Team:CSMU Taiwan/Poster

Poster: CSMU_Taiwan



miRNA.DOC: A novel detection method for Oral Cancer

Presented by team CSMU_Taiwan 2020


Jian-An Pan1, Cheng-Yang Ma1, Yi-Ching Chen1, Hung-Yu Chen1, Huan-Jui Chang1, Dai-Rou Lee1, Hung-Liang Pai1, Tzu-Hsuan Hsiao1, Matilda Key1, Cheng-Ruei Yang1, Shen-Lin Chen2, Hsin-Jung Lee2, Kuan-Lin Chen2, Ting-Yu Lin2, Ting-Yu Lin2, Shao-Chi Lo2, Ho-Lo Huang2, Kuo-Chen Huang2, Dr. Yu-Fan Liu3


1iGEM Student Team Member, 2iGEM Team Advisor, 3iGEM Team PI


Abstract

Visual examination and palpation are current methods for oral cancer detection, but they are dependent on the experience and can result in detection bias1. Therefore, iGEM CSMU_Taiwan developed a new oral cancer detection method, miRNA.DOC, to tackle this problem. We adapted the "Toehold Switch" and glucometer to create a novel detection device2. The toehold switches we designed can detect miRNAs in human saliva and allow the reporter protein, invertase, to be translated. Invertase can break down sucrose into glucose and fructose. After that, the glucometer is used to measure the concentration of glucose and provide quantitative data. We successfully found the best two toehold switches, zr31 and zr146_A, from all the 21 toehold switches we designed after testing their ON/OFF ratios, sensitivities, and specificities. We also measured the glucose concentration under different amounts of the miRNA triggers and verified the positive correlation between the glucose concentration and miRNAs. With the regression curve formulas, we can measure the amount of the miRNA from the glucometer readouts. We hope to provide a quantitative, non-invasive, and accessible method for oral cancer detection with miRNA.DOC.

Introduction

Oral Cancer

According to WHO, oral cancer is all malignancies arising in the soft tissues of the lip, tongue and elsewhere in the oral mucous membranes. It's the eighth most common cancers worldwide. The global incidence rate is about 1.5 cases per 100,000 people3. While in Taiwan, it is 33.64, which is about 22 times higher than average. The relatively high prevalence of oral cancer is caused by a high-risk group in Taiwan who exhibit habits of betel nut chewing.


In addition, the average death age of oral cancer is 56 years old, which is 10 years less than other types of cancer5. One of the main factor that contributes to this is the delayed diagnosis of oral cancer in many patients6. An even more concerning issue is the increasing amount of cases each year. The yearly confirmed cases have doubled over the past ten years in Taiwan7. Therefore, oral cancer is a local and serious problem in Taiwan and needs improvement.

The Problem

According to the research of WHO, oral cancer has high cure rates when detected early and treated with best practices8. However, the current detection method is biased and often lead to delayed diagnosis. As Dr Yu-Feng Huang in CSMU said, "There exist problems in current detection method due to the detection bias."

The current detection method for oral cancer is mainly dependent on visual examinations. Dentist or ENT doctor perform visual examination and palpation to observe if there is any prodrome of oral cancer, such as white and red patch and long-term ulcers. When those oral potentially malignant disorders (OPMD) lesions are observed, those patients will be referred for a biopsy to confirm its malignancy. But, on average, there is only one OSCC case confirmed out of nine biopsies9. This indicates that the current detection method is biased and relies heavily on experience. In other words, the results may vary a lot according to different people conducting the process.

Toehold Switch Mechanism

Toehold switch is a lock that can repress the expression of the reporter protein. Correspondingly, our targeted RNA sequence serves as a key to open the lock.

Toehold switch is mainly composed of three parts, the trigger binding site (TBS) that is complementary to a target RNA sequence, a hairpin loop containing the ribosomal binding site (RBS), and a linker sequence that connects the toehold structure with the protein-coding sequence2.

