Description & Design
Description about Esophageal Cancer
Esophageal cancer is cancer arising from the esophagus. It is the eighth most common cancer and sixth leading cause of cancer mortality in the world. Especially, nearly 90% of esophageal cancer patient are found in china. Chinese patients account for more than a half of all deaths caused by esophageal cancer worldwide. Genetic factors and bad eating habits (e.g. addiction to alcohol, addiction to tobacco, eating too fast and drinking hot liquids) can be the cause of esophageal cancer.
5-year survival rate of esophageal cancer declines sharply as stage of cancer increases. In Stage I, during which cancer cells haven’t transferred, the 5-year survival rate can reach 70%. When it comes to Stage II, this number dramatically drops under 50%. And for Stage III and IV, it is 20% and 10% respectively. Thus early diagnosis plays an important role in the treatment of esophageal cancer.
However, since the early symptoms are not significant and the methods for clinical diagnosis of esophageal cancer are expensive and inconvenient, which makes the early diagnosis become difficult. Prominent symptoms usually appear only when a patient is already in the advanced stage. Early symptoms like swallowing difficulty, pain when swallowing, reduced appetite and loss of weight are often considered a minor illness and easily ignored. The diagnosing method used in hospitals mainly depends on radiology and endoscopic biopsy, which seems to be invasive and uncomfortable. To help solve this dilemma our team aim to build a convenient and harmless method for early diagnosis of esophageal cancer.
miRNA as Potential Biomarkers
Studies have confirmed that miRNA expression is highly concordant cross individuals. And aberrant expression of miRNA may relate to diseases. Some studies have reported that specific miRNAs in tissue and plasma can be discriminatory bio-makers for detecting cancers. However, getting access to tissue of plasma means physical harm to the human body. So we turn to the easily accessible saliva. Since saliva is considered to be a terminal product of blood circulation, components like proteins and RNAs which are present in plasma are also present in saliva. In fact, both coding RNAs and non-coding RNAs, including some miRNAs, have been found in human saliva. Although mRNAs are highly degraded in saliva, miRNAs are stably and abundantly present in saliva. Recently, there are many reports on cancer-related miRNA expression in saliva. Featured miRNA expression are reported to be found in oral squamous cell carcinoma, parotid gland tumors and esophageal cancer, indicating potential salivary miRNA to be biomarkers for detecting these diseases. According to the test done by Zijun Xie group, there are three type of miRNAs significantly upregulated in the whole saliva from the esophageal cancer patient group in contract to normal control group – miR-10b, miR-144, and miR-451 (p value 0.001, 0.012 and 0.002, respectively; AUC 0.762, 0.706 and 0.756, respectively). And four miRNAs are significantly upregulated in saliva supernatants from the esophageal cancer patient group – miR-10b, miR-144, miR-21 and miR-451. Among them, miR-21 is the most frequently reported one for its high performance in specific expression level related to esophageal cancer (according to one of the reports, p < 0.05, AUC = 0.8820, sensitivity = 90.20% and specificity 70.69%; different tests may report different results).
Comprehensively considering the performance of each miRNA, we finally chose miR-144 as our biomarkers for esophageal cancer. To make our detection quick and convenient, we designed synthetic gene pathways based on paper. It will only require some saliva to complete the detection, which will do no harm to human body. And this techniques can be expanded to be used in the detection of many other diseases which has specific miRNA expression pattern in saliva.
To make our detection quick and convenient, we designed synthetic gene pathways. It will only require some saliva to complete the detection, which will do no harm to human body. And this technique can be expanded to be used in the detection of many other diseases which has specific miRNA expression pattern in saliva.
What is Toehold Switch
We choose toehold switch as our miRNA detector. The structure of toehold switch is similar to hairpin, except it has a loop at the top as ‘toehold’. Toehold switch functions as riboregulator through linear-linear interaction between RNAs. When target RNA appears, it will bind one of the toehold switch stems and open the loop, exposing the RBS.
Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 50 end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the gene of interest.
Our Circuit to Detect miRNA-144
There are 3 parts in our circuit in total.
Part 1 includes toehold switch for miRNA-144 and GFP coding sequence. When miRNA-144 exists, the switch is on and mRNA for GFP is transcribed.
Part 2 contains toehold switch for GFP mRNA and T3 RNA polymerase coding sequence (BBa_K346000). Note that, since the maximum length of trigger RNA for toehold switch is about 25nt, so we analyzed GFP mRNA’s structure and choose a small piece from it which ensures binding specificity and stability.
Part 3 is simply a GFP generator (BBa_E0840), with T3 promoter. As we know, T3 promoter can only function when bound with T3 RNA polymerase.
So now it’s clear that, part 2 and part 3 are designed for amplification. They form a positive feedback loop. So the whole process is as follow: when miRNA-144 exists, it triggers part 1 and GFP mRNAs are transcribed. These mRNA on the one hand can be directly translated to GFP and show green fluorescence, on the other hand, can trigger part 2 and T3 RNA polymerase is transcribed and translated, which enables part 3 to work, thus transcribe more GFP mRNAs. And these GFP mRNAs can also be used to active more part 2.