iGEM 2018 — Team Worldshaper-Wuhan

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# Team:Worldshaper-Wuhan/Notebook

* [HOME](https://2018.igem.org/Team%3AWorldshaper-Wuhan)
* [BACKGROUND](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Background)
* PROJECT

  [SOLUTION](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Solution)
  [RESULTS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Results)
  [MODEL](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Model)
  [PROCEDURE](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Procedure)
  [NOTEBOOK](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
* [INTERLAB](https://2018.igem.org/Team%3AWorldshaper-Wuhan/InterLab)

![logo](http://igem2017.worldshaper.cn/2018/Wuhan/images/logo.png)
![logo](http://igem2017.worldshaper.cn/2018/Wuhan/images/logo-3.png)

* HUMAN PRACTICE

  [SILVER](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
  [GOLD](https://2018.igem.org/Team%3AWorldshaper-Wuhan/HP/Gold_Integrated)
  [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Public_Engagement)
  [COLLABORATIONS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)
* PARTS

  [BASIC](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Basic_Part)
  [IMPROVE](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Improve)
* [SAFETY](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Safety)
* [ATTRIBUTIONS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Attributions)

## heading

* ![](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebookhome.jpg)

  NOTEBOOK

  [learn more](#aboutus)

## Notebook

![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/1.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/2.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/3.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/4.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/5.jpg)
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![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/7.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/8.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/9.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/10.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/11.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/12.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/13.jpg)
![notebook](http://igem2017.worldshaper.cn/2018/Wuhan/images/notebook/14.jpg)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Worldshaper-Wuhan/Experiments

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[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AWorldshaper-Wuhan)
[HOME](https://2018.igem.org/Team%3AWorldshaper-Wuhan)

TEAM

[Team Members](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Team)
[Collaborations](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Description)
[Design](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Design)
[Experiments](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Experiments)
[Notebook](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
[InterLab](https://2018.igem.org/Team%3AWorldshaper-Wuhan/InterLab)
[Model](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Model)
[Results](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Results)
[Demonstrate](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Demonstrate)
[Improve](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Improve)
[Attributions](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Parts)
[Basic Parts](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Hardware)
[Measurement](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Measurement)
[Model](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Model)
[Plant](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Plant)
[Software](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Worldshaper-Wuhan)

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### 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/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Worldshaper-Wuhan/Results

* [HOME](https://2018.igem.org/Team%3AWorldshaper-Wuhan)
* [BACKGROUND](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Background)
* PROJECT

  [SOLUTION](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Solution)
  [RESULTS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Results)
  [MODEL](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Model)
  [PROCEDURE](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Procedure)
  [NOTEBOOK](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
* [INTERLAB](https://2018.igem.org/Team%3AWorldshaper-Wuhan/InterLab)

![logo](http://igem2017.worldshaper.cn/2018/Wuhan/images/logo.png)
![logo](http://igem2017.worldshaper.cn/2018/Wuhan/images/logo-3.png)

* HUMAN PRACTICE

  [SILVER](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Human_Practices)
  [GOLD](https://2018.igem.org/Team%3AWorldshaper-Wuhan/HP/Gold_Integrated)
  [PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Public_Engagement)
  [COLLABORATIONS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)
* PARTS

  [BASIC](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Basic_Part)
  [IMPROVE](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Improve)
* [SAFETY](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Safety)
* [ATTRIBUTIONS](https://2018.igem.org/Team%3AWorldshaper-Wuhan/Attributions)

## heading

* ![](http://igem2017.worldshaper.cn/2018/Wuhan/images/resultsHome.jpg)

  RESULTS

  [learn more](#aboutus)

# Our Results

Identification of IL7-AS expression in renal cell carcinoma by TANRIC

Based on evidence that IL7-AS can be induced across multiple human and mouse cell types and has been reported to regulate the expression of interleukin-6 (IL6). IL6 is emerging as a major regulator of renal cell cancer. We asked whether IL7-AS is involved in pathogenesis of renal cell carcinoma. By analyzing data from TANRIC (http://ibl.mdanderson.org/tanric/\_design/basic/query.html), we collected the expression data of IL7-AS in 64 normal tissues and 432 kidney renal clear cell carcinoma tissues. IL7-AS was expressed at higher levels in kidney renal clear cell carcinoma (KIRC) than in normal kidney (Fig. 1).

![Fig. 1 IL7-AS was expressed at higher levels in kidney renal clear cell carcinoma than in normal kidney by TANRIC.](http://igem2017.worldshaper.cn/2018/Wuhan/images/results1.jpg)

Fig. 1 IL7-AS was expressed at higher levels in kidney renal clear cell carcinoma than in normal kidney by TANRIC.

Identification of IL7-AS expression in kidney cancer cell lines

The 769-P and 786-O cell lines are derived from primary clear cell adenocarcinoma. The 293T cell line is originally derived from human embryonic kidney. We examined the expression levels of IL7-AS in 769-P, 786-O and HEK-293T cell lines. IL7-AS expression was markedly higher in 786-O and 769-P cells than in HEK-293T cells (Fig. 2). We also observed that up-regulated IL7-AS expression was correlated with IL6 expressions in these cell lines (Fig. 2).

