Increased of Protein O-Fucosyl Transferase 1 and 2 Genes Expression in Gastric Adenocarcinoma Tissue

Research Article

Clinics of Oncology

ISSN: 2640-1037

Volume 4

Increased of Protein O-Fucosyl Transferase 1 and 2 Genes Expression in Gastric Adenocarcinoma Tissue

Barahouie A1,4, Khosravi A2,3, Jazi MS1,2, Norouzi A5, Jafari SM1,4, Kebria FG6 and Asadi J1,2,4,*

1Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran

2Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran

3Department of Molecular Medicine, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran

4Department of Biochemistry and Biophysics, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran

5Golestan Research Center of Gastroenterology and Hepatology, Golestan University of Medical Sciences, Gorgan, Iran

6Gastroenterology and Hepatology Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran

*Corresponding author:

Jahanbakhsh Asadi, Department of Clinical Biochemistry, Golestan University of Medical Sciences, Gorgan, Iran.

Metabolic Disorders Research Center, Golestan University of Medical Sciences, Herkania Blvd, 01732451653, Gorgan, Iran; E-mail: dr.asadi@goums.ac.ir.

Keywords:

Gastric cancer; Adenocarcinoma; O-Fucosyl transferase genes; Real Time PCR

1. Abstract

Received: 16 Mar 2021

Accepted: 01 Apr 2021

Published: 05 Apr 2021

Copyright:

©2021 Asadi J, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially.

Citation:

Asadi J, Increased of Protein O-Fucosyl Transferase 1 and 2 Genes Expression in Gastric Adenocarcinoma Tissue. Clin Onco. 2021; 4(4): 1-6

1.1. Background: Gastric cancer is one of the most common cancers in the world. Gastric cancer usually occurs at an advanced age (average ≥ 65 years) and has symptoms similar to gastric ulcers and other gastric infections, its early diagnosis is one of the major problems of this type of cancer. Molecular mechanisms initiate cancer and the molecular changes of normal cells compared to cancer cells are very important. dysfunction of Fucosyl transferase enzymes is associated with gastric cancer. Among the fucosyl transferase enzymes, the protein enzymes O-fucosyl transferases plays an important role in the transfer of L-fucose glucose to cell signaling pathway receptors. Therefore, it seems that molecular study and a better understanding of POFUT1&2 enzyme gene expression can help to find prognostic targets and early detection and therapeutic target of gastric cancer.

1.2. Methods: Total RNA was extracted from tumor tissue and tumor margins using TRIzol, and cDNA was made after evaluating the quality and quantity of RNA. Finally, we examined the expression of the protein O-fucosyl transferase 1&2 (at the RNA level) by real-time PCR and measured relative to GAPDH.

1.3. Results: In this study, after examining the expression of O-Fucosyl transferase 1&2 genes in gastric tumor tissue (adenocarcinoma) and normal tissue, it was found that this gene increases the expression in tumor tissue (p _value < 0.05). Curve analysis showed the character of the receiver factor above the curve for POFUT1 (0.76) and POFUT2 (0.76). There was no significant correlation between Pofut1&2 expression level and clinical-pathological features (age and differentiation) of patients but a significant correlation between patients' sex.

1.4. Conclusion: In this study, by examining the gene expression pattern of protein O-Fucosyl transferase 1&2 enzymes in tumor tissue and gastric cancer tumor margin, we concluded that the Pofut1&2 genes in tumor tissue were significantly increased compared to normal tissue. Pofut1&2 can serve as a candidate for a clinically useful diagnostic biomarker and therapeutic target for gastric cancer.

2. Introduction

Gastric cancer is the fifth most common cancer worldwide and the third leading cause of cancer death [1]. Adenocarcinoma comprises most gastric tumors in which cells lining the gastric lining are

involved, while the rest of the tumors are non-Hodgkin's lymphoma, sarcoma, and carcinoid tumors [2]. Early-stage gastric cancer is often asymptomatic or has nonspecific symptoms. Symptoms develop over time, at which time cancer often reaches an advanced stage and may metastasize, which is one of the main reasons for its relatively poor prognosis [3]. Despite the declining incidence rate, the absolute number of new cases is increasing each year, mainly due to the aging of the world's population. More than half of patients with gastric cancer are over 60 years old and the average age at diagnosis is 65 years [1].

