Team:BIT/Description
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Description
Present situation
Real life events around us:
In 1993, the goddess Audrey Hepburn died of colorectal cancer at the age of 63;
In 2016, the famous women's basketball player Chen Luyun died at the age of 38 due to ineffective treatment for colon cancer;
On August 28, 2020, Chadwick Bosman, a famous actor and hero of the film panther, died of colon cancer at the age of 43.
According to the global cancer data report in 2020, colorectal cancer is one of the three cancers with the largest number of new cases in the world,
and the mortality ranks second, second only to lung cancer. [1][2][3]The two points that attract our attention most are the youth of
colorectal cancer and the high survival rate of early detection of colorectal cancer.[4][5][6]
About it become younger in average age:
A study by JAMA Netw Open showed that colorectal cancer is not only younger,
but also has a lower survival rate than colorectal cancer patients diagnosed at the age of 50-51!
The study used data from the National Cancer Database. The included individuals ranged in age from 0 to 90 years.
Among them, patients aged 51 to 55 were used as the control group and defined as late-onset CRC.
Early onset CRC was defined as younger than 50 years of age at diagnosis.
The results showed that in the unadjusted analysis, patients with early-onset CRC had lower 10-year survival than patients diagnosed with CRC aged 51 to 55.
(53.6% vs 54.3% ; P < .001)
uvival rates of early-onset CRC
Suvival rates of later-onset CRC
According to the statistical experimental data, colorectal cancer is younger and the survival rate of young patients is lower.
About survival:
The most commonly used system for staging colorectal cancer is the TNM staging system of the American Joint Commission on cancer (AJCC)——
The depth of tumor invasion in the intestinal wall
The depth of tumor invasion in the intestinal wall, represented by the letter T, is divided into:
T1, T2: relatively early
T3: medium term
T4: it's late
T1, T2: relatively early
T3: medium term
T4: it's late
Relative survival by age
It can be seen from the statistical chart that the earlier colorectal cancer is found and diagnosed, the higher its survival rate.
Based on these two points, the survival rate of young patients is low and the survival advantage of early detection of
colorectal cancer will be greater.
Based on the above research results, early prediction is very important. So, this year's team BIT aims at the early auxiliary screening of colorectal cancer,
and plan to design a large-scale convenient and cheap colorectal cancer marker detection system.
Existing detection methods
We have investigated some current detection methods, which have their own advantages,
but can not meet our needs which a method includes large-scale, convenient, and cheap advantages.
Because the symptoms of colorectal cancer patients are often found in the late stage, it is difficult to treat in time,
the patient tolerance is not high and it is difficult for them to afford such expensive medical expenses, a large-scale, convenient,
and cheap detection method is urgently needed.
In order to achieve the purpose of large-scale and convenient disease prediction earlier and let colorectal cancer enter our normal physical examination,
we decided to develop a new method for early prediction, so that all patients who may have colorectal cancer can have in-depth special examination.
Marker selection
Among many markers, we selected miRNA through data comparison
The reason why we choose miRNA
These advantages make miRNA a good candidate biomarker for the diagnosis of colorectal cancer.
Studies have shown that 31 miRNAs in plasma or serum of patients with colorectal cancer are significantly dysregulated,
of which six up-regulated miRNAs (mir-17-3p, mir-18a, miR-21, miR-29a, miR-92, mir-106a)
and two down-regulated miRNAs are the most common dysregulated miRNAs.[9]
In the selection of specific miRNA, we chose to use joint detection to enhance the specificity.
The reason why we choose miRNA
Three specific miRNAs were selected by data mining.
the specificity of single miRNA diagnosis may be low, and the combined use of multiple targets can improve the diagnostic performance.
The results show that when several miRNAs in serum are combined for the diagnosis of colorectal cancer,
the sensitivity and specificity are greatly improved compared with a single miRNA.