When the targeted RNA sequence binds to the trigger binding sites, it unwinds the lower and upper stem and the RBS and start codon are exposed. Thus, the ribosome can bind to the RBS and start the translation of the reporter protein.

Biomarker

Our Goal - Choice of Biomarkers for Detecting OSCC


Among several potential biomarkers for detection of OSCC, we chose to examine salivary miRNA 21, 31, and 146 due to their relative ease of collection, validated diagnostic accuracy, and prospects of detecting OSCC early on.

Finding our Biomarkers - miRNAs


In our search of suitable biomarkers for detecting OSCC, we consulted professor Cheng-Chia, Yu, an expert in the field of OSCC rapid screening, and gained valuable insight on selecting biomarkers. Professor Yu told us: "It's important to consider whether your biomarkers can distinguish between different stages of oral cancer and their convenience for detection." He further suggested us to choose upregulated miRNAs due to their detection convenience, and select biomarkers that can distinguish patients at different stages. Therefore, we focused on selecting miRNAs which are upregulated in OSCC patients.

Salivary miRNAs - Diagnostic advantages on OSCC


miRNAs play an important role in the post-transcriptional regulation of tumour development, and the alteration of miRNAs in cancer cell illustrates their possibility to become biomarkers for detecting cancer11. In the context of OSCC, the dysregulated miRNAs may be released from the apoptosis or necrosis of cancerous tissue in the oral cavity11. Saliva can preserve this dysregulated profile of miRNAs due to the encapsulation of miRNAs in extracellular bodies and the direct contact of salivary miRNAs with oral cancer tissue12. Furthermore, the collection and analysis of saliva represent a cost-effective way for cancer screening12. As a result, salivary miRNAs encompass the diagnostics potential of miRNAs and the accessibility of saliva, making it a compelling biological sample for detecting OSCC.

Three Selected miRNAs - Improve the Efficacy of Detecting OSCC


Between multiple candidates of salivary miRNAs, we picked miRNA 21, 31, and 146 for their validated properties and diagnostic accuracy on early detection of OSCC. Specifically saying, miRNA 21 and 31 are capable of detecting OPMD (Oral Potentially Malignant Disease) which acts as a predictor of developing OSCC 13,14. On the improvement of diagnostic accuracy, miRNA 146 possesses high sensitivity and specificity on detecting OSCC 15. Additionally, with 9.84 fold of upregulation, miRNA 31 can discriminate OSCC patients from healthy individuals with significant differential expression 14. Consequently, we hope that the incorporation of 3 different miRNAs can effectively improve the robustness for our OSCC detection system.

Toehold Switch Design

Initially, we designed our toehold switches based off of Wang et al.'s (2019)16. They have designed a toehold switch for miRNA 21 which is also one of the miRNAs that we plan to detect. However, we were still having trouble with designing our own version of the toehold switch as the analysis with NUPACK showed unoptimal results. Therefore, we consulted with Alexander Green from Arizona State University and he advised us to design the toehold switch based off of one of his more recent studies on toehold switch for ZIKA virus (2016)17.
We split the design of toehold switches into three sections:
  1. Trigger binding site
    The trigger binding site (TBS) was solely designed upon reverse translating the sequence.
  2. Loop
    We looked at one of Green’s9 and Wang’s paper for our choice of design. In the paper, he designed two seperate loop structures and tested their effects on the ON/OFF ratio.
  3. Linker
    We adapted linkers from Green, Wang, and our randomization.
After they were put together, we ran NUPACK to ensure the spontaneous formation of the secondary structure at 37 degrees Celsius and Vienna RNA Package to predict the interaction between our trigger and the toehold switch. Figure 1. This is the result from NUPACK for one of our designs, it shows spontaneous formation and proper secondary structure for the loop.
Figure 2. This is the result from Vienna RNA package for the same design as the figure above. The black line in the figure indicates the amount of energy required to open the secondary structures of the TBS. The line red indicates the amount of energy required to open the secondary structure after the binding of the trigger.

