Difference between revisions of "Part:BBa K3638003:Experience"
 
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=Applications of BBa_K3638003=
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<div id="write" class=""><p><span> </span></p ><p><strong><span>Results</span></strong><span> </span></p ><p><strong><span>1. The expression of XIST decreased in breast cancer.</span></strong><span> </span></p ><p><span> 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. </span></p ><p><span> 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. </span></p ><p>< img src="file:///C:/Users/Blank/AppData/Local/Temp/msohtmlclip1/01/clip_image002.gif" referrerp
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===effect of pEGFP-miR-155-sponge-1 in breast cancer cells===
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MicroRNA-155 (miR-155) 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.
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[[File:The value of GFP fluorescence in cells.png|600px]]
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<br/>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.  
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[[File:The images of different breast cells transfected with different plasmids.png|500px]]
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[[File:Fig5. The value of GFP fluorescence in cells. Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.png|600px]]
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<br/>Fig5. The value of GFP fluorescence in cells. Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.

Latest revision as of 15:13, 3 October 2020

Applications of BBa_K3638003

effect of pEGFP-miR-155-sponge-1 in breast cancer cells

MicroRNA-155 (miR-155) 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. The value of GFP fluorescence in cells.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. The images of different breast cells transfected with different plasmids.png Fig5. The value of GFP fluorescence in cells. Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.png
Fig5. The value of GFP fluorescence in cells. Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.