Design

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).

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.

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).

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).

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.

Alshaer, W., Zureigat, H., Al Karaki, A., Al-Kadash, A., Gharaibeh, L., Hatmal, M.M., Aljabali, A.A.A., and Awidi, A. (2021). siRNA: Mechanism of action, challenges, and therapeutic approaches. Eur J Pharmacol 905, 174178.

Baidoun, F., Elshiwy, K., Elkeraie, Y., Merjaneh, Z., Khoudari, G., Sarmini, M.T., Gad, M., Al-Husseini, M., and Saad, A. (2021). Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets 22, 998-1009.

Dekker, E., Tanis, P.J., Vleugels, J.L.A., Kasi, P.M., and Wallace, M.B. (2019). Colorectal cancer. Lancet 394, 1467-1480.

Hu, B., Zhong, L., Weng, Y., Peng, L., Huang, Y., Zhao, Y., and Liang, X.J. (2020). Therapeutic siRNA: state of the art. Signal Transduct Target Ther 5, 101.

Lee, S.Y., Yang, C.Y., Peng, C.L., Wei, M.F., Chen, K.C., Yao, C.J., and Shieh, M.J. (2016). A theranostic micelleplex co-delivering SN-38 and VEGF siRNA for colorectal cancer therapy. Biomaterials 86, 92-105.

Traber, G.M., and Yu, A.M. (2023). RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies. J Pharmacol Exp Ther 384, 133-154.