Reporter

Reporter protein selection


Invertase, also called β-D-fructofuranosidase, is an enzyme for the hydrolysis of sucrose into glucose and fructose. It is commonly used as a reporter protein since its product, glucose, can be easily detected with a personal glucose meter (PGM)18, which has a high utilization rate in the public. According to the previous research, Thermotoga maritima Invertase (invertase from Thermotoga maritima) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase.19 Thus, we chose it to be our reporter protein.

Invertase activity test


We used both models and experiments to test the activity of invertase. For the modeling, we used MATLAB to create a model for kinetic investigations of the enzymatic reactions. As for the wet lab experiments, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations. The result of the model and the experiment is shown below.

Figure 3. The initial velocity of the invertase enzymatic reaction under different sucrose concentrations. The green line refers to the regression curve of experimental data, and the blue line refers to the invertase activity model.

The initial velocity of the reaction increases as the concentration of the substrate, sucrose, rises. The trend of experimental data fits our model.

Experiment

For each type of oral cancer-related miRNA, we have designed several toehold switches to detect it. With a series of experiments, we selected the best one for each miRNA and further tested its functionality. Here are our selection criteria for the toehold switch:
  1. High ON/OFF ratio. The protein expression should be high in the ON state (with trigger) and low in the OFF state (without trigger).
  2. High sensitivity. The final glucose signal should be high enough so that a small number of miRNAs can also be detected.
  3. High specificity. The toehold structure should only be opened by its specific trigger. In other words, the ON/OFF ratio of the non-specific miRNAs should be close to 1.
The plasmids of the toehold switches were transcribed and translated with the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) at 37℃ for 2 hours. Afterward, we added 5μl of 0.5M sucrose, and measured the glucose concentration with Bionime Rightest™ GM550 glucose meter after 30 minutes of enzymatic reaction time.
In our experiments, the ON state refers to the conditions with the miRNA triggers; while the OFF state means that there was no miRNA in the environment. We calculated the ON/OFF ratio of the toehold switch, which is defined below:
By measuring the ON/OFF ratios and the glucose concentration values in the ON state, we could find out the most sensitive toehold for microRNA detection.
Moreover, we used miR-191 and miR-233 for our negative selection, which are highly expressed in saliva20, to see whether our toehold switches would be turned on by other miRNAs. By comparing the ON/OFF ratios of the corresponding miRNA with miR-191 and miR-233, we could find out the most specific toehold switch.
With a systematic evaluation with ON/OFF ratio, sensitivity, and specificity, we could select the best toehold switches for detecting miR-21, miR-31, and miR-146.
For the best toehold switches, we further conducted a quantitative examination. We measured the glucose concentrations under different amounts of the miRNA trigger. Understanding the correlation between the two, we can measure the amount of miRNA from the glucose meter readouts and further build up a quantitative system for oral cancer detection.

Result

To select the best toehold switches for detecting miR-21, miR-31, and miR-146, we have carried out a functional test for each toehold switch.
The figure shows the glucose productions of the toehold switches in different states. The blue bar refers to the OFF state (without miRNA); the green bar refers to the ON state (with corresponding miRNA); the yellow bar refers to the state with non-related RNAs (with miR-191); the pink bar refers to the state with non-related RNAs (with miR-223). The ON/OFF ratio of each toehold switch with target microRNAs were listed below. As shown, most of the toeholds had high ON/OFF ratios and great sensitivities, as the glucose concentrations in the ON state are much higher than that in the OFF state. However, the results of the toehold switches for miR-21 detection were different from our expectation, thus we disregarded them for further experiments. As for the specificity, most of the ON/OFF ratios with the non-related microRNAs were close to 1, which means the toehold switches had great specificity to their own target microRNAs.
After a systematic evaluation with ON/OFF ratio, sensitivity, and specificity, we found that zr31 (BBa_K3431023) and zr146_A (BBa_K3431027) were the best toehold switches for miR-31 and miR-146 detection. Therefore, we further conducted a quantitative examination on them.
The glucose production of zr31 and zr146_A in the environment with different amounts of miRNA. As shown, the glucose concentration rises as the amount of the trigger increases, suggesting a positive correlation.

