iGEM 2020 — Team Worldshaper-Wuhan

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Worldshaper-Wuhan/Notebook

Notebook

Home

[Home](https://2020.igem.org/Team%3AWorldshaper-Wuhan)

Project

[Description](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Description)
[Design](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Design)
[Engineering Success](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Engineering)
[Proposed Implementation](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Implementation)
[Modeling](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Model)

Experiment

[Contribution](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Contribution)
[Improvement of an Existing Part](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Improve)
[Notebook](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
[Protocols](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Protocol)
[Safety](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Safety)

Parts

[Parts](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Parts)

Hp

[HP](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
[IHP](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
[Science communication](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Education)

Team

[Team members](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Team)
[Attributions](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Attributions)
[Collaboration](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)

Judging Form

[Judging Form](https://igem.org/2020_Judging_Form?id=3638)

##### First

##### Second

## The experiment note in the Wuhan University

[NOTEBOOK1](https://static.igem.org/mediawiki/2020/4/46/T--Worldshaper-Wuhan--note1.pdf)

## The experiment note in the Optics Valley

[NOTEBOOK2](https://static.igem.org/mediawiki/2020/d/d6/T--Worldshaper-Wuhan--note2.pdf)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Worldshaper-Wuhan/Engineering

Engineering Success

Home

[Home](https://2020.igem.org/Team%3AWorldshaper-Wuhan)

Project

[Description](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Description)
[Design](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Design)
[Engineering Success](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Engineering)
[Proposed Implementation](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Implementation)
[Modeling](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Model)

Experiment

[Contribution](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Contribution)
[Improvement of an Existing Part](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Improve)
[Notebook](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
[Protocols](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Protocol)
[Safety](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Safety)

Parts

[Parts](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Parts)

Hp

[HP](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
[IHP](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
[Science communication](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Education)

Team

[Team members](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Team)
[Attributions](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Attributions)
[Collaboration](https://2020.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)

Judging Form

[Judging Form](https://igem.org/2020_Judging_Form?id=3638)

##### Expression

##### Compete

##### Construction

##### Effect

##### pEGFP-miR-155-sponge-1

##### References

## Results

#### 1. The expression of XIST decreased in breast cancer.

LncRNA is a class of ncRNA whose length is over 200 base pairs. The aberrant expressions of lncRNAs were associated with human diseases, including breast cancer. Recently, several studies indicated that lncRNAs could act as sponges to compete miRNAs, participating in various biological processes [1]. miRNA Sponges contain complementary binding sites to a miRNA of interest, which inhibit miRNA activity. This mechanism gives rise to our idea about fusing a sponge RNA based on the sequences of lncRNA with binding sites complementary to the sequence of miRNA to a plasmid that has reporter gene, EGFP-C1 for instance, which will monitor the expression of miRNA in the cells.
In breast cancer research, several lncRNAs are identified as tumor driving oncogenic lncRNAs, such as H19, LncRNA-Smad7, and few are identified as tumor suppressive lncRNAs, for example GAS5. [2]They are involved in cell growth, apoptosis, cell migration and invasiveness as well as cancer cell stemness. However, the expression of some lncRNA in breast cancer, for example XIST, NEAT1, remains controversial and unclear. According to previous studies, several lncRNAs, including XIST, NEAT1, GAS5, MAL1-AS, SNHG16, were selected for further investigation. We downloaded the data about expression level of XIST, NEAT1, GAS5, and SNHG16 in breast tumors and controls from TANRIC [3] XIST and NEAT1 were expressed at lower levels in breast cancer (Fig.1). The expression of SNHG16 increased in breast tumors, compared with that of control (Fig.1). And GAS5 level was showed no change in breast cancer (Fig.1). Based on the data of Fig.1, we choose XIST to design our microRNA sensor.

![](https://static.igem.org/mediawiki/2020/0/0a/T--Worldshaper-Wuhan--re1.png)

Fig.1 The expression of some lncRNAs in breast cancer by TANRIC.

#### 2. XIST acts as sponges to compete miR-155

We downloaded the sequence of XIST from NCBI [4]and predicted miRNAs which could bind to XIST from TargetScan. We found miR-155 could bind to the sequence of XIST (Fig.2). miR-155 is an oncomiR that is overexpressed in several cancers, including breast cancer. A growing number of studies highlights the role of miR-155 in breast cancer drug resistance development. Given the fact that lncRNA could act as miRNA sponge, we designed the sequence that could bind to miR-155 based on XIST sequence. To improve the binding efficiency, miR-155 sensors contain three binding sites(Fig.3).

![](https://static.igem.org/mediawiki/2020/d/de/T--Worldshaper-Wuhan--re2.png)

Fig.2. The pairing between miR-155 and XIST with binding sites is diagrammed. The miRNA seed region is showed by red.

![](https://static.igem.org/mediawiki/2020/c/c7/T--Worldshaper-Wuhan--re3.png)

Fig.3. The diagram of miR-155 sensor (pEGFP-miR-155-sponge-1)

#### 3. Construction of the plasmids

We constructed two plasmids pEGFP-miR-155-sponge-1 and Phage-pre-miR-155. The plasmids of pEGFP-miR-155-sponge-1 was used to monitor the expression of miR-155 in cells. And Phage-pre-miR-155 was applied to overexpress miR-155 in cells. These plasmids were synthesized by Nanjing Genscript Biotechnology corporation.

#### 4. The effect of pEGFP-miR-155-sponge-1 in breast cancer cells

MicroRNA-155 (miR-155) (kkb website) is a well-known oncogenic miRNA overexpressed in many human cancers, including breast cancer[5]. So we constructed pEGFP-miR-155-sponge-1 and try to test the possibility of detecting miR-155 expression by using this plasmid. In order to explore whether XIST correlates with pEGFP-miR-155 sensor in breast cancer cells with potential target sites. pEGFP–C1 (as negative controls), pEGFP-miR-155-sponge-1 (0.8 ug plasmids for each well) was transfected into human breast cells (MDA-MB 231 cells and MDA-MB 468 cells) in 24-well plate, respectively. After transfection, cells were examined under fluorescence microscopy (Fig.4 A, B, C, D and Table 1). The fluorescence of GFP was decreased in MDA-MB 231 cells and MDA-MB 468 cells transfected with pEGFP-miR-155-sponge-1 compared with controls (Fig. 5). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The endogenous miR-155 could inhibit the expression of GFP in cells transfected with pEGFP-miR-155-sponge-1 (Table 1 and Fig5). The results suggested the GFP value of cells transfected with pEGFP-miR-155-sponge-1 could show the different expression of miRNAs in cells.

![](https://static.igem.org/mediawiki/2020/7/72/T--Worldshaper-Wuhan--re4.png)

Fig 4. The images of different breast cells transfected with different plasmids.

(A). pEGFP–C1 was transfected in MDA-MB 468 cells. (B). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 468 cells. (C). pEGFP–C1 was transfected in MDA-MB 231 cells. (D).pEGFP-miR-155-sponge-1 was transfected in MDA-MB 231 cells.

#### Table 1. The value of GFP fluorescence in cells.

![](https://static.igem.org/mediawiki/2020/2/23/T--Worldshaper-Wuhan--re5.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Worldshaper-Wuhan/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Worldshaper-Wuhan/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)