Project Description

Gastric cancer (GC), also known as stomach cancer, is a widespread and fatal disease. Each year, 7.0 per 100,000 people are diagnosed with GC, and approximately 0.8% of all people will be diagnosed with GC at some point during their lifetime [1]. Furthermore, GC has a very low 5-year survival rate of less than 20% [2]. As shown in Figure 1, GC was the fifth-largest cancer in terms of both incidences and mortality worldwide in 2022.

Symptoms of GC include unexplained weight loss, severe abdominal pain, loss of appetite, nausea, and vomiting [4]. GC progression is divided into three main stages: tumor (T), nodes (N), and metastases (M) [5]. In the T stage, the tumor develops from the mucosa and invades the submucosa, muscle layer, and serosa. In the N stage, the tumor migrates into adjacent lymph nodes. In the M stage, metastasis occurs, and tumor cells migrate to other body organs.

Although GC has a very low 5-year survival rate, timely diagnosis can increase survival rates significantly. Surprisingly, patients diagnosed in the T stage had a 5-year survival rate of 75.4%. This figure is followed by patients diagnosed in the N stage, with a 5-year survival rate of 35.8%. By contrast, patients diagnosed in stage M only had a 5-year survival rate of 7.0% [6]. These numbers highlight the importance of timely GC diagnosis.

Currently, the three main methods for diagnosing GC are endoscopic ultrasound, positron emission tomography scan, and biomarker testing.

EUS is a minimally invasive method to diagnose diseases in the digestive tract, including GC. It is carried out using a long flexible tube inserted into the patient’s digestive tract. Meanwhile, a device that emits and detects sound waves is used to create images. Compared to traditional endoscopy, EUS can provide images with more detail. Moreover, EUS can also provide information about not only the digestive tract but also about the tissues surrounding it [7]. Regarding GC, EUS can help doctors visualize tissues inside the patient’s stomach and determine the depth of tumor invasion as well as the involvement of nearby lymph nodes. Hence, EUS is also used to determine the stage of the patient’s tumor [8]. Furthermore, doctors can carry out biopsy through EUS. Afterward, they can conduct tests or search for biomarkers to diagnose GC. The process of EUS is shown in Figure 2.

However, GC diagnosis through EUS is qualitative and highly observer-dependent, reducing the method’s reliability. Meanwhile, it fails at detecting early-stage tumors that have not developed to an observable size [9].

PET is an imaging test that helps doctors visualize the metabolic activity inside the patient’s body. Unlike traditional computed tomography (CT), PET requires the patient to take or inject a radioactive tracer such as [18F] fluorodeoxyglucose (FDG) beforehand. FDG highly resembles glucose in its structure. Thus, tissues with high radioactivity indicate high FDG uptake and high metabolic activity [10]. High metabolic activity is associated with rapid proliferation and tumor growth. Therefore, PET serves as an accurate and effective way for doctors to visualize tumor growth and metastasis. For instance, the labeled dark regions in Figure 3 indicate GC tumors. M and T indicate tumors in these respective stages.

Although PET is highly effective in detecting tumors, the radioactive tracer may damage the patient’s body. This effect is most damaging to pregnant women and feeding mothers. Meanwhile, PET scans sometimes incorrectly recognize inflammatory regions as tumors, as these regions also have high metabolic activity [12]. In addition, PET requires extensive training and practice. Thus, the outcome is qualitative and highly observer-dependent. The above problems can lead to false-positive and false-negative results, reducing PET’s reliability in practice.

Biomarkers are biological molecules in patient tissues or body fluids that serve as signs to indicate particular illnesses. GC can be screened or diagnosed by biomarkers from the blood, saliva, urine, stool, and gastric juice. Biomarkers for GC include has-miR-1260b found in blood tissues, carcinoembryonic antigen (CEA) protein and mRNA preprolactin (PPL) found in the saliva, miR-376c found in the urine, G protein-coupled receptor 87 (GPR87) protein found in the stool, and BARHL2 DNA found in gastric juice [13]. Tests for different biomarkers vary in their invasiveness and accuracy. Based on their biochemical nature, biomarkers can provide an early diagnosis for diseases, including GC.