Future

Experiment


  • Validating freeze-drying
    • We will prove the feasibility to freeze-dry the reagents and our designed toehold switches onto the wall of the reaction tube. Also, we need to compare the freeze-dried version with our current version and modify the reagents accordingly.

  • Salivary Purification and RNA amplification
    • We will design and conduct experiments on saliva purification and RNA amplification prior to using the kit.

  • Finding threshold of the expression of miRNAs
    • We need to collect samples from oral cancer patients with different stages of the disease and conduct experiments to find out the threshold of the expression of miRNAs for each stage. 

Application


  • Hospitals and clinics
    • Doctors in the hospitals and clinics can use miRNA.DOC to deduce the amount of miRNA from the data shown on the glucometer and have a more objective screening process for better diagnostics.

  • High-risk group's workplace
    • Doctors can bring miRNA.DOC to high-risk group's workplace and use miRNA.DOC to screen for the workers who are too busy to see the doctors.

  • Home screening
    • We aim to lower the entry level requirement for using miRNA.DOC and allow patients to document the result from every test and track oral cancer development for doctors to assess the situation easily.

Commercialization

  • Taiwan Instrument Research Institute, National Applied Research Laboratories (TIRI, NARLabs )
    • They shared different collaboration patterns between research teams and biotechnology companies to choose from. After consideration, we believed that industry-academia cooperation was a better choice for us and thus we planned to cooperate with the glucose meter leading brand Bionime.

  • Product Launch
    • We visited Bionime, biotech company for glucometers, and demonstrated our product. Consultant Tsai gave us lots of positive feedback, and he also said he could assist us in industry-academia cooperation and find resources for our future research.

  • Blueprint


  • Timeline


    • Human Practices

    PROMOTE iGEM, SYNTHETIC BIOLOGY, and ORAL CANCER

    We hosted several events and activities to promote the core topics of the competition. We listed some of them below to highlight our achievement.

    ．Interview at The National Education Radio
    We introduced the severity of oral cancer in Taiwan, the current detecting procedure, and how we planned to improve it by miRNA.DOC. Furthermore, as our first attempt to meet up with people outside the scientific field, this radio interview brought us new perspectives and ideas on how to give our audience a more intact insight into the disease.


    ．Patient Experience Activity
    This patient experience activity highlighted the value of empathy. We learned from videos filmed by Taichung Veterans General Hospital about an active range of motion exercise for the mouth and specific cleaning methods for oral cancer patients. Consequently, during the club expo at CSMU, we demonstrated the two techniques to CSMU students and hope that they could understand more about the challenges the oral cancer patients face every day.

    ．Social Media Accounts
    We managed our social media accounts on Facebook and Instagram. Our weekly posts covered topics including details of our project, information on oral cancer, activities beyond the lab, experimental guidelines, and etc.. For every post, we drew illustrations, included both English and Chinese captions, and kept the caption lively and brief. Our followers have doubled since the launch of this project. We are confident to say that our followers are truly learning from our posts and that we are showing excellence in spreading scientific knowledge.

    ．I've Gotta PhD
    We collaborated with iGEM NCKU_Tainan and set up a Facebook page, I've gotta PhD, to allow every iGEM team to create health-related work. We came up with seven topics and formulated concrete guidelines to help other iGEM teams to participate. By now, we've received 13 pieces of fantastic work from 10 different iGEM teams all over the world. Each team targeted different topics and provided sufficient information to instill the concept of health into Internet users and better provide viewers different perspectives on health issues.



    TEAMING UP WITH OTHERS

    As iGEM is a worldwide community, we got to collaborate with other teams and boosted mutual learning. With iGEM_TU Delft, we created a video to educate the public about synbio. With iGEM_Ming-Dao, we offered them guidance on experimental skills and shared our practical experience in iGEM. With iGEM_CCU, we made a picture book introducing viruses. With iGEM_NCKU, we started the project of I've Gotta PhD for the purpose of promoting SDGs and better health.