High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems ™ , USA) was used to obtain cDNA from isolated total RNAs from head and neck tissues RNA contents were added to the solution mixture in different concentrations to obtain a 100 ng/µL mixture.
The TaqMan Gene Expression Master Mix Kit (Applied Biosystems, USA) with probes was used to determine RASSF4 assay primers and their properties while TaqMan Gene Expression Assay, GAPDH was used for the control.

SNPs genotyping
The Taqman SNPs genotyping assay kit (Applied Biosystems, USA) was used to detect the rs884879 and rs7896801 in Real Time PCR Instrument (Applied Biosystems 7500 Fast Instrument, Applied Biosystems StepOnePlus ™ ). Probes for RS884879; FAM-labeled probe was used to detect the C allele. The VIC-labeled probe was used to determine the T allele. The probes for RS7896801 were used to detect the FAM-labeled probe A allele. The VIC-labeled probe was used to determine the G allele.

Statistical analysis
The statistical analysis was performed using the SPSS 21.0 Package program. The limit of significance was taken as p<0.05. χ2 and Fisher's Exact tests were used to evaluate the differences between the genotype, and allele incidence among the groups. Student's t-test, One-way Annova, and Mann Whitney U-tests were used to compare the demographic data between the groups.

Results
The study group consisted of 68 patients diagnosed with head and neck cancer, and 70 healthy individuals. There was no statistical difference regarding the age between the study groups (p>0.05).
No significant difference was observed in terms of RASSF4 gene expression in tumor and healthy tissue (p>0.05) ( Table 1).
The RASSF4 rs7896801 and rs884879 genotype and allele distributions were examined between the patients and controls, the frequency of rs7896801 G allele carrying was 2.4 fold higher in the patients (p=0.015, χ2=5.95, OR=2.4, 95% CI=1. . The frequency of carrying AA genotype increased significantly in the control group (p=0.015, χ2=5.95, OR=1.8, 95% CI=1.10-3.18) The examination of the RASSF4 rs884879 genotype and allele distributions between study groups showed that the frequency of carrying CC genotype increased 2.2 times in the healthy group compared to the patient group (p=0.031, χ2=4.64, OR=2.2, 95% CI=1.03-5.23). Significant increase was detected in the frequency of carrying T allele in the patient group (p=0.048, χ2=3.92, OR=2.5, 95% CI=0.98-6.74) ( Table 2).
No significant result was detected in accordance with the RASSF4 rs7896801 and rs884879 genotype distributions in the examination of RASSF4 serum levels (p>0.05). Also, there was no significant difference was detected in accordance with the tumor staging between early, and advanced stage tumors in examination of the RASSF4 gene expressions and serum RASSF4 levels (p>0.05) ( Table 3).

Discussion
Various aspects of head and neck cancer have been extensively studied, however, the role of mutations and mRNA expression levels were included in a small number of studies. Oncogenes such as PIK3CA, KIT, RAS were most susceptible to mutations; BRAF, PDGFRA, ABL1 and EGFR mutations were found to occur less frequently in the nasopharyngeal carcinoma study of Zhang et al. The comparison of the frequency of mutations in subgroups of patients with or without recurrence or metastasis showed that the frequency of recurrent and developing mutations were increased in nasopharyngeal cancer patients [15].
RAS/RAF/ERK pathway is known to have an important role in tumor development. KRAS, HRAS and NRAS mutations occur at least in one third of all human cancers. KRAS mutations are the most common mutations [16][17][18]. However, Zhang et al. found KRAS mutations less than expected in nasopharyngeal cancer compared to NRAS, and HRAS mutations [15].
Studies with RASSF4 are usually epigenetic inactivation studies caused by methylation or deletions. Decreasing RASSF4 mRNA expression and the hypermethylated promoter region of RASSF4 was found in head and neck squamous cell carcinoma, and nasopharyngeal carcinoma [19,20]. We did not find any significant differences in RASSF4 expression    [21]. Although RAS protein was detected in a nasopharyngeal cancer study, Porter et al. found no structural change in the RAS gene [22]. Herman et al. reported that promoter methylation plays an important role in function loss of some tumor suppressor genes [23]. Loss of RASSF1 expression in human cancer has been associated with abnormal promoter methylation in at least 37 tumor types. RASSF1A has the potential to be an ideal cancer biomarker because the methylation of RASSF1A occurs significantly in various tumor types compared to normal tissues [24][25][26]. RASSF2 promoter methylation is seen to correlate with lymph node metastasis in lung cancer cell lines, gastric cancer and nasopharyngeal carcinoma [27][28][29]. In a recent study, it was demonstrated that loss of RASSF4 promotes Multiple Myeloma progression by disconnecting RAS from its proapoptotic pathways [30]. Eckfeld et al. found no association in examining the coding sequence of RASSF4 in terms of inactivating mutations [7]. In our study, We observed that rs7896801 G and rs884879 T alleles play as a risk factor for head and neck cancer. Besides, we did not show any association among tumor staging and RASSF4 expression and serum RASSF4 levels.
This is the first study which examined the RASSF4 gene expression, serum levels, and SNP variants in Turkish patients with head and neck cancer. In our study, no significant significance was observed in terms of gene expression, but SNPs that may be risk factors for head and neck cancer were suggested.
We suggest that these preliminary data will lead to further studies for RASSF4 protein, especially in SNP studies.