Significant Upregulation of the HMGA1 and HMGA2 Genes in Iranian Patients Suffering from Gastric Cancer

Background: Gastric cancer is one of the most common malignancies worldwide, leading to high mortality. The HMGA family (High-mobility group) consists of regulatory proteins of chromatin structure and plays a crucial role in tumorigenesis. This study aimed to investigate changes in the expression of these proteins in the affected population of Khuzestan, Iran.

Materials and methods: a total of sixty tissue samples, including thirty gastric cancer tumors and thirty non-tumors (margins), were obtained from the cancer institute in Tehran. After extracting RNA and assessing its quantity and quality by nanodrop and agarose gel electrophoresis, we employed Real-time PCR to evaluate the expression of HMGA1 and HMGA2 genes. Statistical analysis was performed using GraphPad Prism 9.2.0.332 statistical software, along with t-test and ANOVA statistical methods.

Results: The results revealed that the relative expression of the HMGA1 gene in tumor samples increased significantly by 17 folds compared to non-tumor samples (P-value=0.0001). Similarly, the expression of the HMGA2 gene in tumor samples increased significantly by 15 folds compared to normal samples (P-value=0.0038).

Conclusion: The study demonstrated an increase in the expression of HMGA1 and HMGA2 genes in cancer cells of the affected population in Khuzestan, Iran. Understanding the mechanism behind this can contribute to early diagnosis and eventually prediction in high-risk individuals. Therefore, it is recommended to further investigate the role of these genes in future studies.

Gastric cancer is considered the fourth most common cancer globally, with both environmental and genetic factors contributing to its occurrence [1-3]. According to GLOBOCAN’ 2020 findings, 1,089,103 new cases of gastric cancer detected annually, accounting for 5.6% of total cancers, and approximately 768,793 deaths, representing 7.7% of the total deaths [4, 5]. More than 50% of newly affected people are diagnosed in developed countries such as South and Central America, East Asia [China and Japan], and Eastern Europe [6]. The 5-year survival rate is estimated to be below 30% in most regions of the world, emphasizing the need for diagnostic and treatment methods [7]. Factors such as age, high salt intake, Helicobacter infection, and diet are involved in gastric cancer, considered the world's third deadliest cancer [23, 24]. The diagnosis of this disease and its stage is done using methods such as CT, laparoscopy, and endoscopic ultrasound [23, 25]. Despite many efforts to control gastric cancer, the high rate of metastasis and drug resistance has practically inhibited the progress of this path [22]. For this reason, the requirement to find faster diagnostic and treatment methods is very noticeable. High mobility group (HMG) are a large family of non-histone proteins that have a high movement speed in electrophoresis due to their low molecular weight. The HMGA proteins play a crucial role in the gene regulation by binding to DNA’s minor groove and modifying chromatin structure [26, 27]. They are divided into three groups: HMGA-A, HMGA-B, and HMGN, each with its subgroups [8-10]. The HMG superfamily influences normal cell development and tumor growth by binding to AT-rich regions in DNA’s minor groove [11]. Studies indicate that the HMGA1 and HMGA2 families are involving in transcription regulation, cell cycle progression, embryogenesis, differentiation, aging, and tumor progression [12-14]. The HMGA1 (HMGI/Y) and HMGA2 (HMGI/C) are two functional subgroups that indirectly regulate protein-DNA interactions through epigenetic mechanisms [15]. They also serve as biomarkers for various malignancies, including gastric cancer [16, 17]. The HMGB family, which includes three subgroups HMGB1, HMGB2, and HMGB3 respectively, participates in many molecular processes as replication, transcription, recombination, and DNA repair [18]. Studies have also shown the relationship between the HMGN family and diseases such as diabetes, breast cancer, thyroid cancer, and resistance to treatment in liver cancer [19-21]. Analyses of the bladder, gastrointestinal tract, ovarian and breast cancers show an increase in the HMGA1 gene expression. This increased expression was also evident in lung cancer [31]. Some investigations conducted in different populations showed a significant relationship between the expression of HMGA1 and HMGA2 genes and gastric cancer. The pathways of HMG effect on gastric cancer are usually through interaction with microRNAs such as MicroRNA-495, the c-myc pathway, etc. [29]. Cytogenetic studies of endometriosis as a common female disease showed that HMGA rearrangement may be involved in its pathological complications and tumorigenesis [30]. Studies show that long-non-coding RNAs like HCP5 (HLA complex P5) can even cause drug resistance in gastric cancer through miR-519d/HMGA1 [32]. The latest findings also show that insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) can reduce the survival of GC patients through interaction with HMGA1. The IGF2BP2 directly effects on HMGA1 and leads to metastasis in the GC [33]. The HMGA2 and the HMGB3 could be important biomarkers in diagnosing and treating gastric cancer [34]. Because this issue was not addressed in the affected population of Khuzestan province (Iran), we aimed to investigate the eventual involvement of the mentioned genes in domestic patients suffering with gastric cancer.

