Design

Gastric cancer (GC), also known as stomach cancer, is a widespread and fatal disease. It has a 5-year survival rate of less than 20% and is the fifth-largest cancer in terms of both incidences and mortality [1]. Symptoms of GC include unexplained weight loss, severe abdominal pain, loss of appetite, nausea, and vomiting [2]. Gastric cancer is divided into three stages: tumor (T), nodes (N), and metastases (M) [3]. 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.

However, timely diagnosis can increase survival rates significantly. Patients diagnosed in the T, N, and M stages have 5-year survival rates of 75.4%, 35.8%, and 7.0% respectively [4]. These figures highlight the importance of timely GC diagnosis.

Currently, the three most widely used methods to diagnose GC are endoscopic ultrasound (EUS), positron emission tomography scan (PET-CT), and biomarker testing.

EUS is a minimally invasive technique to diagnose diseases in the digestive tract (including GC). When conducting the EUS test, the doctor inserts a long flexible tube with a device that emits and detects sound waves into the patient’s digestive tract. EUS uses sound waves to provide images of the digestive tract and tissues surrounding it [5]. However, GC diagnosis through EUS is qualitative and highly observer-dependent, reducing the method’s reliability, and it fails at detecting early-stage tumors that have not developed to an observable size [6].

PET-CT is an imaging test that helps doctors visualize the metabolic activity inside the patient’s body. It requires the patient to take or inject a radioactive tracer such as [18F] fluorodeoxyglucose (FDG) beforehand. PET serves as an accurate and effective way for doctors to visualize tumor growth and metastasis. Although PET is highly effective in detecting tumors, the radioactive tracer may damage the patient’s body, which is most damaging to pregnant women and feeding mothers. PET scans sometimes incorrectly recognize inflammatory regions as tumors. Thus, the outcome is qualitative and highly observer-dependent.

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 [7]. Biomarkers can provide an early diagnosis for diseases, including GC, based on their biochemical nature. However, due to the complexity of the human body and the indirect relationship between GC and biomarkers, the accuracy may be significantly reduced.

Our project aims at developing a novel diagnostic method based on the G3BP1 protein, a biomarker overexpressed in GC. To achieve this, we will utilize the HSU mRNA degradation activity of G3BP1. Details will be provided below.

To achieve our goal, we designed two experimental plasmids: pCMV-EGFP-EIF3B-HSU and pMIR-EIF3B-HSU. The pCMV-EGFP-EIF3B-HSU plasmid contains the GFP gene with a highly structured 3’UTR (HSU) region from the EIF3B gene connected to its downstream. The pMIR-EIF3B-HSU plasmid contains the luciferase gene with an HSU region from the EIF3B gene connected to its downstream. When transcribed, the mRNA for GFP and luciferase will have HSU structures. Meanwhile, both plasmids contain an origin of replication and antibiotic-resistance genes to serve experimental purposes. The plasmid maps for these two plasmids are shown in Figure 1.

When plasmids were transfected into cells, the cellular machinery will transcribe the GFP or luciferase genes into RNAs with HSU structures. In healthy cells, these mRNAs will last for a relatively long period and be sufficiently translated to produce large amounts of GFP or luciferase proteins. In desired conditions, a high concentration of GFP proteins will produce a high fluorescence value, and a high concentration of luciferase enzymes will show high activity. In GC cells, G3BP1 proteins will rapidly degrade the mRNAs, so the mRNAs will last shortly and be minimally transcribed. Subsequently, a small amount of GFP or luciferase proteins will be produced by translation. In desired conditions, a low concentration of GFP proteins will show a low fluorescence value, and a low concentration of luciferase enzymes will show low activity. Fluorescence values and luciferase activity can be monitored by devices, namely luminometers and fluorescence microscopes. In our experiments, we will use the GES cell line for healthy cells and the MGC-803 and AGS cell lines for tumor cells. The experimental outline is shown in Figure 2.

Additionally, we also used the plasmid backbones: pCMV-EGFP and pMIR. Meanwhile, we also constructed two plasmids with non-functional 3’UTR regions: pCMV-EGFP-EIF3B-MUT and pMIR-EIF3B-MUT. These four plasmids serve as control groups for the HSU plasmids. The plasmid maps for these plasmids are shown in Figure 3.

Additionally, we also used the plasmid backbones: pCMV-EGFP and pMIR. Meanwhile, we also constructed two plasmids with non-functional 3’UTR regions: pCMV-EGFP-EIF3B-MUT and pMIR-EIF3B-MUT. These four plasmids serve as control groups for the HSU plasmids. The plasmid maps for these plasmids are shown in Figure 3.

Because these four plasmids contain no HSU structure, G3BP1 will not have an effect on the expression of GFP or luciferase genes. Thus, there should not be a significant difference between the expression level of these proteins in all cell lines.

RNA is the only class of molecule that serves both storage and enzymatic functions. While nucleotide sequences store genetic information, RNA structures developed through folding enable them to interact with other biomolecules. Such natures provide RNA molecules with great flexibility and maneuverability. As explained previously, our project utilizes the secondary structure of HSU mRNA to diagnose GC. Therefore, future researchers can easily build upon our findings to make further developments or modifications.

First, we will amplify our genes of interest, constructed by companies, using PCR technology. Second, we will purify our gene of interest from other DNA fragments using gel electrophoresis and gel recycling. Third, we will restriction enzymes to cut stick ends on both our genes of interest and the plasmid backbones we purchased. Fourth, we will use ligase enzymes to construct our plasmids. Fifth, we will transfer the plasmids into DH5α E. coli bacteria through heat. Sixth, we will select bacteria containing the correct plasmids through culturing in an Ampicillin-containing LB medium and agar LB petri dishes. Seventh, we will lyse the bacteria and collect the plasmids. Eighth, we will transfect the plasmids into our cell lines, simulating GC and healthy stomach cells. Ninth, we will culture stomach cells in a DMEM medium. Lastly, we will test the luciferase activity and fluorescence activity of the cells.

To make our project a reality, we will make a test kit containing two types of plasmids and the needed reagents. The pMIR-EIF3B-HSU plasmid carries a luciferase reporter, and the pCMV-EGFP-EIF3B-HSU carries a GFP reporter.

First, the doctor will use a biopsy to collect stomach epithelial cells from the patient. Afterward, the cells will be cultured until they reach a concentration of 60%. Next, the doctor can use neofect reagents in our test kits to transfect the two plasmids into the patient’s stomach cells. The cells will be cultured for another 48 hours for gene expression.

Regarding the cells transfected with the pMIR-EIF3B-HSU plasmid, the mRNA codes for the luciferase protein and has a highly structured 3’UTR (HSU) region from the EIF3B gene [8]. G3BP1 is an HSU mRNA-degrading protein overexpressed in GC cells [9]. In healthy cells, low G3BP1 concentration leads to slow luciferase mRNA degradation, producing a large amount of luciferase proteins. In GC cells, high G3BP1 concentration leads to rapid luciferase mRNA degradation, producing a small amount of luciferase proteins. To visualize the luciferase concentration, doctors will lyse the cells to release luciferase proteins and add its substrate. A luminometer will be used to test luciferase activity quantitatively. The same process will be repeated using Renilla substrate as a control group to adjust the meaning. Lastly, it will be compared against our threshold value for diagnosis. The process is shown in Figure 4.