Engineering success

Colorectal cancer (CRC) is among the most prevalent malignancies globally and ranks as the fourth leading cause of cancer-related deaths. According to the American Cancer Society, the lifetime risk of developing CRC is approximately 1 in 23 for males and 1 in 25 for females. Notably, the mortality rate for CRC patients under 55 years of age has been increasing by approximately 1% annually since the mid-2000s(Baidoun et al., 2021). It is estimated that CRC will cause around 53,010 deaths in 2024(Dekker et al., 2019). Advances in biomedical research have significantly enhanced our understanding of cancer, driving the exploration of novel therapeutic strategies for CRC. Among these, small interfering RNA (siRNA) therapy has shown remarkable potential in the cancer treatment(Li et al., 2020; Montero et al., 2023).

In our study, TEAD4 was found to be highly expressed in CRC patients(Guo et al., 2022). We designed siRNAs targeting TEAD4. Transfection of these siRNAs into CRC tumor cells resulted in the inhibition of TEAD4 expression. As TEAD4 plays a critical role in modulating the downstream Hippo pathway, which is known to promote tumor growth and metastasis, we assessed the impact of TEAD4 knockdown on cell proliferation, migration, and reactive oxygen species (ROS) levels in CRC cell lines.

To find the most efficient siRNA that applies to the targeted protein TEAD4, we would have to first enter the website international public database NCBI (https://www.ncbi.nlm.nih.gov/). And we need to search up the key word “TEAD4”, (Figure 1A) and get the TEAD4 transcript 1 isoform (Figure 1B). Then click on the TEAD4 transcript 1 isoform, we got the TEAD4 sequence. (Figure 1C&1D).

The first step in constructing shRNA targeted to TEAD4 involves designing a specific sequence that can effectively knock down the TEAD4 gene.

We designed the siRNA against TEAD4 by a special database (https://www.invivogen.com/sirnawizard/). After we input the TEAD4 gene name and sequence (Figure 2A), the database would output all the possible siRNA sequence (Figure 2B). We chose two pairs of siRNAs with the least off-target possibility.

Concerned with the fact that siRNA that is easy to be degraded in the cell, the shRNA plasmid can be more stable and keep releasing the siRNA, which would knock down the expression of the TEAD4 more effectively. Then, we constructed sh-TEAD4-1 and sh-TEAD4-2 plasmids.

Once the shRNA sequence is designed, oligonucleotides corresponding to the sense and antisense strands of the shRNA are synthesized. These oligonucleotides are chemically synthesized and then annealed to form a double-stranded DNA template with sticky ends compatible with the cloning vector pLKO.1 plasmid. The map of the original pLKO.1 plasmid was listed below (Figure 3).

The backbone plasmids were cut by two specific restriction endonuclease enzymes, AgeI and EcoRI. the linear pKLO.1 plasmid and the sequence of the siRNA were ligated by T4 DNA ligase. Then the recombinant plasmid containing the shRNA sequence were transformed into the DH5α cell and cultured in the LB culture medium with the ampicillin (Figure 4A).

To test whether the target fragments were inserted in the plasmid successfully, Part sequences of the siRNA were amplified by the Polymerase Chain Reaction (PCR) technique. And the results were identified on the 1% Agarose Gel (Figure 4B). Moreover, the recombinant plasmids were also confirmed by using Sanger sequencing (Figure 4C).

To evaluate TEAD4 protein expression, successful knockdown of TEAD4 is indicated by a significant reduction in its protein levels compared to control cells. Then the western blot analysis was used to assess TEAD4 protein expression and to validate the knockdown efficiency. The results demonstrated a significant reduction in TEAD4 protein levels following transfection with shRNA plasmids (Figure 5), confirming the high specificity and effectiveness of our siRNA.

The Cell Counting Kit-8 (CCK-8) assay is a practical and widely used method to evaluate cellular toxicity and proliferation. The intensity of color development correlates with the rate and extent of cell proliferation.

To further assess proliferative capacity, SW480 cells were transfected with sh-TEAD4-1 and sh-TEAD4-2 plasmids at varying dosages (0 µg, 0.5 µg, 1 µg, 2 µg) (Figure 6).

Statistical analysis revealed no significant changes in proliferation within the NC group after treatment with varying plasmid dosages. In contrast, sh-TEAD4-1 plasmid at different concentrations significantly inhibited CRC proliferation, with no notable differences observed between the 1 µg and 2 µg treatments, suggesting that the inhibition was not dose-dependent.

