Difference between revisions of "Team:MIT/Experiments/miRNA"

 
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<h1 style="color:#ff9715; text-align: center; font-size: 40px; line-height: 40px;">microRNA: Detecting a Cell Specific Profile</h1>
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<h1 style="color:#ffffff; background-color:#ff9715; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> How can we detect endometriosis using microRNA? </h1>
 
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<img src = "https://static.igem.org/mediawiki/2016/f/ff/T--MIT--miRNAmechanism.PNG" style = "padding:10px; width: 800px; height = 500px; float: left; border:10px;">
 
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> What can we learn from miRNA? </h1> 
 
 
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<p style="font-family: Verdana;"> <img src = "https://static.igem.org/mediawiki/2016/d/d9/T--MIT--miRNA_mechanism.PNG" style = "padding:10px; width: 400px; height = 400px; float: left; border:10px;"> <strong><font size = "2">
 
 
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miRNA are small units of RNA that play a regular role in gene regulation. miRNA binds and cleaves messenger RNA, inhibiting gene expression. There are hundreds of miRNA, and their profile and activity vary between different cell types.
 
 
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<h2 style="color:#ff9715; text-decoration:underline"> What is miRNA?</h2>
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microRNA (miRNA) are small units of RNA <b>found in all mammalian cells</b> and play a regular role in <b>gene regulation</b>. miRNA is single stranded and only about 20 nucleotides long. miRNA forms a RNA-induced silencing complex (RISC) with other proteins and guides RISC to the complementary sequence on the mRNA (the miRNA target site). RISC cleaves messenger RNA and inhibits gene expression post transcriptionally.
 
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<img src = "https://static.igem.org/mediawiki/2016/7/70/T--MIT--miRNA_profile.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
 
<img src = "https://static.igem.org/mediawiki/2016/7/70/T--MIT--miRNA_profile.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
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This makes it is possible to tell the difference between cells from the uterus and liver based on miRNA profiles. Furthermore, cells from the uterus of women with endometriosis have been found to have dysregulated miRNA in comparison to cells from the uterus of women who do not have endometriosis().</p>
 
 
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<FONT COLOR="FF9733" size="5" face="verdana" >Found in all mammalian cells</FONT>
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<h2 style="color:#ff9715; text-decoration:underline"> Why is miRNA a good biomarker?</h2>
 
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There are hundreds of different miRNAs that can make up a specific cell's miRNA profile. The profile and activity of miRNA varies between different <b>cell types </b> and different <b>cells states</b>.This makes it is possible to tell the difference between cells from the uterus and liver, and the difference between healthy and diseased cells based on miRNA profiles.
 
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<FONT COLOR="ff0080" size="5" face="verdana" >Found in all mammalian cells</FONT>
 
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<FONT COLOR="0059b3" size="5" face="verdana" >Unique to cell states</FONT>
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> How can our circuit sense miRNA? </h1> 
 
  
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<img src = "https://static.igem.org/mediawiki/2016/c/c2/T--MIT--miRNA_ts_setup.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
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<h2 style="color:#ff9715; text-decoration:underline"> What is the miRNA profile of Endometriosis?</h2>
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<img src = "https://static.igem.org/mediawiki/2016/b/bb/T--MIT--miRNA_candidates.PNG" style = 'padding: 5px'; width: 250px; height = 250px; float: right; border:5px;'></p>
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<p>Endometrial cells from women with endometriosis have been found to have dysregulated miRNA compared to endometrial cells from women who do not have endometriosis.(<a href=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744474/>1,</a><a href=http://molehr.oxfordjournals.org/content/13/11/797.full>2,</a><a href=https://www.ncbi.nlm.nih.gov/pubmed/26370665>3</a>). In order to study the uses of these miRNAs in our circuit, eight candidates were chosen based on fold difference in miRNA level between the the endometrial biopsy from women with and without endometriosis. The eight candidates are shown in the table to the right:</p>
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<img src = "https://static.igem.org/mediawiki/2016/7/75/T--MIT--endometriosis_profile.PNG" style = 'padding: 5px 10px 5x 10px;       width: 400px; height = 400px; float: left; border:10px;'>
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<figcaption><a href=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744474/>(1)</a> One of the graphs observed in order to choose miRNA candidates to characterize. The three miRNAs, miR-34c-5p, miR-9, miR-34-b are shown to be downregulated and have a significant fold difference (*p=0.05 **p<0.05, error bars= standard error of mean) compared to their respective levels in healthy eutopic endometrial cells. These measurements were from an endometrial biopsy and taken using quantitative polymerase chain reaction (qPCR), similar to the other sources we also studied.</figcaption>
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<b> Eight miRNAs candidates were chosen to be characterized in order to determine their gene regulatory ability for the purposes of increasing the specificity of our circuit.</b>
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<h1 style="color:#ffffff; background-color:#ff9715; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> How can our circuit sense miRNA activity? </h1> 
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<img src = "https://static.igem.org/mediawiki/2016/0/0e/T--MIT--miRNA_targetsites2.PNG" style = 'padding: 5px 10px 5x 10px; width: 500px; height = 500px; float: right; border:10px;'>
  
