Contribution
Our contribution experiment is based upon the YiYe-China iGEM project from 2021(BBa_K3822001). The project aims to validate a new method of early detection of colorectal cancer, using an RNA toehold switch system as a detector, to detect methylation in the important colorectal cancer oncogenes TFPI2 and SDC2.
We’d like to refresh literature background of this project and re-measure the detector so that everyone can understand it better and may use it in future projects.
1. New supplementary literature
Based on the reading of a large number of published papers, we added more information about the alteration of s TFPI2 and SDC2 methylation in colorectal cancer and from patients compared with healthy people.
1.1 In the first paper[1], the author used methylation of SDC2 and TFPI2 to defined three methylator phenotypes of colorectal cancer.
1.1.1 Background
Methylated SDC2 and TFPI2 are widely used for colorectal cancer (CRC) detection. However, they often miss some CRCs, which directly diminishes the sensitivity. Further investigations of the underlying mechanisms leading to the missed samples will facilitate developing more eligible methylation markers.
Fig. 1 Methylation status of SDC2 and TFPI2 in TCGA cohort. A, B β values of SDC2 and TFPI2 in CRCs and normal controls in TCGA dataset (A) and GEO dataset (B). C, D The histograms and density distributions of methylation β-value (C) and M-value (D) on TCGA CRC. The red and green curves in panel D indicated the distribution of M values fitted by the Gaussian mixed model.
1.1.2 Methods
CRC samples from TCGA and GEO datasets were divided into three groups, High-methylation/ High-methylation (HH), High-methylation/Low-methylation (HL), and Low-methylation/Low-methylation (LL) according to the methylation status of SDC2 and TFPI2 promoters. Variations in age, tumor location and microsatellite instable were then assessed between the three groups and verified in our custom cohort.
1.1.3 Results
Samples of HL group preferred to derive from left-sided CRCs (P < 0.05). HH samples showed the highest microsatellite instability and mutation load (mean nonsynonymous mutations for HH/HL/LL: 10.55/3.91/7.02, P = 0.0055). Almost all mutations of BRAF, one of the five typical CpG island methylator phenotype (CIMP) related genes, were observed in HH group (HH/HL/LL: 51/0/1, P = 0.018). Besides, older patients were frequently found in HH group. Expression analysis identified 37, 84, and 22 group-specific differentially expressed genes (DEGs) for HH, HL, and LL, respectively. Functional enrichment analysis revealed that HH-specific DEGs were mainly related to transcription regulation, while LL-specific DEGs were enriched in the biological processes of extracellular matrix interaction and cell migration.
1.1.4 Conclusion
The current study revealed that the performance of methylation-based markers might be affected by tumor location, patient age, mutation load and MSI, and these respective sides should be considered when developing new methylation markers for CRC detection.
1.2 In the second paper[2], the author used methylation profile of SDC2, TFPI2, WIF1, and NDRG4 genes in stool DNA, to screen of gastric and colorectal cancer in high-risk populations
1.2.1 Background
Because of poor compliance or low sensitivity, existing diagnostic approaches are unable to provide an efficient diagnosis of patients with gastric and colorectal cancer. Here, we developed the ColoCaller test, which simultaneously detects the methylation status of the SDC2, TFPI2, WIF1, and NDRG4 genes in stool DNA, to optimize the screening of gastric and colorectal cancer in high-risk populations.
1.2.2 Methods
A total of 217 stool samples from patients with gastrointestinal cancer and from patients with negative endoscopy were prospectively collected, complete with preoperative and postoperative clinical data from patients. The methylation of these samples was detected using ColoCaller, which was designed by selecting CpGs with a two-step screening strategy, and was interpreted using a prediction model built using libSVM to evaluate its clinical value for gastric and colorectal cancer screening.
1.2.3 Results
Compared to pathological diagnosis, the sensitivity and specificity of the ColoCaller test in 217 stool DNA samples were 95.56% and 91.86%, respectively, for colorectal cancer, and 67.5% and 97.81%, respectively, for gastric cancer. The detection limit was as low as 1% in 8 ng of DNA.
1.2.4 Conclusion
In this study, we developed and established a new test, ColoCaller, which can be used as a screening tool or as an auxiliary diagnostic approach in high-risk populations with gastric and colorectal cancer to promote timely diagnosis and treatment.
2. New experiment data
2.1 Methods
To confirm the validity of the toehold switch system as a nucleic acid detector, we inoculated recombinant bacteria that contained two plasmids constructed with toehold RNA, trigger RNA, and mCherry as the fluorescent reporter gene. After adding the incubated bacterial solution to a 96-well microplate (100 µl per well), we tested its fluorescence using a microplate reader and fluorescence microscope.
2.2 Results
The results from our experiment showed that the fluorescence value of the experimental group (containing both the toehold and trigger plasmids) was significantly higher (avg. 90,811.25) than that of the two control groups (toehold group avg. 54,611.75; trigger group avg. 58,897), and the negative control (ddH2O) in the cell-free testing environment.
Figure 2. Results collected from fluorescence analysis of recombinant toehold switch system bacteria groups. 1A. is a graph of the fluorescence values of the three groups, 1B, 1C, and 1D are the fluorescence microscopy images of the toehold, trigger, and toehold + trigger groups, respectively.
Figure 3. The results from the testing of the toehold switch system in a cell-free protein expression testing environment. From left to right, the tubes represent the toehold group, the trigger group, the toehold + trigger group, and ddH20. Only the toehold + trigger group emitted visible red fluorescence.
2.3 Conclusion
The fluorescence value of the toehold + trigger group was significantly higher than that of all three control groups, which is consistent with the experimental results obtained in the YiYe-China 2021 project. Thus, we can also conclude that this toehold switch system using mCherry as a fluorescent reporter gene is an effective nucleic acid detector, that can be applied to detect methylated oncogenes in colorectal cancer for an early diagnosis method.
Reference
[1] Ma Liang,Gong Jian,Zhao Meimei et al. A Novel Stool Methylation Test for the Non-Invasive Screening of Gastric and Colorectal Cancer.[J] .Front Oncol, 2022, 12: 860701.
[2] Lei Ruixue,Zhao Yanteng,Huang Kai et al. The methylation of SDC2 and TFPI defined three methylator phenotypes of colorectal cancer.[J]. BMC Gastroenterol, 2022, 22: 88.