Patients and tissue specimens

We collected a total of 60 tissue samples, including 30 gastric cancer tumor samples and 30 non-tumor samples (tumor margins), from the tumor tissue bank of Imam Khomeini Hospital in Ahvaz (Khuzestan, Iran). Patients from southwest Iran (26 men & 6 women) had not undergone chemotherapy or radiotherapy before sampling. Patients’ clinicopathological features are presented in Supplementary Table 1. The provided tissues were kept in a freezer at -80 degrees Celsius. We ensure that all procedures were performed in compliance with relevant laws and institutional guidelines in line with the declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects - and have been approved by the appropriate institutional committee of Shahid Chamran University of Ahvaz, Ahvaz, Iran (EE/99.3.2.39641/scu.ac.ir). The informed consent was recorded from all patients who participated in this study.

Table 1: Clinicopathological characteristics of patients participating in the research

qRT‐PCR: We extracted total RNA from tissues and cells following the manufacturer's instructions for the Trizol reagent (Invitrogen, USA). Incubation with 3 U/mg DNase R (Epicenter Technologies) was done for 20 minutes at 37°C. We assessed RNA quantity and quality using a nanodrop device (ND-100, Thermo) and agarose gel electrophoresis. Subsequently, we synthesized cDNA using the Prime Script™ II 1st strand cDNA Synthesis Kit (Takara, Japan). We performed qRT‐PCR with a real‐time PCR system (Light Cycler 96, Roche, Germany) using SYBR Green Master Mix (Takara, Japan). The expression levels of HGMA1 and HGMA2 genes were measured using the 2−ΔΔCT (Livak) method. The primer sequences were designed by Allele ID® software and were obtained from Genecopoeia (Table 2). The specificity of the primers was checked by primer blast from the NCBI database. The human ACTB gene primers were used for amplification of the housekeeping gene as the normalizer to the two target human genes (HMGA1 & HMGA2). Thermal cycling conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 secs, annealing at 62°C for 30 secs, and extension at 72°C for 30 sec. The results were normalized to the Actin Beta gene (ACTB) expression.

Table 2: Primers used in Real-time PCR

Ethical consideration: The Islamic Azad University of Dezfoul financially supported the present study; furthermore, the Ethics Committee of the University approved this work [ethics code: IR.IAU.D.REC.1401.038]. This article does not contain any studies with human participants or animals.

Statistical analysis: We utilized the t-test and ANOVA to compare the tumor and normal groups, and the results were considered statistically significant with a P≤ 0.05. Analysis was conducted using PRISM 8.0 software.

The HMGA1 expression characteristics in GC

Comparison of the relative expression of the target gene in tumor samples compared to non-tumor samples showed that the relative expression of the HMGA1 gene in tumor samples was 17 times higher and increased significantly (P-value=0.0001) (Figure 1). The Fold change (2^-ΔΔCt) was estimated at 6.17 for HMGA1 Gene-related data.

The HMGA2 expression characteristics in GC

Comparison of the relative expression of the target gene in tumor samples compared to non-tumor showed that the relative expression of the HMGA2 

gene in tumor samples compared to non-tumor increased 15 times significantly (P-value=0.0038). The fold change (2^-ΔΔCt) was estimated at 9.14 for HMGA2 gene-related data.

The relationship between gene expression changes

This research measured the correlation coefficient to evaluate the expression changes of two HMGA1 and HMGA2 genes. For this purpose, each gene’s fold change was compared with another gene’s fold change. The obtained results are given in Table 3.

