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===Applications of BBa_K2514000=== | ===Applications of BBa_K2514000=== | ||
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+ | <html><head><meta http-equiv="Content-Type" content="text/html; charset=utf-8" /><meta http-equiv="Content-Style-Type" content="text/css" /><meta name="generator" content="Aspose.Words for .NET 17.1.0.0" /><meta name="referrer" content="never"><title></title></head><body><div><p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:14pt"><span style="font-family:'Times New Roman'; font-weight:bold">Followed exp</span><span style="font-family:'Times New Roman'; font-weight:bold">erience is supplemented by 202</span><span style="font-family:'Times New Roman'; font-weight:bold">2</span><span style="font-family:'Times New Roman'; font-weight:bold"> iGEM YiYe-China</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-weight:bold"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:20pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-size:10pt; background-color:#ffffff">Part </span><span style="font-family:'Times New Roman'; font-size:10pt; background-color:#ffffff">BBa_K2514000</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">is a part constructed by the iGEM Worldshaper-Wuhan team in 2017.</span><span style="font-family:'Times New Roman'"> This plasmid contains complementary binding sites to miR-21, which can </span><span style="font-family:'Times New Roman'">monitor the expression of miR-21</span><span style="font-family:'Times New Roman'">. </span><span style="font-family:'Times New Roman'">The </span><span style="font-family:'Times New Roman'">purpose of the project (Worldshaper-Wuhan 2017) is to establish a miRNA sensor which can offer a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><a name="OLE_LINK1"></a><a name="OLE_LINK2"></a><a name="OLE_LINK3"></a><a name="OLE_LINK4"><span style="font-family:'Times New Roman'; font-weight:bold">In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work:</span><span style="-aw-bookmark-end:OLE_LINK1"></span><span style="-aw-bookmark-end:OLE_LINK2"></span><span style="font-family:'Times New Roman'; font-weight:bold"> </span></a></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-weight:bold"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">1. </span><span style="font-family:'Times New Roman'">Based</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">on</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">the</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">reading</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">of</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">a large number of </span><span style="font-family:'Times New Roman'">published papers,</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">we added more information about the alteration of miR-21 expression in the serum and tissues of colorectal cancer patients, compared with </span><span style="font-family:'Times New Roman'">healthy</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">people. </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:11pt"><span style="font-family:'Times New Roman'; font-size:10.5pt">In Fig</span><span style="font-family:'Times New Roman'; font-size:10.5pt">.</span><span style="font-family:'Times New Roman'; font-size:10.5pt">1</span><span style="font-family:'Times New Roman'; font-size:7.33pt; vertical-align:super">[1]</span><span style="font-family:'Times New Roman'; font-size:10.5pt">, a total of 10 adenomas as benign lesions with moderate dysplasia were resected by endoscopic mucosal resection (EMR). Ten specimens of normal colorectal mucosal tissues were evaluated as normal controls. The specific regulation of miRNA-21 expression in CRC tissues </span><span style="font-family:'Times New Roman'; font-size:10.5pt">and controls </span><span style="font-family:'Times New Roman'; font-size:10.5pt">was investigated by one-step qRT-PCR analysis.</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> The level of miRNA-21 is significantly increased in the tissues of colorectal cancer patients, compared with that of controls (Fig.1)</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="-aw-bookmark-end:OLE_LINK3"></span><span style="-aw-bookmark-end:OLE_LINK4"></span><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:94.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><img src="https://static.igem.org/mediawiki/parts/1/1a/YiYe_China_1.png" width="252" height="164" alt="" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline" /></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:68.25pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:11pt"><span style="font-family:'Times New Roman'; font-size:10.5pt">In Fig.2</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> </span><span style="font-family:'Times New Roman'; font-size:7.33pt; vertical-align:super">[</span><span style="font-family:'Times New Roman'; font-size:7.33pt; vertical-align:super">2</span><span style="font-family:'Times New Roman'; font-size:7.33pt; vertical-align:super">]</span><span style="font-family:'Times New Roman'; font-size:10.5pt">,</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> the expression levels of miRNA-21 was detected in the serum of colorectal cancer patients (</span><span style="font-family:'Times New Roman'; font-size:10.5pt">n=48</span><span style="font-family:'Times New Roman'; font-size:10.5pt">) and healthy people (</span><span style="font-family:'Times New Roman'; font-size:10.5pt">n=48</span><span style="font-family:'Times New Roman'; font-size:10.5pt">). In comparison to the control group, the miRNA-21 expression level</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> was upregulated</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> in serum</span><span style="font-family:'Times New Roman'; font-size:10.5pt"> from </span><span style="font-family:'Times New Roman'; font-size:10.5pt">CRC patients (p < 0.05).</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:18pt; text-align:center; widows:0; orphans:0; font-size:9pt"><span style="font-family:'Times New Roman'; color:#ff0000"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:94.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><img src="https://static.igem.org/mediawiki/parts/f/ff/YiYe_China_2.png" width="131" height="255" alt="https://static.igem.org/mediawiki/parts/f/ff/YiYe_China_2.png" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline" /></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">Taken together, these studies fu</span><span style="font-family:'Times New Roman'">rther supported that upregulation of miR-21 was detected in tissues and serum from CRC patients.