Glycosylation is the main modification of proteins after translation. N or O-glycans play a major role in protein synthesis and then modulate their functional activity [4] and the formation of receptor-ligand complexes for cell and cell interactions [5]. It is often found as an N-glycan carbohydrate compound in environmental situations, but it is also linked to the N-acetyl glucosamine nucleus. It is involved in the adhesion of selectin-dependent leukocytes, the stability of the maternal-fetal interface, and the formation of Lewis blood group antigen [6]. O-fucosylation is an unusual change after the translation of proteins catalyzed by two glycosyltransferases, protein O fucosyl transferase 1 (POFUT1) and protein O-fucosyl transferase 2 (POFUT2). POFUT1 modulates domains such as epidermal growth factor (EGF-like) and POFUT2 modifies thrombospondin modification (TSR) [7]. Increased evidence suggests the role of POFUT1 in controlling the balance between undifferentiated and differentiated normal cells [8]. The human glycoprotein POFUT1 is composed of 393 arm amino acids of chromosome 20, near the centromere [9]. POFUT1 is an ER-resident enzyme that allows focusing on S or T in C2X4 (S / T) C3 consensus motifs, where C2 and C3 are the second and third cysteines of the 6 protected species in EGF-like domains [10]. The most important known target is the POFUT1 Notch receptor, with four similarities in humans, NOTCH1 to NOTCH4, which contain between 29 and 36 EGF-like domains with 14 to 20 O-fucosylation consensus sites [11, 12]. Proper glycosylation of notch receptors, especially their O-fucosylation, is necessary for subsequent optimal degradation, which results in the release of NICD (Notch intracellular domain), which is displaced to control transcription of target genes in the nucleus [13, 14]. In mice, Pofut1 knockout is lethal. Embryos die in the middle of digestion with severe defects in somitogenesis, carcinogenesis, neurogenesis, and their phenotype is similar to that of low-impact embryos in the Notch signaling pathway [11]. O-fucose helps stabilize EGF-like, which participates in a new ER quality control pathway, essential for correcting the targeting of the Notch to the cell membrane and its interaction with ligands [15]. Recent evidence demonstrates the implications of Notch in tumor pathology. In acute T-cell lymphoblastic leukemia (T-ALL), the NOTCH1 gene is mutated in at least 65% of cases [16]. An extrauterine Notch1 expression induces epithelial-mesenchymal transmission in cancer growth and metastasis in human breast can-

cer [17]. In gastric cancer cells, activation of the Notch pathway also causes cell proliferation and metastasis, here phosphorylated via STAT3 and TWIST [18]. Overexpression of POFUT1 was also observed in oral squamous cell carcinoma and was associated with increased tumor size [19]. In gastric cancer, increased POFUT1 expression is associated with some clinical features such as higher TNM staging and tumor differentiation modes [20]. The POFUT1 gene is localized in the 20q11.21 region, which is often amplified in tumor cells such as the breast [21], acute myeloid leukemia [22], colorectal cancer with poor prognosis [23], and gastric cancer [24]. POFUT2 modifies thrombospondin type 1 (TSPs) repeats [25]. Thrombospondin I (TSPs) are small proteins (40 to 60 amino acids) found in secreted proteins and high cell-surface proteins that are involved in various cellular activities including cell binding, Extracellular Matrix regeneration (ECM), migration, Reproduction, and apoptosis are active. Many proteins containing TSPs require post translation modification with the unusual Glucoseβ1-3Fucose disaccharides for proper positioning and function. These disaccharides bind only to properly folded TSPs that are O-focalized by the enzyme Pofut2. Pofut2 deficiency impairs the secretion of target proteins and causes severe gastrolith defects in mouse embryos [26]. Our prior study of esophageal cancer showed that POFUT1&2 was more expressed in tumor tissue than in controls [27]. Although data from all of these studies suggest that Pofut 1&2 may also play an important role in the development of these cancers, and many studies have reported an association between O-fucosylation change and other invasive tumors, our knowledge of the impact O-fucosylation on gastric cancer remains unknown. Therefore, we started this study to evaluate the expression of POFUT1&2 in gastric cancer and to determine its potential value as a new biomarkers and therapeutic target for this cancer.

3. Methods

This study was approved by the Golestan Ethics Committee University of Medical Sciences (IR.GOUMS.REC.1398.235) and informed written consent was obtained from all patients.

3.1. Patient Samples

In the present study, 25 tumor tissues and 25 healthy tumor margins were collected during 10 months (December 2019 to September 2020) from Sayyad Shirazi Hospital (Gorgan, Iran). Tumor margin samples were collected from the same individuals from whom the tumor samples were taken. These samples were taken from seemingly normal tissue at a distance from the tumor site. Written informed consent was obtained from all patients. All of these specimens were confirmed by the pathology laboratory of the hospital (Table 2).

3.2. Real-Time PCR Assay (qRT-PCR)

Total RNA extraction was performed by Trizol (Invitrogen Cat. No.15596-026). cDNA synthesis was performed using the RevertAid First Strand cDNA synthesis kit (Thermo Scientific # k1621).

According to the manufacturer's instructions. Sequences of primers were obtained from published papers and purchased from Denazist Co. The list of Primers is shown in (Table 1).

Real-time PCR was performed by the qPCR Kit (Amplicon A325402–25 2x qPCR Master Mixed Green) using the Applied

Biosystems StepOnePlus Realtime PCR system. The thermal cycle conditions were optimized for Pofut1 at 95°C for 5 min, 38 cycles at 95°C for 20 Second, 61°C for 20 Second, 72°C for 20 Second and a final extension of the extension at 72°C for 2 min. To normalize gene expression, GAPDH was used as a household gene.

Table 1: Primers used in the RT-PCR reactions.

