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sh-TEAD4-2

Usage and Biology

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[1]. It is estimated that CRC will cause around 53,010 deaths in 2024 [2]. 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. In our study, TEAD4 was found to be highly expressed in CRC patients [3]. 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.

Result

1. Construction of the shRNA targeted to TEAD4

we constructed sh-TEAD4-2 plasmids. The backbone plasmids were cut by two specific restriction endonuclease enzymes, AgeI and EcoRI. Part sequences of the siRNA were amplified by the Polymerase Chain Reaction (PCR) technique. Then the linear pKLO.1 plasmid and the sequence of the siRNA were ligated by T4 DNA ligase. The recombinant products were transformed into the DH5α cell and cultured in the LB culture medium with the ampicillin (Figure 1). To test whether the target fragments were inserted in the plasmid successfully, the recombinant products were analyzed on the 1% Agarose Gel (Figure 2). Moreover, the recombinant plasmids were also confirmed by using Sanger sequencing (Figure 3).

                           
                           Figure 1 Solid Plate of sh-TEAD4 Plasmid
                           
                          
              Figure 2 Electrophoresis of sh-TEAD4 PCR Identification of the Plasmids

                        
                Figure 3 The sh-TEAD4-2 plasmid validated by Sanger sequencing

2 siRNA targeted to TEAD4 inhibited the TEAD4 expression

At first, we used the western blot to detect the TEAD4 protein expression and validate the knockdown efficiency. We found the TEAD4 protein was inhibited after transfected with shRNA (Figure 4). The results confirmed good specificity and of our siRNA

                    
    Figure 4 The protein expression of TEAD4 after transfected with sh-TEAD4-1 and sh-TEAD4-2 plasmid 

3.1 siRNA targeted to TEAD4 inhibited cell proliferation by CCK-8 experiment

We then held a CCK8 experiment to test the cell proliferation after transfection with sh-TEAD4-1plasmid using different concentration. The depth of color gets pastel while the amount of cell gets lower, and vise versa, showing a proportional relationship between the number of cells and the depth of color. There weren’t any change of depth of color with controlled group, while the sh-TEAD4 plasmids successfully inhibited the expression of TEAD4 as the dosage get larger, representing our success in inhibition of shRNA against TEAD4.

                           
        Figure 5 the proliferation change after transfected with shRNA targeted to TEAD4

Through statistical analysis, it has been proven that the proliferation did not show any significant change after treating with different amount of plasmid in the NC group. After treating with CRC cells with different amount of sh-TEAD4-2 plasmid, all high dosage group showed significant decrease of proliferation compared with low dosage group (0 ug). Moreover, the higher the concentration of the transfected plasmid, the more palpable the impeding effect (Figure 5). Hence, the results showed that proliferation capability of SW480 cell were decreased in a dose dependent manner.

3.2 siRNA targeted to TEAD4 increased cell ROS level

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 biomacromolecule. The other main source is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is expressed on the plasma membrane. 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, exceeding the amount that the cell can process. This would lead to the oxidative stress in the cell, causing the cell’s apoptosis. 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 used the DCFH-DA as a probe. When it enters the cell membrane, hydrolyzed by the esterase, DCFH-DA would transform to DCFH staying in the cytoplasm and emitting the fluorescence. Detecting the extent of the fluorescence, we can determine the ROS level. In our experiment (Figure 6-7), we transfected the PLKO.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 ug, it would raise the ROS level. We used different amount of sh-TEAD4 plasmids, 0µg, 0.5µg, 1µg, and 2µg, to treat SW480. Compared with the 0µg treatment, other groups have statistical significance in increasing ROS level of the colorectal cancer cell. When the amount of the sh-TEAD4s increase, the ROS level showed by the fluorescent intensity also increase. This suggests that sh-TEAD4-2 plasmid can promote the ROS level in CRC. Moreover, sh-TEAD4 amount could increase the ROS level in a dosage dependent manner.

           
           Figure 6 the ROS level detection after transfected with shRNA targeted to TEAD4

                             
                      Figure 7 the statistical diagram of ROS level detection 

3.3 siRNA targeted to TEAD4 inhibited cell migration by transwell experiment

Transwell experiment is a tool used to measure the migration ability of cells. There are three kinds of plasmids that we infected into CRC cells, NC (pLKO.1), sh-TEAD4-1, respectively. Then we infect 0, 0.5µg, 1µg, 2µg of plasmid into the CRC cells. Through statistical analysis, it has been shown that there was no significant difference in the migration ability of the NC group; We also used different amount of sh-TEAD4 plasmids, 0µg, 0.5µg, 1µg, and 2µg, to treat SW480. Compared with the 0µg treatment, other high dosage groups have statistical significance in inhibiting the migration ability of the colorectal cancer cell. When the amount of the sh-TEAD4s delivered into SW480 increase, the migrated cells also decrease. This suggests that the sh-TEAD4-1 plasmids can inhibit the migration level in CRC. Moreover, sh-TEAD4 amount could decrease the migration ability in a dosage dependent manner (Figure 8-9).

                            
                 Figure 8 the migration ability detection after transfected with shRNA targeted to TEAD4
                            
                          
                      Figure 9 the statistical diagram of migration ability detection 

Conclusion

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. 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.

Reference

Reference

1.Baidoun F et al. Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets. 22, 998-1009 (2021).

2.Dekker E, Tanis PJ, Vleugels JLA, Kasi PM, Wallace MB. Colorectal cancer. Lancet. 394, 1467-1480 (2019).

3.Guo Y et al. CK2-induced cooperation of HHEX with the YAP-TEAD4 complex promotes colorectal tumorigenesis. Nat Commun. 13, 4995 (2022).

Sequence and Features