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">2.</span><span style="font-family:'Times New Roman'; font-size:10pt; font-weight:bold; background-color:#ffffff"> We tested the function of miRNA-21 sponges containing </span><span style="font-family:'Times New Roman'; font-size:10pt; font-weight:bold; background-color:#ffffff">complementary binding sites to miR-21 from Part </span><span style="font-family:'Times New Roman'; font-size:10pt; font-weight:bold; background-color:#ffffff">BBa_K2514000</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:31.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">To confirm the effect of miRNA-21 sponges (pEGFP-miRNA-21-sponge-6s) in cells, we transfected pEGFP-miRNA-21-sponge-6s (0.25</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">ug /well) and Control vectors (0.25</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">ug /well, as negative control) into 293T cells in 24 well-</span><span style="font-family:'Times New Roman'">plates</span><span style="font-family:'Times New Roman'">, respectively. Then the GFP fluorescence was observed under fluorescence microscopy.</span><span style="font-family:'Times New Roman'"> The fluorescence of GFP in cells transfect with pEGFP-miRNA-21-sponge-6s was lower in that of controls. The result confirmed that the fluorescence of GFP could monitor the expression of miRNA-21 in cells (Fig. 3). </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:73.5pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><img src="https://static.igem.org/mediawiki/parts/d/d4/YiYe_China_3.png" width="466" height="247" alt="" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline" /></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-weight:bold">Conclusion</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">1. Compared with the control group, the fluorescence was significantly reduced, which was consistent with the experimental results of the Worldshaper-Wuhan team.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">2. Fluorescence of GFP can monitor the expression of miRNA-21 in cells.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">3. The expression level of miRNA-21 </span><span style="font-family:'Times New Roman'">could be </span><span style="font-family:'Times New Roman'">an indicator </span><span style="font-family:'Times New Roman'">for </span><span style="font-family:'Times New Roman'">colorectal cancer.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'"> </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-weight:bold">References </span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">[1]</span><span style="font-family:'Times New Roman'"> </span><span style="font-family:'Times New Roman'">Yang, Yang, et al. "MicroRNA-21 controls hTERT via PTEN in human colorectal cancer cell proliferation." Journal of physiology and biochemistry 71.1 (2015): 59-68.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'">[2] Ghareib A F, Mohamed R H, el-Fatah A, et al. Assessment of serum MicroRNA-21 gene expression for diagnosis and prognosis of colorectal cancer[J]. Journal of Gastrointestinal Cancer, 2020, 51(3): 818-823.</span></p><p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"><span style="font-family:'Times New Roman'; font-weight:bold"> </span></p></div></body></html> |
Revision as of 12:02, 10 October 2022
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K2514000
Followed experience is supplemented by 2022 iGEM YiYe-China
Part BBa_K2514000 is a part constructed by the iGEM Worldshaper-Wuhan team in 2017. This plasmid contains complementary binding sites to miR-21, which can monitor the expression of miR-21. The purpose of the project (Worldshaper-Wuhan 2017) is to establish a miRNA sensor which can offer a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer.
In order to provide useful support and contribution to future iGEM, we have carried out the following two aspects of work:
1. Based on the reading of a large number of published papers, we added more information about the alteration of miR-21 expression in the serum and tissues of colorectal cancer patients, compared with healthy people.
In Fig.1[1], a total of 10 adenomas as benign lesions with moderate dysplasia were resected by endoscopic mucosal resection (EMR). Ten specimens of normal colorectal mucosal tissues were evaluated as normal controls. The specific regulation of miRNA-21 expression in CRC tissues and controls was investigated by one-step qRT-PCR analysis. The level of miRNA-21 is significantly increased in the tissues of colorectal cancer patients, compared with that of controls (Fig.1)
In Fig.2 [2], the expression levels of miRNA-21 was detected in the serum of colorectal cancer patients (n=48) and healthy people (n=48). In comparison to the control group, the miRNA-21 expression level was upregulated in serum from CRC patients (p < 0.05).
Taken together, these studies further supported that upregulation of miR-21 was detected in tissues and serum from CRC patients.
2. We tested the function of miRNA-21 sponges containing complementary binding sites to miR-21 from Part BBa_K2514000
To confirm the effect of miRNA-21 sponges (pEGFP-miRNA-21-sponge-6s) in cells, we transfected pEGFP-miRNA-21-sponge-6s (0.25 ug /well) and Control vectors (0.25 ug /well, as negative control) into 293T cells in 24 well-plates, respectively. Then the GFP fluorescence was observed under fluorescence microscopy. The fluorescence of GFP in cells transfect with pEGFP-miRNA-21-sponge-6s was lower in that of controls. The result confirmed that the fluorescence of GFP could monitor the expression of miRNA-21 in cells (Fig. 3).
Conclusion
1. Compared with the control group, the fluorescence was significantly reduced, which was consistent with the experimental results of the Worldshaper-Wuhan team.
2. Fluorescence of GFP can monitor the expression of miRNA-21 in cells.
3. The expression level of miRNA-21 could be an indicator for colorectal cancer.
References
[1] Yang, Yang, et al. "MicroRNA-21 controls hTERT via PTEN in human colorectal cancer cell proliferation." Journal of physiology and biochemistry 71.1 (2015): 59-68.
[2] Ghareib A F, Mohamed R H, el-Fatah A, et al. Assessment of serum MicroRNA-21 gene expression for diagnosis and prognosis of colorectal cancer[J]. Journal of Gastrointestinal Cancer, 2020, 51(3): 818-823.