However, the indirect relationship between GC and biomarkers, as well as the complexity of the human body, significantly reduces the biomarkers’ accuracy. While biomarkers in gastric juice have the closest relationship with GC, many biomolecules, including DNA, are rapidly degraded in gastric juice due to its high acidity and the presence of some digestive enzymes [14].

Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1) is an oncogene with various important roles in GC cells. On a molecular level, it can trigger liquid-liquid phase separation (LLPS), thereby promoting the assembly of stress granules (SGs). In GC cells, SGs produced with the help of G3BP1 promote cell proliferation, resist apoptosis, and resist chemotherapy [15]. It has been widely reported that G3BP1 expression is upregulated in GC cells [16-17]. These results implied that G3BP1 can serve as a biomarker for gastric cancer.

Secondary mRNA structure is formed through intramolecular complementary base pairing within an mRNA molecule to achieve minimum free energy (−ΔG/nt) and thus become more thermodynamically stable. The secondary structure of an mRNA molecule depends on various factors, including base sequence, protein binding, and guanine-cytosine content. An mRNA secondary structure is considered to have a highly-structured 3’UTR (HSU) if its −ΔG/nt≥0.3. Alternatively, an mRNA secondary structure is considered to have a poorly-structured 3’UTR (PSU) if its −ΔG/nt ≤0.2 [18]. Computer software such as mFold or ViennaRNA can deduce the secondary structures of an mRNA strand and calculate their respective −ΔG/nt values [19]. An example of mRNA secondary structure is shown in Figure 4.

Specifically, G3BP1 targets HSU mRNAs over PSU mRNAs. At the molecular level, G3BP1 binds to double-stranded RNA (dsRNA) commonly found in HSU regions. Afterward, it recruits related proteins to degrade the mRNA molecule. Therefore, by monitoring the HSU mRNA concentration of a cell, we can indirectly detect the expression of G3BP1, which may diagnose GC patients. This process is shown in Figure 5.

As described previously, G3BP1 expression is upregulated in GC and can bind to HSU regions to facilitate mRNA degradation. Subsequently, we can measure HSU mRNA-degrading activity to reflect its expression level of G3BP1, which offers a promising approach for early diagnosis of gastric cancer.

To achieve this goal, we designed two systems. A GFP system and a fluorescence system.According to reports, G3BP1 binds to the HSU fragment in EIF3B mRNA and regulates its mRNA stability[20]. In both systems, we fused an HSU region from the EIF3B gene used to the 3’ UTR region of the reporter gene and constructed plasmids. Afterward, we transfected the plasmids into cells to produce mRNA. Upon cell culturing, measured fluorescence value from GFP or luciferase activity to measure the level of G3BP1 expression in cells. The experimental outlines for the two systems are shown in Figure 6.

We fused nucleic acid sequences containing an HSU region into a plasmid containing GFP or luciferase reporter (such as Fluc), transfected these plasmids into cells, and monitored the expression level of G3BP1 in cells by detecting the fluorescence level of GFP and luciferase activity.

As shown in Figure 6, in normal gastric mucosal cells, high levels of GFP or luciferase activity were detected because the low expression G3BP1 couldn’t degrade mRNAs containing HSU. On the other hand, in GC cells, the high level of G3BP1 has a stronger degradation effect on HSU mRNA, resulting in the low expression of GFP or fluorescence intensity.

In summary, our YiYe-China team aims to develop a “sensor” for gastric cancer screening, which is based on the degradation ability of G3BP1 on mRNA containing HSU, to detect the content of G3BP1 in the body of GC patients, thereby quantitatively reflecting the progress of the disease in real-time and providing strong support for early diagnosis of gastric cancer. This novel, efficient, and inexpensive detection method will pave the way for the accurate diagnosis and clinical staging of gastric cancer and completely change the new intellectual methods for this topic.