![Fig. 2 Compared to HEK-293T, IL7-AS was highly expressed in 786-O and 769-P cells as shown by real-time PCR](http://igem2017.worldshaper.cn/2018/Wuhan/images/results2.jpg)

Fig. 2 Compared to HEK-293T, IL7-AS was highly expressed in 786-O and 769-P cells as shown by real-time PCR

IL7-AS promotes kidney cancer cell migration

Cell migration is a central process in the development and maintenance of tumor. We cloned the full length of IL7-AS, two truncated sequences of IL7-AS (IL7-AS-S1 and IL7-AS-S2) into PCDNA3.1 (Fig. 3) and transfected these plasmids to 786-O cells. Through in vitro scratch wound healing assay, overexpression of IL7-AS promoted cell migration of 786-O cells (Fig. 4 and 5). In order to determine which domain is essential for the migration of IL7-AS, we cloned two truncated sequences of IL7-AS (IL7-AS-S1 and IL7-AS-S2). Overexpression of IL7-AS-S1 or IL7-AS-S2 increased the migration of 786-O cells (Fig. 4 and 5). These results suggested that IL7-AS-S1 and IL7-AS-S2 contain key structural domains. IL7-AS may play an important role in migration of renal cell carcinoma.

![Fig. 3 Scheme of three IL7-AS isoforms on human chromosome 8](http://igem2017.worldshaper.cn/2018/Wuhan/images/results3.jpg)

Fig. 3 Scheme of three IL7-AS isoforms on human chromosome 8

![Fig.4](http://igem2017.worldshaper.cn/2018/Wuhan/images/results4.jpg)
![Fig.4](http://igem2017.worldshaper.cn/2018/Wuhan/images/results5.jpg)

Fig. 4 Overexpression of IL7-AS-S2, IL7-AS-S1 and IL7-AS promotes 786-O cells migration. 786-O cells were transfected with pcDNA3.1-IL7-AS-S2, pcDNA3.1-IL7-AS-S1 or pcDNA3.1-IL7-AS for 24 h. The cells growth areas were calculated with Image J at the time points of 0, 6, 12 and 24 h. Bar=100 μm. Data represents means ± SE from 3 independent experiments. \*p<0.05 versus non-transfected cells.

In order to investigate whether cell migration induced by IL7-AS-S2 depends on the amount of IL7-AS-S2 transfection. We transfected the different concentrations of IL7-AS-S2 (1ug, 2ug, 4ug) into 786-0 cells. Both three concentrations of IL7-AS-S2 can infect the migration of 786-0 cells (Fig. 5）.

![Fig.5](http://igem2017.worldshaper.cn/2018/Wuhan/images/results6.jpeg)
![Fig.5](http://igem2017.worldshaper.cn/2018/Wuhan/images/results7.jpeg)

Fig. 5 Different concentrations of IL7-AS-S2 promotes 786-O cells migration. 786-O cells were transfected with pcDNA3.1-IL7-AS-S2 (1 ug, 2 ug, 4 ug) for 24 h. The cells growth areas were calculated with Image J at the time points of 0, 6, and 12 h. Bar=100 μm. Data represents means ± SE from 3 independent experiments. \*p<0.05 versus non-transfected cells.

IL7-AS contains a complicated secondary structure

In silico prediction of lncRNA secondary structure is another useful method to assign putative functions to non-coding transcripts, based upon the widely held assumption that highly folded structures impart functionality through binding interactions with proteins/nucleotides. Characterization of IL7-AS, IL7-AS-S1 and IL7-AS-S2 using RNA fold minimum free energy estimations predicted a highly folded secondary structure with several hairpin loops, which imply interact with protein (Fig. 6).

A. IL7-AS

![IL7-AS](http://igem2017.worldshaper.cn/2018/Wuhan/images/il7-as.jpeg)

B. IL7-AS-S1

![IL7-AS-S1](http://igem2017.worldshaper.cn/2018/Wuhan/images/il7-as-s1.jpeg)

C. IL7-AS-S2

![IL7-AS-S2](http://igem2017.worldshaper.cn/2018/Wuhan/images/il7-as-s2.jpeg)

Fig. 6 The optimal secondary structure with a minimum free energy. The secondary structures of IL7-AS; IL7-AS-S1 and IL7-AS-S2 were predicted by the RNAfold webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The structure is colored according to base-pairing probabilities. For unpaired regions, the color denotes the probability of being unpaired.

Bioinformatic prediction of IL7-AS-S2 function

LncRNAs perform the function through interacting with proteins. Considering IL7-AS-S2 can influence the cell migration, we want to predict proteins which can interact with IL7-AS-S2. Using the catRAPID omics algorithm (http://service.tartaglialab.com/page/catrapid\_omics\_group), we determined whether IL7-AS-S2 interacts with proteins. Results showed that the related RNA-binding proteins were involved in RNA-splicing/processing (CWC15, RU2A), and protein synthesis (EIF3G, SBDS) (table 1). Based on the proteins predicted, we estimated that IL7-AS-S2 may regulate RNA splicing or protein synthesis of some tumor related genes though interacting with several RNA-splicing or protein synthesis associated proteins.

Table 1. Predicted proteins interacted with IL7-AS-S2

![Table1](http://igem2017.worldshaper.cn/2018/Wuhan/images/results9.jpg)

Conclusions and perspectives

The results reported here demonstrate that IL7-AS may play an important role in pathogenesis of renal cell carcinoma. We found that IL7-AS was expressed at higher levels in kidney renal clear cell carcinoma than in normal kidney. Moreover, IL7-AS expression was higher in renal cell carcinoma associated cells than in human embryonic kidney cells. IL7-AS promoted the migration of renal cell carcinoma related cells. Our results for the first time to evaluate the possibility of IL7-AS in renal cell carcinoma as a novel biomarker for diagnosis and a potential target for therapy in the future.