Table 3: P-value and Fold Change data analysis of HMGA-1 and HMGA-2 gene

Based on the related HMGA1 and HMGA2 expression data, co-expression analysis was performed to calculate the correlation between the two mRNAs of the genes. Co-expression analysis shows significantly positive co-expressed between two genes. The correlation coefficient between the two genes was calculated at 0.245 and the P-value was 0.193. The relationship between gene expression and clinicopathological characteristics. We investigated the relationship between HMGA1 and HMGA2 gene expression changes and clinicopathological characteristics of gastric cancer samples using the t-test, ANOVA, and p = 0.05. The results are given in Table 4. According to the results, there was no significant correlation between HMGA1 gene expression changes and the clinicopathological characteristics of gastric cancer samples. Regarding the HMGA2 gene, a significant correlation was observed in distant metastasis and invasion around the nerve and the tumor stage. But no significant relationship was observed in other clinicopathological characteristics. Because the number of sample distributions regarding the depth of invasion was not enough (refer to Table 4); statistical analysis to find the relationship between the mean relative expression of the tested genes and this feature could not be performed.

Table 4: The relationship between gene expression and clinicopathological indicators of gastric cancer samples (* is statistically significant).

Because of different nature of cancer types, the genetic architecture of tumors is very different. Therefore, types of cancer must be considered differently. In addition, the epigenetic effects in the development of tumors should not be ignored, as factors such as lifestyle, diet and even geographical conditions can support the development of tumor cells. In order to understand individual steps of the tumor cells, a direct examination of the tumor tissue can sometimes be informative in terms of genetic changes. For this reason, we aimed to investigate the eventual genetic changes, particularly the changes of the expression pattern of two oncogenes, in gastric tumors. The results of the present study showed that comparing the relative expression levels of the HMGA1 and the HMGA2 genes in tumor samples increased significantly by 17 folds (P-value=0.0001) and 15 folds (P-value = 0.0038), respectively. Obtained results are of interest because they were conducted for the first time in Iranian patients with gastric cancer and clearly demonstrate the progressive role of these oncogenes in gastric cancer. The massive increase in gene expression of both target genes in this work suggests that the development of gastric tumors could be accompanied at least by their Upregulation. 

Our tumor samples were mostly from pre-metastasis stage, which shows that the increase in both oncogenes occurred at earlier stages. Age of onset and gender of patients were apparently insignificant, apart from minor deviations (table 1). Previously, a study of 55 cases of gastrointestinal neuroendocrine tumors showed overexpression of the HMGA1 and HMGA2 genes in gastrointestinal neuroendocrine tumors compared to normal tissue. The results of the mentioned report are consistent with ours and this agreement shows the importance of these genes in this type of cancer [35]. Genetically, there are three groups of genes responsible for the pathogenesis of tumor growth known as the oncogenes, the tumor suppressor genes, and the repair genes, respectively.  Imbalance between the mentioned gene groups usually leads to malignancy, where some of those code for transcription factors [36]. The HMGA1 and HMGA2 genes are knowingly an oncogene and regulate chromatin modeling and remodeling and. Their oncogenic effect influences many cellular processes from proliferation to apoptosis [37]. There are numerous studies that demonstrate the existence of regulating miRNAs or lncRNAs of both oncogenes [38]. Therefore, we suggest that the inhibition of upstream regulators of these two genes could slow down or even stop the progression of the tumor cells. In addition, and as a diagnostic tool, the regulators can be determined in the first phases of cancer development and create a prognosis. 

The expression of HMGA1 and HMGA2 genes in tumor tissues from individuals with gastric cancer showed a significant increase of 15- to 17-fold compared to normal marginal tissues. This clearly reflects the previous reports of the significant alterations of the two genes in various cancers and shows the importance of these oncogenes in cellular malignancy. The study highlights a significant increase in the expression of both oncogenes in tumor tissues and highlights the need to explore their mechanisms in oncogenic cells. Further studies in different populations may shed light on the involvement of these genes in the invasion and metastasis of gastric cancer cells. Considering the importance of the subject, we suggest to identify the mechanism of action of the HMGA1 and the HMGA2 in tumor and non-tumor tissues and their pathway in the invasion and metastasis of gastric cancer cells in different populations be examined more precisely and completely by larger tumor samples in different stages and on different cell lines driven from human gastric tumor.

Our special thanks go to Shahid Chamran University of Ahvaz, Ahvaz, Iran for supporting this work.