 
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Our circuit utilizes the natural function of miRNA to produce a differential output depending on whether the cell has dysregulated miRNA. This is achieved by attaching 4 tandem sites complementary to the affected miRNA following a gene of interest. </p>
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Our circuit utilizes the natural function of miRNA to regulate gene expression. Depending on the miRNA activity in a cell, different levels of our desired gene will be expressed. This allows our circuit to produce a differential output depending on whether the cell has dysregulated miRNA. This is achieved by attaching 4 tandem sites complementary to the affected miRNA following a gene of interest. This is called the miRNA target site (miRNA-ts).
 
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Are our target sites sensitive to miRNA levels? </h1>  
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<b>As miRNA activity increases, the output of the desired gene decreases.</b>
  
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<a href="https://2016.igem.org/Team:MIT/Experiments/miRNA/more_background">Read more about our decision to use miRNA sensors</a>
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<h1 style="color:#ffffff; background-color:#ff9715; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> Are the target sites sensitive to miRNA levels? </h1>
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<img src = "https://static.igem.org/mediawiki/2016/6/60/T--MIT--tHESC_siRNA_final1.PNG" style = 'padding: 5px'; width: 300px; height = 400px; float: right; border:5px;'>
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These 4 tandem sites for miRNA binding were tested by coding for them distal to the gene for red fluorescent protein. We were able to see a ten fold repression upon increasing the concentration of siRNA from 0 to 1 nM. Saturation appeared at about 10 nM.
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These 4 tandem sites for miRNA binding were tested by inserting them after a red florescent gene (mKate) in the 3' untranslated region. We were able to see a ten fold repression upon increasing the concentration of siRNA from 0 to 1 nM. Saturation appeared at about 10 nM.
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<a href="https://2016.igem.org/Team:MIT/Experiments/miRNA/more_experiments">Read more about our experiment testing miRNA target site sensitivity</a>
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More information on testing target site sensitivity. </p>
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<h1 style="color:#ffffff; background-color:#ff9715; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> Does miRNA activity in endometrial cells respond to hormones? </h1>
  
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<p style="font-family: Verdana;"> <img src = "https://static.igem.org/mediawiki/2016/3/3b/T--MIT--tHESC_profile.PNG" style = "padding:10px; width: 250px; height = 250px; float: right; border:10px;">
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<img src = "https://static.igem.org/mediawiki/2016/b/b5/T--MIT--miRNA_profile_final.PNG" style = "padding:10px; width: 550px; height = 600px; float: left; border:10px;">
 
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Why use a miRNA sensor? </h1>
 
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The two most common methods for looking at the miRNA profile of a cell line are by miRNA microarray and quantitative polymerase chain reaction (qPCR). However, it has been indicated that the physical amount of miRNA present doesn't always have a strong correlation to the repression of the associated gene. By using a miRNA sensor, we can directly measure the activity of the miRNA, which the aspect of miRNA being used in the circuit.
 
 
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<i>"We detected the expression of more than 310 miRNAs (Fig. 2a). Our library included sensors for 165 of these miRNAs (188 when considering families), but we detected the suppression of only 67 sensors (Fig. 2b). Thus, 59% of the expressed miRNAs that we sampled did not have suppressive activity."</i> Mullokandov et. al Nature 2012
 
 
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The eight miRNA candidate target sites were characterized in TERT-immortalized Human Endometrial Stromal Cells (tHESC).
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Unlike more common cell lines like MCF7 and HEK293, tHESC is not a highly characterized cell line.This meant that the levels of our eight miRNA candidates were completely unknown in tHESC. We set out to characterize our miRNA target sites (miRNA-ts) in tHESC using a miRNA sensor.
 
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<a href="http://press.endocrine.org/doi/10.1210/en.2003-1606?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed">What is tHESC?</a>
 
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<a href="https://2016.igem.org/Team:MIT/Experiments/miRNA/more_experiments">Read more about our experiment to characterize miRNA targets sites in tHESC</a>
 
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Using a miRNA Sensor </h1> 
 
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The sensor, courtesy of Jeremy Gam, consists of a red fluorescent protein, mKate, which is constitutively controlled by the hef1a promoter. After mKate, you will notice here are four yellow blocks. These represent four miRNA target site domain repeats. After the target sites is a blue fluorescent protein also controlled by the hef1a promoter. When there is miRNA present, it will bind to the miRNA target site on the mKate mRNA and repress the expression of mKate.
 
