iGEM 2024 — Team yiye-wuhan

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# Notebook

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# Engineering success

1.siRNA is a Promising Therapeutic Approach for Colorectal Cancer (CRC)

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.

2. siRNA design

2.1 Retrieve the TEAD4 mRNA Sequence

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).

![](https://static.igem.wiki/teams/5407/engineering-1.jpg)

Figure 1 Retrieve the TEAD4 mRNA Sequence

(A) Search of TEAD4 in the NCBI (B) Choose the TEAD4 transcript (C-D) The TEAD4 description and
sequence

2.2 Design the siRNA Sequence targeted to TEAD4

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.

![](https://static.igem.wiki/teams/5407/engineering-2.jpg)

Figure 2 Design of siRNA Sequences Targeting TEAD4

3. Construction of shRNA Targeted to TEAD4

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).

![](https://static.igem.wiki/teams/5407/engineering-3.jpg)

Figure 3 Map of pKLO.1 plasmid

The sequence and map of pKLO.1 plasmid originated from addgene.

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).

![](https://static.igem.wiki/teams/5407/engineering-4.jpg)

Figure 4A Pick up the single clone from solid plate

![](https://static.igem.wiki/teams/5407/engineering-5.jpg)

Figure 4B Electrophoresis of PKLO.1-sh-TEAD4 PCR Identification of the Plasmids

1: pLKO.1（241bp）2: marker 3: PCR products of siTEAD4-1 and siTEAD4-2 coloning（293bp）

![](https://static.igem.wiki/teams/5407/engineering-6.jpg)
![](https://static.igem.wiki/teams/5407/engineering-7.jpg)

Figure 4C The sh-TEAD4-1 and sh-TEAD4-2 plasmid sequence validated by Sanger sequencing.

Upper: sh-TEAD4-1 sequence validated in the composite plasmid, the core sequence targeted to the
Untranslated Region (UTR) of TEAD4; Lower: sh-TEAD4-2 sequence validated in the composite plasmid,
the c

4.siRNA-Mediated Knockdown of TEAD4 Expression

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.

![](https://static.igem.wiki/teams/5407/engineering-8.jpg)

Figure 5 The detection of TEAD4 knockdown efficiency

(TEAD4 protein expression was determined by western blot analysis. GAPDH protein was used for equal
loading assessment)

5.1 TEAD4-Targeted siRNA Inhibits Cell Proliferation the CCK-8 Assay

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).

![](https://static.igem.wiki/teams/5407/engineering-9.jpg)
![](https://static.igem.wiki/teams/5407/engineering-10.jpg)

Figure 6 the proliferation ability after transfected with shRNA targeted to TEAD4
(upper is the chromogenic reaction of CCK8 experiment in 96 well plate. Relative cell viability was
normalized by the control pLKO.1 group \*\*\*means p<0.01, \*\*\*\*means p<0.001 by Student’s t-test
significance, ns means none significance) <p class="small">The sequence and map of pKLO.1 plasmid
originated from addgene

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.

5.2 TEAD4-Targeted siRNA Increases Cellular ROS Levels

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.

![](https://static.igem.wiki/teams/5407/engineering-11.jpg)

Figure 7 the ROS level detection after transfected with shRNA targeted to TEAD4

(Representative images of the total view of the ROS were shown, sw480 cell were transfected with
PLKO.1 plasmid, sh-TEAD4-1 and sh-TEAD4-2 composite plasmid that could silence TEAD4 gene
expression. Data were collected from 10 fields of three independent experiments.Scale bar- 250 μm)

![](https://static.igem.wiki/teams/5407/engineering-12.jpg)

Figure 8 the statistical diagram of ROS level detection

(\*\*\*means p<0.01, \*\*\*\*means p<0.001 by Student’s t-test significance, ns means none
significance)

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.

5.3 siRNA Targeted to TEAD4 Inhibits Cell Migration in Transwell Assays

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.

![](https://static.igem.wiki/teams/5407/engineering-13.jpg)

Figure 9 Transwell migration assay of control pLKO.1 and TEAD4 shRNA sw480 cells. (Representative images
of the total view of the transwell were shown, sw480 cell were transfected with PLKO.1 plasmid,
sh-TEAD4-1 and sh-TEAD4-2 composite plasmid that could silence TEAD4 gene expression, data were
collected from 10 fields of three independent experiments)

![](https://static.igem.wiki/teams/5407/engineering-14.jpg)

Figure 10 the statistical diagram of migration ability detection
(\*\*\*means p<0.01, \*\*\*\*means p<0.001 by Student’s t-test significance, ns means none significance)

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.

A

![](https://static.igem.wiki/teams/5407/engineering-15.jpg)

B

![](https://static.igem.wiki/teams/5407/engineering-16.jpg)

Figure 11 A. The Main Experimental Procedure; B. The summary diagram of the main experiment

Reference

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.

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.

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