Design
Colorectal cancer
Colorectal cancer, also known as bowel cancer, is a cancer that develops in the tissues of the colon or
rectum(Dekker et al., 2019). Colorectal cancer is the third most common cancer worldwide, accounting for
approximately 10% of all cancer cases and is the second leading cause of cancer-related deaths worldwide.
Nowadays, it is the world's most deadly cancer with almost 900,000 deaths annually in the world (Baidoun et
al., 2021). Current treatments for colorectal cancer include endoscopic and surgical local excision,
downstaging preoperative radiotherapy and systemic therapy, extensive surgery for locoregional and
metastatic disease, local ablative therapies for metastases, and palliative chemotherapy, targeted therapy,
and immunotherapy. Although these treatment options have doubled overall survival for advanced disease to 3
years, the survival of patients with metastasised disease is still worse. Recently, with an appropriate
delivery system, the siRNA therapy has already achieved a remarkable feat by revolutionizing the treatment
arena of cancers(Lee et al., 2016).
siRNA therapy
Small interfering RNA, siRNA, is a kind of double-strand RNA, having the length of 20-24 base pair. In the
nucleus, it interferes with the specific gene, which inactivate a certain process in the path. In the
typical drug utilizing siRNA, the double-strand RNA is cleavage into the siRNA. Wrapped by endosomal and
lysosomal entrapment, the siRNA is able to reach the cytoplasm where the siRNA is released from the
medicine. After this, interacting with multiple proteins, it would form RNA-induced silencing complex, RISC.
In the process, the passenger strand would be removed, and the antisense strand would continuously
incorporate with the RISC. Last, the RISC would help to replace the target mRNA with the siRNA strand,
downregulating the specific gene expression. The replaced segment of the mRNA would be degraded in the
cytoplasm (Alshaer et al., 2021; Hu et al., 2020). As another targeted path in our research, the hippo-path
plays essential role in the cell’s proliferation, differentiation, and survival by controlling a series of
proteins, including LAST1/2, YAP/ TAZ, and TEAD4. Only when YAP/TAZ can interact with TEAD4, the expression
of the specific gene would start. Among these proteins, the TEAD4 is an ideal biomarker for the cancer,
which is overexpressed in the colorectal cancer patients. In the normal cell, the YAP/TAZ can be
phosphorylated, which can be recognized by the cell’s metabolic system to degrade them. In this case, the
YAP/TAZ wouldn’t combine with TEAD4 to express certain gene(Traber and Yu, 2023). However, when the
hippo-path acts abnormally, the YAP/TAZ wouldn’t be phosphorylated, which would not be degraded. Therefore,
excessive number of YAP/TAZ would interact with TEAD4, causing the overexpression of the specific gene that
controls the proliferation process of the cell. The combination of the reactions would lead to increasing
rate of cancer reduction.
Part Design
Utilizing the mechanism of the siRNA and TEAD4 mentioned above, our project designs the siRNA to
downregulate the expression of the TEAD4 in the colorectal tumor cell SW480. Theoretically, when the siRNAs
were transfected into SW480 cells, the TEAD4 expression was inhibited. TEAD4 expression could affect
downstream Hippo pathway. Threrefore, when SW480 cells were treated with siRNAs, the proliferation,
migration and invasion ability were inhibited. The tumor cells tend to lose the cancer cell character after
siRNA treatment, which are expected to achieve favorable curative effect in clinical practice (Figure 1).
Figure 1 The working model of our research
In our research pipeline, there are three main steps enabling the siRNA to knockdown the TEAD4 expression.
First, the siRNA targeted to TEAD4 and plko.1 backbone plasmid we used in the experiment. By cutting the
plko.1 and siRNA sequence with same restriction enzyme, we joined the two parts with T4 DNA Ligase and
finally got the shRNA recombinant product. Second, SW480 cells were transfected with the recombinant
plasmid. When the plasmid, the recombinant product, entered the cytoplasm, the small hairpin RNA, shRNA
derived from the plasmid. After this, after the processing of the Dicer enzyme, the shRNA was cut into siRNA
pieces, releasing the siRNA stably, which was a long-acting process. This prolonged the duration of the
TEAD4 expressed gene’s downregulation. Third, to determine the effectiveness of the method mentioned above,
we did several experiments to examine the effect of the siRNA knocking down the TEAD4 expression. We
detected the proliferation ability by cell counting kit-8(CCK-8) test and the migration ability by transwell
migration assay and the internal oxidative stress by Reactive Oxygen Species Assay (Figure 2).
Figure 2 The flowchart of our research
In summary, we intended to treat colorectal cancer by a new bio-therapy method-siRNA. SiRNA has proven to be
an adaptable and powerful therapeutic strategy where advancements in chemistry and pharmaceutics continue to
bring the drugs into the clinic. Although siRNA display a promising effect in curing CRC due to the
stability and low cost, the risk of off-target effects should not be ignored.
Reference
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Dekker, E., Tanis, P.J., Vleugels, J.L.A., Kasi, P.M., and Wallace, M.B. (2019). Colorectal cancer. Lancet
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