Part:BBa_K3730012

miRNA sponge, to reduce specific types of free hsa-miR-199a, 195 and 22 level in cell

The transcription of this miRNA sponge sequence will allow you to combine the free hsa-miR-199a,hsa-miR-195 and hsa-miR-22 and store them. They are consist of three parts.

Our design to realize specific replication of viruses is based on the difference of miRNA between HCC cell (HEPG2 cell line) and normal cell (HEK293T cell line).
This part, which includes sponges, is able to make this difference more significant by absorbing the free miRNA (hsa-miR-199a, hsa-miR-195 and hsa-miR-22) in the cytosol. Thus, this part consists of three types of sequences, absorbing the miRNA respectively.

In our experiment, we used eGFP-3U ( know more about 3U: BBa_K3730013 ), whose expression was regulated by miR-199a, miR-195, and miR-22. When the quantity of free miRNAs increased, the expression of eGFP decreased.
In the following figures, spe stands for sponge-empty, which is the plasmid without sponge sequences, as the control group.

Figure. 1 Fluorescence intensity after cotransfection of 3U and Sp/Spe in Hek293T and HepG2.
(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge refers to sponge-pcDNA3.1-P2A-C, and sponge empty refers to sponge-empty-pcDNA3.1-P2A-C. )

According to Fig. 1 (a), in Hek293, when the ratio of the 3u and the pSp or pSp null is 1:3, the expression of the eGFP was significantly lowered. Fig. 1 (b) shows that in HepG2, the significance was shown also when the ratio was 1:3. LSD test also showed the same results.
Meanwhile, as shown in Table. 1, when the ratios of the two plasmids (sponge or sponge empty and 3u) were regarded as a covariate, the difference between the experimental group and the control group is significant, which indicates that sponges are able to increase the expressions of the eGFP.
However, as the sponges also increased the expression of that gene in Hek293T, which represent normal cells, we are bound to improve the specificity of the sponge parts.

For full results and description please turn to the result page of our wiki.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 289
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 289
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 289
  Illegal BamHI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 289
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 289
  
• 1000
  COMPATIBLE WITH RFC[1000]