Table 2: Relationship between expression of POFUT1&2 in gastric cancer patients and clinicopathological features of patients (n=25).

Data presented as number of patients. a, Spearman's rho correlation. b, Independent sample test. NS, not statistically significant (P>0.05).

4. Statistical Analysis

Statistical analysis was performed using SPSS 25 software (SPSS Inc). Differences in POFUT1&2 expression between gastric cancer tissues and adjacent non-tumor tissues were analyzed using Mann-Whitney test and Independent sample test and Spearman’s Rank-Order Correlation. To determine the sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and gene-related cut-off value, the area under the receptor yield characteristic curve (ROC) was used. P <0.05 was considered statistically significant.

5. Result

To identify differences in POFUT1&2 gene expression, we performed POFUT1&2 expression with real-time PCR-based functional in tumor samples and cancerous healthy margins. There was a significant difference in the level of POFUT1&2 expression between tumor tissues and tumor margins. POFUT1 gene expression showed a Cycle of Threshold (CT) a maximum of 32.38 and a minimum of 27.22 in tumor tissue (mean = 29.8), and a maximum of 32.04, and a minimum of 28 in the healthy margin (mean = 31.22). In this comparison, it was found that the amount of POFUT1 in tu-

mor tissue increased significantly compared to the healthy margin (P value = 0.006) (Figure 1a). And too, POFUT2 gene expression showed a maximum of 24.83 and a minimum of 18.47 in tumor tissue (mean = 21.05), and a maximum of 26.69, and a minimum of 18.4 in the healthy margin (mean = 21.5). It was found that the amount of POFUT2 in tumor tissue increased compared to the healthy margin (P value = 0.001) (Figure 1b).

The relation between POFUT1&2 expression and clinicopathological features such as age, sex, and differentiation of tumors were analyzed as well (Table 2). The data showed no significant correlation between POFUT1&2 expression levels with age and differentiation but a significant correlation between patient's sex. Receptor Operational Characteristics Analysis (ROC) used to describe the discrimination accuracy of a diagnostic test or predictive model. Determining the best evaluation for POFUT1&2 gene expression in tumor samples compared to healthy margins. Analysis to evaluate the ability of POFUT1&2 as markers of gastric cancer showed that the area under the ROC curve for POFUT1 was (0.76) (Figure 2a) and for POFUT2 was (0.66) (Figure 2b) indicating the potential of POFUT1&2 as biomarkers for this malignancy. Relative expression was calculated using ΔCT the method.

6. Discussion

Comprehensive mechanisms by which tumors begin to develop are crucial because early detection of cancer can initiate treatment and increase a patient's chances of recovery. Fucosylation promotes the development of malignant tumors [29]. Recently, many studies show that increasing the expression of O-fucosylation is important in the invasion and metastatic behavior of cancers [28, 30-33]. O-fucosylation associated with the expression of the protein O-fucosyltransferase1 (POFUT1) and protein O-fucosyltransferase2 (POFUT2). Therefore, understanding the molecular mechanisms of O-fucosylation, especially O-fucosyltransferase enzymes, may be used to improve effective treatment strategies for cancers.

Our findings show that genes POFUT1&2 are expressed at the level of mRNA in Gastric tissue (tumors and tumor margins).

Quantitative gene expression analysis showed that increased expression of POFUT1&2 was found in tumor tissue. In humans, 87 targets of POFUT1 have been reported [34, 35]. POFUT1 and Notch have been described in two types of cancer. Notch receptors

were shown that their O-fucosylation is necessary for their interaction with ligands and therefore Notch signaling [36]. In breast cancer, overexpression of POFUT1 and NOTCH1 was associated with lymph node metastasis and advanced tumor stage [37] and in hepatocellular carcinoma, overexpression of POFUT1 causes abnormal activation of the NOTCH way in HES1, which increases cell migration and proliferation [38]. Moreover, NOTCH signaling and transcription factors are important regulators in the progression of gastric cancer [18]. It is hypothesized that POFUT1 may act through the NOTCH signaling pathway during the progression of gastric cancer.

Studies demonstrated that POFUT2 specifically adds O-fucose to TSPs [39]. The potential importance of O-fucosylation of TSRs in regulating TGFβ signaling is underscored [29]. POFUT2 is predicted to modify forty-nine targets, including members of Thrombospondin, ADAMTS (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeats) [25]. The secreted ADAMTS proteases cleave a variety of ECM substrates and have important

roles in modulating the structure and function of the ECM components and other secreted molecules. Several ADAMTs bind to fibrillins, which form tissue microfibrils that provide important mechanical ECM properties and regulate TGFβ signaling [29]. In the present study increased POFUT2 expression may play a role in tumor metastasis and progression in Gastric cancer by increasing the TGFβ signaling pathway, which should be further explored in future studies.

7. Conclusion

In conclusion, our findings showed that POFUT1&2 is overexpressed in gastric cancer and can serve as a candidate for a clinically useful diagnostic biomarker and therapeutic target for gastric cancer.

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