NOTCH Target Gene HES5 Distinct Anti-Tumorigenic Roles in Gastric Carcinoma

doi:10.21203/rs.3.rs-857387/v1

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NOTCH Target Gene HES5 Distinct Anti-Tumorigenic Roles in Gastric Carcinoma

https://doi.org/10.21203/rs.3.rs-857387/v1

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I. Background:Gastric cancer (GC) is ranked the third greatest cause of mortality globally and the second common cancer in Iran. To date, many pathways including HES family have been found to be linked to cellular proliferation, differentiation and cancer. HES5, a transcription factor that binds to DNA, is known as a downstream protein in Notch pathway. Therefore, we aimed to investigate the association between HES5 expression and GC.

II. Methods and Results:In this study,75 gastric cancer patients were included. After RNA extraction and cDNA synthesis, the expression rate of HES5 was evaluated by quantitative Real-time PCR. Also, presence of H. Pylori infection was verified using specific primers set for H. pylori, and PCR on extracted DNA. The results showed a remarkable decrease in HES level in malignant tissues when compared to the neighboring non-cancerous tissues (Normal) (P< 0.0001). Moreover, an inverse relation between down regulation of HES 5 gene and H.Pylori infection was identified. In conclusion

III. Conclusions: These findings emphasize that HES5 may notably suppress the tumor development.

General Biochemistry

Molecular Biology

Gastric cancer

HES

expression

H. Pylori.

Gastric cancer (GC) is one of the most prevalent cancers in the world and ranked as the third leading cause of cancer-related fatalities globally (1).GC is estimated as the most common cancer in Iran. Regarding the increasing frequency of Helicobacter pylori (H.pylori) infection, poor diet habits and insufficient early screening methods, GC remains one of the leading cause of death among gastric intestinal(GI) cancers in Iran(2). Recent genetic profiling approaches of The Cancer Genome Atlas (TCGA) identified critical signaling pathways that might be implicated in the development of human GC. Although, understanding how these pathways drive GC cell development is critical in order to determine how they may be targeted for GC therapy. The Hairy/Enhancer-of-split (HES) gene family encode seven basic-Helix-Loop-Helix proteins in human contain Hes1–7. HES factors have three conserved domains: the Orange domain, the WRPW domain, harboring a WRPW motif (Trp-Arg-Pro-Trp) and the basic helix–loop–helix (bHLH) domain (3, 4). These genes ,encode a group of nuclear proteins that suppress the transcription in cells through either active or passive manners (5). HES genes regulate the proper schedule of cellular differentiation and conservation of stem and progenitor cells by antagonizing the effects of bHLH activators(3, 4).

HES5, as a member of HES family, acts as a DNA-binding transcription factor that functions as a downstream protein and effector of human Notch pathway (6). HES5 is involved in neural stem cell maintenance, regulating mammalian neuronal differentiation and neuronal stem cell self-renewal (7, 8). It is also implicated in cell differentiation and elevating tumorigenesis in cancers including prostate, non-small- cell lung, ovarian ,liver, and neuroblastoma (8-12). According to these studies, HES5 may play a role in the onset and progression of cancers.

HES5's expression pattern and biological functions in human gastric cancer remain unclear at the present moment. In this study, we investigated the link between HES5 and gastric cancers to specify whether it is involved in the mechanism of malignancy and if there is a correlation between its expression and clinicopathological data.

Tissue samples

All tissue samples investigated in this study gathered from Omid and Ghaem hospitals of Mashhad University of Medical Sciences (MUMS), Iran. Prior to surgery, all patients did not undergo any therapeutic intervention such as chemotherapy and radiation. Fresh tissues were promptly transmitted to the RNA later solution (Qiagen, Hilden, Germany) and kept at −20 °C until RNA extraction.

Inclusion criteria included not having had any therapy prior to surgery and histological confirmation of more 70% of tumor cells in resected samples. Samples' histopathological features such as size, tumor location, and grade were documented. The Union International Cancer TNM classification criteria were used to identify the surgical stages of tumoral samples.(13). Informed consent forms were obtained from all the patients.