For example, the combined diagnosis of miRNA-21, mirna-92a, mirna-181b and mirna-203 can increase the sensitivity and specificity of
colorectal cancer detection to 93% and 91% [10]respectively. Therefore, considering comprehensively,
miR-21, mir-92a and miR-29a [11][12]were selected for the joint diagnosis of colorectal cancer.
Our Project
For specific miRNAs, our project developed an ultrasensitive miRNA detection method based on loop-mediated isothermal amplification ( LAMP ) and CRISPR/Cas12a trans-cutting technology.
Form the double stem-loop DNA&CRISPR/Cas12a
In order to integrate the technical route and operation method of early screening of CRC constructed by the project and overcome the problems of complex process,
low patient tolerance and unsuitable for large-scale early screening of traditional detection methods, our project constructs an early screening platform for CRC to realize the rapid,
portable and simple operation of the “sample-to-response” integrated detection system for CRC,
which provides effective intelligent interactive IVD equipment for large-scale early screening.
Equipment use process
Equipment modeling diagram
After testing, the system is simple to operate. It can complete the early screening of CRC within 1h and the sensitivity can reach fM level.
In addition, our equipment also has the ability to detect different miRNAs, which makes it possible to construct a standardized cancer detection platform.
Innovation
Bio:
• We developed a LAMP + CRISPR/Cas12a combined detection system. By designing a pair of specific stem-loop DNA probes,
LAMP primer design process can be greatly simplified. The DNA probe can be connected with the target miRNA to form a
double stem-loop DNA template, triggering an efficient LAMP reaction and achieving specific amplification with the help of highly specific ligase.
• LAMP products contain a large number of newly generated miRNA-specific sequences. The crRNA can guide CRISPR/Cas12a system to specifically recognize these miRNA-specific sequences and activate the side-chain cleavage activity of Cas12a, which produce obvious fluorescence signals. There are positively correlated with the original miRNA content. Therefore, LAMP + CRISPR system can detect target miRNAs with high sensitivity and specificity.
• After verifying the feasibility of LAMP + CRISPR combined detection system, we further optimized the reaction system. We use LAMP freeze-dried ball to replace the traditional LAMP reagent and prove that the amplification effect lyophilized ball system is comparable to that of the original system, which greatly simplified the operation difficulty of the experiment.
• LAMP products contain a large number of newly generated miRNA-specific sequences. The crRNA can guide CRISPR/Cas12a system to specifically recognize these miRNA-specific sequences and activate the side-chain cleavage activity of Cas12a, which produce obvious fluorescence signals. There are positively correlated with the original miRNA content. Therefore, LAMP + CRISPR system can detect target miRNAs with high sensitivity and specificity.
• After verifying the feasibility of LAMP + CRISPR combined detection system, we further optimized the reaction system. We use LAMP freeze-dried ball to replace the traditional LAMP reagent and prove that the amplification effect lyophilized ball system is comparable to that of the original system, which greatly simplified the operation difficulty of the experiment.
Hardware:
• We integrate the biological detection process by using a sliding chip. We pre-embedded freeze-dried ball reagent in the slip chip. So we can reduce the complexity of the injection process and the possibility of false positive or false negative due to non-standard experimental operation.
• We use machine to slide chips so we can prevent less uncertainty from manual operations and make them easy to use.
• High integration of detection process is realized by constructing supporting instruments, which avoids the trouble caused by using large-scale experimental equipment such as microplate reader and PCR instrument. It is conducive to the use of large-scale early screening.
• We construct a software system based on WeChat mini-app to realize the visualization of results and Bluetooth interactive control, which is conducive to the realization of intelligent human-computer interaction process. It is suitable for the end user experience.
• We use machine to slide chips so we can prevent less uncertainty from manual operations and make them easy to use.
• High integration of detection process is realized by constructing supporting instruments, which avoids the trouble caused by using large-scale experimental equipment such as microplate reader and PCR instrument. It is conducive to the use of large-scale early screening.
• We construct a software system based on WeChat mini-app to realize the visualization of results and Bluetooth interactive control, which is conducive to the realization of intelligent human-computer interaction process. It is suitable for the end user experience.
