Current Colorectal Cancer Screening
Data from Global Cancer Statistics 2020 shows that the combination of colon and rectum cancer holds for approximately 10% (1,880,725) and 9% (915,880) of all cancer incidence and death in the world respectively [1].
In Indonesia, colorectal cancer stands at rank 4th and 5th of the most incidence and deaths respectively in Indonesia. It holds approximately 8.4% (33,427) and 7.6% (17,786) of all cancer incidence and death in Indonesia respectively [2].
Whereas, data from the Jogja Cancer Registry indicate that CRC ranks as the second among the most frequently encountered cancer cases in Yogyakarta [3].
Despite the slow progression of CRC, taking approximately 10-15 years [4], a majority of CRC patients, including in Yogyakarta are diagnosed at advanced stages due to the difficulties in diagnosis even though early diagnosis plays a crucial role in determining the success of therapy [3].
Fecal samples offer various biomarkers for CRC detection. Fecal Occult Blood Test (FOBT) and Fecal Immunochemical Test (FIT) screening method use blood as biomarker in the stool. However, both have low sensitivity and specificity especially at premalignant stages of CRC [5]. Another widely used method is colonoscopy, but it comes with its own problems as it is more invasive and requires technical experts to perform the procedure [6]. Something that is really hard to find in rural areas in Yogyakarta.
Our Proposed Biomarkers
One of the most promising biomarkers for CRC is RNA in the form of miRNA. They have been found to be dysregulated in various types of cancer, making them potential biomarkers for cancer screening and diagnosis [7]. Different miRNAs such as miR-92a and miR-21 exhibit upregulation during the early adenoma and advanced adenoma stages, respectively [8] (Figure 1.). Data from previous research shows that the sensitivity of miR-21 and miR-92a are ranging from 56-86.05% and 72-92.07% respectively. Whereas the specificity of miR-21 and miR-92a are ranging from 48.3-81.08% and 51.7-73% [9,10,11]. Although, these numbers were based on the stool sample and the lumen sample should hold for better results compared to the stool. Thus, both of these miRNA have the potential to serve as biomarkers, facilitating the early detection of CRC with an appropriate detector.
Figure 1. miRNA upregulation profile on each stage of CRC [7]
Overview of Loop-Initiated RNA Activator (LIRA) system
miRNA is typically detected using the qPCR method which is the current gold standard, but qPCR is a relatively expensive method of detection in terms of reagent and the instrument required for screening especially in rural areas [12]. Thus, a new method is needed as a detector for miRNA as the biomarker of CRC. One of the novel detector systems developed for specific nucleic acid sequences is LIRA. Loop-Initiated RNA Activator (LIRA) is a novel nucleic acid detection method based on RNA secondary structure. LIRA utilizes the concept of protein translation, wherein translation cannot commence if the ribosome fails to bind to the Ribosome Binding Site (RBS). By closing the RBS sequence in its secondary structure and opening it in the presence of a specific sequence, LIRA can be employed as a detector for specific nucleic acid biomarkers. LIRA could be designed with various inputs and gates, including an OR gate, which facilitates the output or the expression of a reporter gene upon the presence of either of the two input sequences [13] (Figure 2.). Consequently, LIRA can be developed into a biodevice for detecting specific early-stage CRC biomarkers, such as miR-21 in the early adenoma phase and miR-92a in the advanced adenoma phase. However, in order to express LIRA, a safe and non-toxic biological host or chassis is required in the lumen.
Figure 2. LIRA OR Gate Mechanism [13]
Overview of Escherichia coli Nissle 1917
Figure 3. ColDBlu against cancer cell
Escherichia coli Nissle 1917 or EcN is a bacterium that potentially could be used as chassis for human uses. It was developed by Dr. Alfred Nissle from the World War 1 era and since then has been marketed with the brand name Mutaflor® for Inflammatory Bowel Disease (IBD) treatment [14]. Today, EcN has been discovered to have anticancer activity by specifically colonizing and altering the signaling pathways of CRC cells [15] [16]. In addition, the two endogenic plasmids of EcN could be engineered and maintained without the need of antibiotic selection [17]. These properties of EcN make it a suitable chassis for use in CRC.
The Practical Utilization of The LIRA as Biodevice System
Figure 4. Proposed mechanism of LIRA with miR-21 and miR-92a as its input
Figure 5. Proposed LIRA transcription unit with aeBlue as its reporter
With the concepts of synthetic biology, we developed a living early screening system with an engineered EcN to become the host of a LIRA biodevice that could detect miR-21 and miR-92a as the early biomarkers of CRC.
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[14] Behrouzi A, Mazaheri H, Falsafi S, Tavassol ZH, Moshiri A, Siadat SD. Intestinal effect of the probiotic Escherichia coli strain Nissle 1917 and its OMV. Journal of Diabetes & Metabolic Disorders. 2020 Jun;19:597-604
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[16] Alizadeh S, Esmaeili A, Omidi Y. Anti-cancer properties of Escherichia coli Nissle 1917 against HT-29 colon cancer cells through regulation of Bax/Bcl-xL and AKT/PTEN signaling pathways. Iranian Journal of Basic Medical Sciences. 2020 Jul;23(7):886.
[17] Kan A, Gelfat I, Emani S, Praveschotinunt P, Joshi NS. Plasmid vectors for in vivo selection-free use with the probiotic E. coli Nissle 1917. ACS synthetic biology. 2020 Dec 10;10(1):94-106.
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