RNA extraction, cDNA synthesis and quantitative real-time-PCR

RNA extraction from all samples was performed using RNeasy Mini kit (Qiagen, Germany) based on the manufacture protocol. RevertAid first strand cDNA synthesis kit (Fermentas, Lithuania) was utilized for the mRNA reverse transcription. Quantitative Real-time PCR was carried out using a Light cycler (Roche, Germany) utilizing HES 5gene-speciﬁc primer sets on and RealQ Plus Master Mix Green (Ampliqon, Denmark). Primer sequences are mentioned on table 2. The thermal profile consisted of 15 minutes at 95℃ followed by 40 cycles of 25seconds at 95℃, 25seconds at 60℃, and 25 seconds at 72℃ was applied. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) expression was employed as a normalizer for data analysis (14). All experiments were carried out in duplicate. Over expression was defined as a fluorescence intensity of mRNA expression in tumor tissue that was greater than twice the average of the adjacent normal tissues. Underexpression was also defined as less than twofold expression, and the difference between them was regarded as no change.

DNA extraction and detection of H. Pylori infection

Genomic DNA was extracted from normal and tumoral gastric samples by the enzymatic method. 10mg of tissue was lysed in 200 μl of digestion buffer, 20 μl of proteinase K, and 5 μl RNase at 55 °C for 3 hours’ incubation. Phenol and chloroform were added to the mixture for purification. The DNA pellet was observed after two volumes of 100 % ethanol and 0.1 volume of 3 M sodium acetate were added to the supernatant. The pellet was then washed by ethanol 70% and was immersed in TE buffer subsequently. PCR was done on extracted DNA using specific primers set for H. pylori genes of UreC (glmM), 16S rRNA, and virulence factor of CagA according to the table 2 sequences.

Statistical analysis

The statistical analysis was performed using SPSS 22 statistical package (SPSS, Chicago, IL). The correlations between the expression of the gene and histopathological features of patients, including age, sex, grade, stage, and lymph node metastasis, were analyzed based on the requirement, using either χ2 or Fisher exact tests. The Independent sample T-test and ANOVA were used to identify the correlation between gene expression and different categorical data. P values of less than 0.05 were deemed statistically significant.

Study Population

The clinicopathological features of 75 gastric cancer patients involving 55 males and 20 females are shown in table 1. The general mean age ± SD (the standard deviation) of all patients at the time of diagnosis was 63.03 ± 10.98 years, and mean size ± SD of tumor samples was 6.46 ± 3.11 cm. Most cases (54 out of 75-72%) were in stages II/III of the tumor but others (15/75, 20%) were in stage IV and no case was in the first stage. 61/75 (81.3%) of tumor samples had lymph node metastasis. Based on the histopathological analysis, tumors located in the cardia position were 31/75 (41.3%), while 44/75 (58.7%) found in noncardia regions of the stomach. About 52 tumor samples (69.3%) were classifiedas intestinal form, whereas19 (25.3%) and 4 (5.3%) tumors were classified as diffuse and mixed types, respectively. All tumors were investigated for H. pylori (Helicobacter pylori) infection according to specific primers for three genes of 16s rRNA, UreC, and CagA. 33 of the tumor tissue samples (44%) were positive for CagA.

Expression analysis of HES5

Relative comparative qRT-PCR indicated that log2 fold change of HES5 was decreased at mRNA level in 54.7% (48/86) (P< 0.0001) compared to the adjacent normal tissues, in comparison 20% (44.18) of the samples showed overexpression or normal levels of HES5, respectively. The profile of gene expression in all patients is displayed in Figure 1 as a scatter plot. The minimum and maximum expression fold changes showed in figure 1B. Moreover, the average HES5 expression level of gastric cancer tissues was1.53 ± 4.23, as well as the average level of matched adjacent non-cancerous tissues, was 3.36 ± 4.81 and -1.74 ± 3.06, respectively. We identified that the HES level in malignant tissue was considerably lower than neighboring non-cancerous tissues (Normal) (P< 0.0001).

Correlation between hes-5 and histopathological features

Table 1 lists all of the clinicopathological characteristics of the patients. Overall analysis showed significant correlation between expression of HES 5 gene and location of tumor (p= 0.05). Furthermore, there was a significant correlation between overexpression of HES5 and gender (p= 0.039).

