Contribution

Part BBa_K25140001 is a part constructed by the iGEM Worldshaper-Wuhan team in 2017. This plasmid contains complementary binding sites to miR-21, which can monitor the expression of miR-21. The purpose of the project (Worldshaper-Wuhan 2017) is to establish a miRNA sensor which can offer a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer.

In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work:

1. Based on the reading of a large number of published papers, we added more information about the alteration of miR-21 expression in the serum and tissues of colorectal cancer patients, compared with healthy people.

In Fig.1[1], a total of 10 adenomas as benign lesions with moderate dysplasia were resected by endoscopic mucosal resection (EMR). Ten specimens of normal colorectal mucosal tissues were evaluated as normal controls. The specific regulation of miRNA-21 expression in CRC tissues and controls was investigated by one-step qRT-PCR analysis. The level of miRNA-21 is significantly increased in the tissues of colorectal cancer patients, compared with that of controls (Fig.1)

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Fig.1. The expression of miRNA-21 in CRC tissues and controls. miRNA-21 was significantly upregulated in CRC tissues (3.78±0.26) compared with control group (1.15±0.16) [1].

In Fig.2 [2], the expression levels of miRNA-21 was detected in the serum of colorectal cancer patients (n=48) and healthy people (n=48). In comparison to the control group, the miRNA-21 expression level was upregulated in serum from CRC patients (p < 0.05).

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Fig 2. The expression of miRNA-21 in the serum from CRC and healthy people. The level of miRNA-21 was significantly increased in CRC serum (3.46±1.32), compared with control group (1.23±0.38).

Taken together, these studies further supported that upregulation of miR-21 was detected in tissues and serum from CRC patients.

2. We tested the function of miRNA-21 sponges containing complementary binding sites to miR-21 from Part BBa_K25140001

To confirm the effect of miRNA-21 sponges (pEGFP-miRNA-21-sponge-6s) in cells, we transfected pEGFP-miRNA-21-sponge-6s (0.25 ug /well) and Control vectors (0.25 ug /well, as negative control) into 293T cells in 24 well-plates, respectively. Then the GFP fluorescence was observed under fluorescence microscopy. The fluorescence of GFP in cells transfect with pEGFP-miRNA-21-sponge-6s was lower in that of controls. The result confirmed that the fluorescence of GFP could monitor the expression of miRNA-21 in cells (Fig. 3).

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Fig 3. The images of 293T cells transfected different plasmids. A. pEGFP-C1 transfected. B. pEGFP-miRNA-21-sponge-6s transfected. (200╳)

Conclusion

1. Compared with the control group, the fluorescence was significantly reduced, which was consistent with the experimental results of the Worldshaper-Wuhan team.

2. Fluorescence of GFP can monitor the expression of miRNA-21 in cells.

3. The expression level of miRNA-21 could be an indicator for colorectal cancer.

References

[1] Yang, Yang, et al. "MicroRNA-21 controls hTERT via PTEN in human colorectal cancer cell proliferation." Journal of physiology and biochemistry 71.1 (2015): 59-68.

[2] Ghareib A F, Mohamed R H, el-Fatah A, et al. Assessment of serum MicroRNA-21 gene expression for diagnosis and prognosis of colorectal cancer[J]. Journal of Gastrointestinal Cancer, 2020, 51(3): 818-823.