Project advantages and application prospects
References：
[1] WANG F，CHEN ZN，ZHANG YX． Effects of PCDH10 gene overexpression on proliferation and migration of color- ectal cancer cells[J].Journal of Zhengzhou University ( Medical Sciences) ，2020，55( 5) : 653
[2] Marley A Ｒ，Nan H． Epidemiology of colorectal cancer [J]．International Journal of Molecular Epidemiology and Genetics，2016，7( 3) : 105 － 114．
[3] Kolligs F T． Diagnostics and epidemiology of colorectal cancer[J]．Visceral Medicine，2016，32( 3) : 158 － 164．
[4] WHO. Global Health Estimates 2020: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2019[Z/OL].[2021-02-20]. https://www.who.int/data/gho/data/themes/mortality-andglobal-health-estimates/ghe-leading-causes-of-death.
[5] IARC.The Section of Cancer Surveillance[Z/OL]. [2021-02-20]. https://www.iarc.who.int/research-sections-csu/
[6] BRAY F, FERLAY J, SOERJOMATARAM I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.
[7] Ji D, Qiao M, Yao Y, et al. Serum-based microRNA signature predicts relapse and therapeutic outcome of adjuvant chem- otherapy in colorectal cancer patients[J]. E Bio Medicine, 2018, 35:189-197.
[8] Ragusa M, Barbagallo C, Statello L, et al. Non-coding land- scapes of colorectal cancer[J]. World J Gastroenterol, 2015, 21(41) : 11 709-11 739.
[9] Giráldez MD, Lozano JJ, Ramírez G, et al. Circulating microRNAs as biomarkers of colorectal cancer: results from a genome-wide profiling and validation study [J]. Clin Gastroenterol Hepatol, 2013, 11(6) : 681-688.
[10] Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissuespecific microRNAs from mouse[ J]. Curr Biol, 2002, 12(9): 735-739.
[11] Schemer AJ, Okayama H, Harris CC. The role of microRNAs in colorectal cancer. Cancerjournal．2012．18B): 244-252.
[12] Reddy KB. MicroRNA(miRNA)in cancel. Cancer Cell International. 2015. 15(1):68-76.
[2] Marley A Ｒ，Nan H． Epidemiology of colorectal cancer [J]．International Journal of Molecular Epidemiology and Genetics，2016，7( 3) : 105 － 114．
[3] Kolligs F T． Diagnostics and epidemiology of colorectal cancer[J]．Visceral Medicine，2016，32( 3) : 158 － 164．
[4] WHO. Global Health Estimates 2020: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2019[Z/OL].[2021-02-20]. https://www.who.int/data/gho/data/themes/mortality-andglobal-health-estimates/ghe-leading-causes-of-death.
[5] IARC.The Section of Cancer Surveillance[Z/OL]. [2021-02-20]. https://www.iarc.who.int/research-sections-csu/
[6] BRAY F, FERLAY J, SOERJOMATARAM I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.
[7] Ji D, Qiao M, Yao Y, et al. Serum-based microRNA signature predicts relapse and therapeutic outcome of adjuvant chem- otherapy in colorectal cancer patients[J]. E Bio Medicine, 2018, 35:189-197.
[8] Ragusa M, Barbagallo C, Statello L, et al. Non-coding land- scapes of colorectal cancer[J]. World J Gastroenterol, 2015, 21(41) : 11 709-11 739.
[9] Giráldez MD, Lozano JJ, Ramírez G, et al. Circulating microRNAs as biomarkers of colorectal cancer: results from a genome-wide profiling and validation study [J]. Clin Gastroenterol Hepatol, 2013, 11(6) : 681-688.
[10] Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissuespecific microRNAs from mouse[ J]. Curr Biol, 2002, 12(9): 735-739.
[11] Schemer AJ, Okayama H, Harris CC. The role of microRNAs in colorectal cancer. Cancerjournal．2012．18B): 244-252.
[12] Reddy KB. MicroRNA(miRNA)in cancel. Cancer Cell International. 2015. 15(1):68-76.