The majority of cases (31 out of 75 (41. 3%)) were located in non-cardia position. There was a significant association between the tumor location and mRNA expression levels.61 out of 75 of the cases (81.3%), had lymph node metastasis. The majority of cases (70.6%) were on stages III/IV and 65.3% of all patients showed middle grade. Evaluation of H.Pylori infection in tumoral samples showed that 33 out of 75 samples had positive signs of infection. Moreover, an inverse correlation between H.Pylori infection and downregulation of the HES 5 gene was detected (p=019). In contrast to sex, location and, H.pylori infection, there was no significant correlations between HES 5 mRNA expression and tumor stage, lymph node involvement, size of the tumor, and grade.

Gastric cancer is estimated to be caused by genetic, epigenetic or environmental factors that disrupt potentially oncogenic signaling pathways. Previous studies suggested that oncogenic cellular pathways, such as Wnt/β-catenin, Hedgehog, growth factor receptor (EGFR), nuclear factor-κB and Notch pathways are involved in gastric carcinogenesis (15). Activation or deactivation of each element of theses pathways result in increased cellular proliferation, evasion of apoptosis or arrest of cell cycle check points. The malfunctioning Notch signaling cascade is one of these pathways that have been linked to a variety of cancers. NOTCH ligands and receptors appear to function as tumor suppressors or oncogenes depending on the type of cancer. The HES family transcription repressor members are among the most prevalent target genes contributing to the canonical NOTCH pathway response, although their role in gastric cancer is unknown. In this study, we assessed the level of HES5 gene expression in gastric tumor cells to investigate the possibility of HES family interference in tumors progression of gastric carcinoma patients.

Gathering data from the previous study reveals the oncogenic function of HES5 via NOTCH signaling pathway. The comparison of lung cancer tissues to adjacent non-tumor tissues as well as three cell lines, SPCA1, H1299 and A549, indicated that HES5 might be a significant factor in the lung cancer cell proliferation (9). Another study in cervical carcinoma suggest that Hes1/Hes5 gene would inhibit the cell differentiation by down-regulating the Hash1(16). Also, increased expression of HES5 in ovarian cancer cells might be considered as a prognostic marker (12).

Another study also indicated that HES5 has both oncogenic and suppressive tumorigenic functions in different organs. According our results, tumoral cells showed downregulation of HES5 compared to normal cells, while other data suggested HES5 silencing as an early event in prostate tumorigenesis and proposed this gene as an advantageous biomarker for targeted intervention strategies(11). In line with our results, it has been demonstrated that down regulation of HES1 and HES5 limits the neural differentiation and preserve neural stem cells in the embryonic brain (17). Additionally, a significant correlation between HES5 and the age of our patients were observed, probably this gene is related to aggressiveness. Therefore we concluded that there is an inverse correlation between age and the levels of HES5 mRNA expression. Hence, we speculate that downregulation of HES5 could be a key factor in gastric cancer progression.

These findings emphasize that HES5 may act as a double-edged sword in cancerous cells. This study was the first evaluation of HES5 expression in gastric tumoral cells which showed a significant suppression effect in malignant tissue compared to normal cells. Future studies are required to clear the connection between HES5 and NOTCH pathways or other genes.

GC: Gastric cancer

H.pylori: Helicobacter pylori

GI: gastric intestinal

TCGA: The Cancer Genome Atlas

HES: Hairy/Enhancer-of-split

bHLH: basic helix–loop–helix

Funding

No funding.

Conflicts of interest/Competing interests

The authors declare that they have no conflict of interests.

Ethics approval

The study was approved by the Ethics Committee of Mashhad Universities of Medical Sciences, Iran (IR.MUMS.fm.REC.1397.75).

Consent to participate

Each patients was informed about the study and consent was obtained from them.

Acknowledgements.

Not applicable

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Table1: The clinicopathological data of 75 gastric cancer patients.

Table2: The sequences and the annealing temperature of primers

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08 Sep, 2021
First submitted to journal
28 Aug, 2021

You are reading this latest preprint version

NOTCH Target Gene HES5 Distinct Anti-Tumorigenic Roles in Gastric Carcinoma

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