  
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<a href="https://2016.igem.org/Team:MIT/Experiments"><h1 style="color:#FFFFFF; background-color:#ff9715;; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: trebuchet MS"> Back to Experiments Home</h1></a>
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<p> References
 
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1.Burney, RO, et al. Molecular Human Reproduction (2009). 15(10):625-631.
 
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2.Pan, Q. Xiaoping, L. Toloubeydokhti, T. Chegini, N. Basic Science of Reproductive Medicine (2007). 13(11):797-806.
 
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3.Joshi, NR, et al. Human Reproduction (2015). 30(12):2881-91.
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Probing tHESC for miRNA Levels </h1>
 
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Unlike more common cell lines like MCF7 and HEK293, tHESC is not a highly characterized cell line. This meant that the levels of our eight miRNA candidates were completely unknown in tHESC. We set out to characterize our miRNA target sites (miRNA-ts) in tHESC using a miRNA sensor.
 
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<a href="https://2016.igem.org/Team:MIT/Experiments/miRNA">What is tHESC?</a>
 
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Latest revision as of 01:11, 20 October 2016

microRNA: Detecting a Cell Specific Profile

How can we detect endometriosis using microRNA?




















What is miRNA?


microRNA (miRNA) are small units of RNA found in all mammalian cells and play a regular role in gene regulation. miRNA is single stranded and only about 20 nucleotides long. miRNA forms a RNA-induced silencing complex (RISC) with other proteins and guides RISC to the complementary sequence on the mRNA (the miRNA target site). RISC cleaves messenger RNA and inhibits gene expression post transcriptionally.



Why is miRNA a good biomarker?


There are hundreds of different miRNAs that can make up a specific cell's miRNA profile. The profile and activity of miRNA varies between different cell types and different cells states.This makes it is possible to tell the difference between cells from the uterus and liver, and the difference between healthy and diseased cells based on miRNA profiles.


Found in all mammalian cells


Unique to cell type


Unique to cell states


What is the miRNA profile of Endometriosis?

Endometrial cells from women with endometriosis have been found to have dysregulated miRNA compared to endometrial cells from women who do not have endometriosis.(1,2,3). In order to study the uses of these miRNAs in our circuit, eight candidates were chosen based on fold difference in miRNA level between the the endometrial biopsy from women with and without endometriosis. The eight candidates are shown in the table to the right:




(1) One of the graphs observed in order to choose miRNA candidates to characterize. The three miRNAs, miR-34c-5p, miR-9, miR-34-b are shown to be downregulated and have a significant fold difference (*p=0.05 **p<0.05, error bars= standard error of mean) compared to their respective levels in healthy eutopic endometrial cells. These measurements were from an endometrial biopsy and taken using quantitative polymerase chain reaction (qPCR), similar to the other sources we also studied.






Eight miRNAs candidates were chosen to be characterized in order to determine their gene regulatory ability for the purposes of increasing the specificity of our circuit.


How can our circuit sense miRNA activity?

Our circuit utilizes the natural function of miRNA to regulate gene expression. Depending on the miRNA activity in a cell, different levels of our desired gene will be expressed. This allows our circuit to produce a differential output depending on whether the cell has dysregulated miRNA. This is achieved by attaching 4 tandem sites complementary to the affected miRNA following a gene of interest. This is called the miRNA target site (miRNA-ts).

As miRNA activity increases, the output of the desired gene decreases.

Read more about our decision to use miRNA sensors







Are the target sites sensitive to miRNA levels?

These 4 tandem sites for miRNA binding were tested by inserting them after a red florescent gene (mKate) in the 3' untranslated region. We were able to see a ten fold repression upon increasing the concentration of siRNA from 0 to 1 nM. Saturation appeared at about 10 nM.

Read more about our experiment testing miRNA target site sensitivity



Does miRNA activity in endometrial cells respond to hormones?


















The eight miRNA candidate target sites were characterized in TERT-immortalized Human Endometrial Stromal Cells (tHESC). Unlike more common cell lines like MCF7 and HEK293, tHESC is not a highly characterized cell line.This meant that the levels of our eight miRNA candidates were completely unknown in tHESC. We set out to characterize our miRNA target sites (miRNA-ts) in tHESC using a miRNA sensor.

What is tHESC?

Read more about our experiment to characterize miRNA targets sites in tHESC

Back to Experiments Home

References
1.Burney, RO, et al. Molecular Human Reproduction (2009). 15(10):625-631.
2.Pan, Q. Xiaoping, L. Toloubeydokhti, T. Chegini, N. Basic Science of Reproductive Medicine (2007). 13(11):797-806.
3.Joshi, NR, et al. Human Reproduction (2015). 30(12):2881-91.