Upon treatment of CRC cells with varying amounts of sh-TEAD4-2 plasmid, higher dosage groups exhibited a significant reduction in proliferation compared to the low dosage group (0 µg). Furthermore, the inhibitory effect became more pronounced with increasing plasmid concentrations. These results indicate that the proliferative capacity of SW480 cells decreased in a dose-dependent manner.

The reactive oxygen species, ROS, is a kind of cell’s metabolic product generating by the metabolism of oxygen. One main source of it is the substrate end of the inner mitochondrial membrane. As the metabolic by-product, ROS is considered as the vicious bio-macromolecule. In the normal condition, the amount of ROS stays in a low level. However, when the cell meets stimulus, the ROS level would increase dramatically. This would lead to the oxidative stress in the cell and causing cell death. ROS level is an important marker of cellular oxidative damage caused by normal physiological function and environmental factors. Therefore, it’s necessary to measure the ROS level in our experiment.

We utilized DCFH-DA as a fluorescent probe. Upon entering the cell membrane, DCFH-DA is hydrolyzed by intracellular esterase to form DCFH, which remains in the cytoplasm and emits fluorescence. By detecting the intensity of fluorescence, we can quantitatively assess the ROS levels within the cells.

In our experiment, we transfected the PLKO.1, sh-TEAD4-1, and sh-TEAD4-2 plasmid into the SW480 cancer cell, and we detected the ROS level in these cells. In the treatment of various PLKO.1 amount, the data has no statistical significance, showing no notable change in the ROS level. However, when the amount of the PLKO.1 amount reached 2 μg, it would raise the ROS level（Figure 7-8）.

We treated SW480 cells with varying amounts of sh-TEAD4 plasmids (0 µg, 0.5 µg, 1 µg, and 2 µg). Compared to the 0 µg group, the other groups exhibited a statistically significant increase in ROS levels within the colorectal cancer cells. As the amount of sh-TEAD4 increased, the ROS levels, indicated by fluorescent intensity, also rose. This suggests that the sh-TEAD4-1 and sh-TEAD4-2 plasmids can elevate ROS levels in CRC cells. Furthermore, the increase in ROS levels is dose-dependent on the amount of sh-TEAD4 used.

The Transwell assay is a useful technique to assess the migratory capability of cells. CRC cells were transfected with three different plasmids: a negative control (NC, pLKO.1), sh-TEAD4-1, and sh-TEAD4-2. These cells were then treated with varying concentrations of the plasmids 0 µg, 0.5 µg, 1 µg, and 2 µg.

Statistical analysis revealed that there was no significant difference in the migration ability of cells in the NC group across the different plasmid concentrations. However, in the SW480 cells treated with sh-TEAD4 plasmids, a significant reduction in migration was observed compared to the 0 µg treatment group. This inhibitory effect on cell migration was dose-dependent, with higher concentrations of sh-TEAD4-1 and sh-TEAD4-2 resulting in fewer migrated cells （Figure 9-10）.

These results suggest that both sh-TEAD4-1 and sh-TEAD4-2 effectively inhibit the migratory capacity of CRC cells, and this inhibition is more pronounced with increasing plasmid concentrations.

We explored the impact of shRNA Targeting TEAD4 on the proliferation, migration, and ROS levels in SW480 cell line. Our results reveal that TEAD4 knockdown significantly reduces cell proliferation and migration, while concurrently increasing ROS levels in CRC cells (Figure 11). These findings underscore the therapeutic potential of TEAD4 as a target for RNA interference (RNAi) strategies in colorectal cancer, presenting promising opportunities for clinical applications.

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

Guo, Y., Zhu, Z., Huang, Z., Cui, L., Yu, W., Hong, W., Zhou, Z., Du, P., and Liu, C.Y. (2022). CK2-induced cooperation of HHEX with the YAP-TEAD4 complex promotes colorectal tumorigenesis. Nat Commun 13, 4995.

Li, P.P., Yan, Y., Zhang, H.T., Cui, S.H., Wang, C.H., Wei, W., Qian, H.G., Wang, J.C., and Zhang, Q. (2020). Biological activities of siRNA-loaded lanthanum phosphate nanoparticles on colorectal cancer. J Control Release 328, 45-58.

Montero, J.C., Del Carmen, S., Abad, M., Sayagues, J.M., Barbachano, A., Fernandez-Barral, A., Munoz, A., and Pandiella, A. (2023). An amino acid transporter subunit as an antibody-drug conjugate target in colorectal cancer. J Exp Clin Cancer Res 42, 200.