Part:BBa_K4789004

Usage and Biology

Our project aim to effectively diagnose the cervical cancer patients. LncRNAs are generally more than 200 nucleotides long but they are rarely able to translated into protein. In cervical cancer research, lncRNA MALAT1 are identified as tumor driving oncogenic lncRNA [1]. lncRNAs could bind to specific miRNA and act as sponges to compete miRNAs[2]. This mechanism gives rise to our idea about fusing a sponge RNA based on the sequences of lncRNA MALAT1 with binding sites complementary to the sequence of miRNA to a plasmid that has reporter gene, which will monitor the expression of miRNA in the cells.

Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Recently, a new FR, named Peppers, contain a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red.They could bind with a GFP fluorophore-like synthetic dye, (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl acetonitrile (HBC)(fig 1). Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA’s transcription, localization and translation[3].

We designed this part with pepper fluorescence to monitor the expression of miR-22 in cells. The “Pepper” plasmid containing the part sequence of LncRNA MALAT1 in order to reflect miRNA expression in vivo. lncRNAs can interact with miRNAs as “sponges”. And the fluorescence reflect the miRNA expression in the cervical cancer in turn. We tested the sensitivity and specificity of the vectors in Hela cells. In the future, the miRNA-LncRNA MALAT1 complex could be used to screen and detect the cervical cancer. The patients could benefit from our work. The model of the plasmid was listed below (Fig 1).

                   
                        Fig 1. The diagram of miR-22-sponge-pepper

Result

1.1 Function verification of miR-22 sensor

In order to test the ability of miR-22 sensor, we transfected miR-22-sponge-pepper and pre-miR-22 (overexpress miR-22 in cells) into Hela cells. The control group only transfected with miR-22-sponge-pepper (2 ug), the experimental group transfected with both miR-22-sponge-pepper (2 ug) and pre-miR-22 (1 ug). 48 hours later, we add 2 μm HBC fluorescent dye into per well. After incubation for 2 hours, cells were harvested and the green fluorescence was measured by plate reader (SpectraMax i3). The result showed that miR-22 could inhibit the fluorescence of Pepper in cells transfected with miR-22-sponge-pepper (Fig 2). The result suggested that miR-22 sensor can detect the alteration of miR-22 expression in cells.

               
        Fig 2. The images of Hela cells transfected with different plasmids. 
          (A) miR-22-sponge-pepper were transfected into Hela cells. 
          (B) miR-22-sponge-pepper and pre-miR-22 were transfected into Hela cells

1.2 The sensitivity of miR-22 sensor

To further test the sensitivity of miR-22-sponge-pepper (miR-22 sensor) as a monitor to detect the expression of miR-22, Hela cells were transfected with the same amount of miR-22-sponge-pepper and different amount of pre-miR-22 (0 ug, 0.5 ug, 1ug, 2ug) (Fig 3). Down-regulation of green fluorescence value was observed in cervical cancer cells transfected with different concentration of pre-miR-22 compared with control cells (Table 1). Moreover, the fluorescence was significantly decreased in a dose dependent manner (p<0.05, Fig 4). Based on the values in cell treated with different concentration of miRNA-22, the standard curve of the relationship between fluorescence and pre-miR-22 amount were made by EXCEL. The correlation coefficient (R2 value) of miRNA-22 was 0.9958. The linear fitting graph equation is y=-86524x+2E-06 (Fig 5).

            
            Fig 3.Hela cells were transfected with different miR-22 in 24-well plates.
                      
                  Table 1 The value of fluorescence of miR-22 sensor
           

           
                Fig 4. The value of green fluorescence in cells.

           
                Fig 5. The standard curve of miR-22-sponge-pepper in Hela cells

Conclusion

Besides, wo also detect the sensitivity of miR-145-sponge-pepper (https://parts.igem.org/Part:BBa_K4789005). Taken together, both miR-22-sponge-pepper and miR-145-sponge-pepper can act as “sensor” to monitor the cervical cancer progression. When it comes to the sensitivity and fluorescence intensity, miR-22 sensor is more sensitive than miR-145 as a monitor.

Reference

1. Hao Chenjun,Lin Shaodan,Liu Ping et al. Potential serum metabolites and long-chain noncoding RNA biomarkers for endometrial cancer tissue.[J] .J Obstet Gynaecol Res, 2023, 49: 725-743.

2. Tornesello Maria Lina,Faraonio Raffaella,Buonaguro Luigi et al. The Role of microRNAs, Long Non-coding RNAs, and Circular RNAs in Cervical Cancer.[J] .Front Oncol, 2020, 10: 150.

3. Chen Xianjun,Zhang Dasheng,Su Ni et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.[J] .Nat Biotechnol, 2019, 37: 1287-1293.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 867
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 867
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 576
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 867
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 867
  
• 1000
  COMPATIBLE WITH RFC[1000]