Part:BBa_K2514000

pSB1C3-miR-21-sponge-6s

Our project constructed a mir-21 sponge containing marker gene of GFP for the detection and inhibition mir-21 in the cell lines, and offers a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer in the future. miRNA sponge, which contains multiple target sites complementary to a miRNA of interest, is used to be a inhibitor of miRNA. Here, we proposed miRNA sponge could be a monitor to reflect the change of miRNA expression in cells. After transfection of the miR-21 sponge, sponge RNAs can attract miR-21, inhibiting the expression of eGFP reporter. The expression of miR-21 should be monitored by measuring the expression of eGFP reporter. Here, we designed two different mir-21 sponges with different binding sits to test the efficiency of sponges. One miR-21 sponge contains two miR-21 binding sites with 3-nt spacers for bulged sites. Another one mir-21 sponge contains six miR-21 binding sites with 3-nt spacers for bulged sites. We wanted to compare the accuracy and the effectiveness of different miR-21 sponges contained different binding sites for measuring the expression of miR-21 in colorectal cancer cells.

Followed experience is supplemented by 2022 iGEM YiYe-China

 

Part BBa_K2514000 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)

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).

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_K2514000

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).

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]