Team:Worldshaper-Wuhan/Engineering
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.
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).
Fig.2. The pairing between miR-155 and XIST with binding sites is diagrammed. The miRNA seed region is showed by red.
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.
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.
Fig5. The value of GFP fluorescence in cells. Different cells were transfected with pEGFP-C1 or pEGFP-miR-155-sponge-1 for 48 h.
5. The effect of pEGFP-miR-155-sponge-1 as a monitor to detect the expression of miR-155
In order to explore whether the expression of miR-155 correlates with the GFP value of pEGFP-miR-155-sponge-1 in breast cancer cells, breast cancer cells were cotransfected with pEGFP-miR-155-sponge-1 and pre-miR-155 (overexpression of mature miR-155). After transfection, cells were examined under fluorescence microscopy (Fig. 6 A, B, C, D). The fluorescence of GFP was decreased in MDA-MB 231 cells and MDA-MB 468 cells transfected with pre-miR-155, compared with that of controls (Fig. 6). In addition, we also measured the value of GFP fluorescence by plate reader (SpectraMax i3). The down-regulation of GFP fluorescence was observed in breast cancer cells transfected with pre-miR-155, compared with controls (Fig. 7). Taken together, these results reveal pEGFP-miR-155-sponge-1 could act as a monitor to detect the expression of miR-155.
Fig 6. The images of different breast cells transfected with different plasmids.
(A). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 468 cells. (B). pEGFP-miR-155-sponge-1 and pre-miR-155 were cotransfected in MDA-MB 468 cells. (C). pEGFP-miR-155-sponge-1 was transfected in MDA-MB 231 cells (D). pEGFP-miR-155-sponge-1 and pre-miR-155 were cotransfected in MDA-MB 231cells.
Table 2. The value of GFP fluorescence in cells.
Fig 7. The value of GFP fluorescence in cells. Different cells were cotransfected with pEGFP-miR-155-sponge-1 and pre-miR-155 for 48 h.
To further test the effect of pEGFP-miR-155-sponge-1 as a monitor to detect the expression of miR-155, cells were transfected with pEGFP-miR-155-sponge-1 with different concentration of pre-miR-155. We used the plasmid of pre-miR-155 to quantify miR-155 expression and calculate the copy numbers of miR-155 by using the formula listed below. The down-regulation of GFP fluorescence was observed in breast cancer cells transfected with different concentration of pre-miR-155 compared with normal cells (Fig. 7). Taken together, these results reveal a combination of pEGFP-miR-155 sensor and XIST. copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
Table 3 The value of eGFP fluorescence in MDA-MB 468 cells
The standard curve of pEGFP-miR-155-sponge-1 was made by EXCEL (Fig8). We find that the value of fluorescence is dependent on the copy numbers of miR-155 in cells. Based on the formula, the correlation coefficient (R2 value) of pEGFP-miR-155-sponge-1 in MDA-MB 468 cells was 0.9988.
Fig 8. The standard curve of pEGFP-miR-155-sponge-1 in MDA-MB 468 cells
References:
[1] Zhou S, Wang L, Yang Q, Liu H, Meng Q, Jiang L, Wang S, Jiang W. Systematical analysis of lncRNA-mRNA competing endogenous RNA network in breast cancer subtypes. Breast Cancer Res Treat. 2018 Jun;169(2):267-275. doi: 10.1007/s10549-018-4678-1. Epub 2018 Feb 1. PMID: 29388017.
[2] Peixin Dong,1,*† Ying Xiong,2,† Junming Yue,3,4,† Sharon J. B. Hanley,1 Noriko Kobayashi,1 Yukiharu Todo,5 and Hidemichi Watari1,*Exploring lncRNA-Mediated Regulatory Networks in Endometrial Cancer Cells and the Tumor Microenvironment: Advances and Challenges Cancers (Basel). 2019 Feb; 11(2): 234.
[3] http://ibl.mdanderson.org/tanric/_design/basic/query.html
[4] NR_001564, https://www.ncbi.nlm.nih.gov/nuccore/NR_001564.2
[5] Zi-Ming Du, Li-Fu Hu, Hai-Yun Wang, Li-Xu Yan, 1 Yi-Xin Zeng, Jian-Yong Shao * and Ingemar Ernberg *Upregulation of MiR-155 in Nasopharyngeal Carcinoma is Partly Driven by LMP1 and LMP2A and Downregulates a Negative Prognostic Marker JMJD1APLoS One. 2011; 6(4): e19137.