Team:CSU CHINA/Demonstrate
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Demonstration
√ Specific function of synthetic promoter in TNBC cell line.【Module1】
√ High efficiency of miR-BD binding the endogenous miRNA.【Collaboration with SYSU_CHINA】
√ High efficiency of synthetic sponge inhibiting the endogenous miRNA. 【Module2】
√ Specific damaging effect of the whole system towards TNBC cells. 【Module1&2&3】
First-Phase Preparation
We identified genes that were differentially expressed in human triple negative breast cancer (TNBC) cells compared to healthy tissues from the publicly available The Cancer Genome Atlas (TCGA) database [1] using R (version 3.0.2). We followed standard procedures to calculate differentially expressed genes between cancer cases compared to controls using t test. We conclude nine TFs (transcription factors), namely ESR1, E2F1, FOXM1, MYBL2, PITX1, PTTG1, UHRF1, FOXA1, GATA3, which express statistically different between TNBC and normal tissues (Figure1).
Figure1:(A) The heat map compared the expression of several TFs (transcription factors) between carcinoma and normal tissues in different types of cancer.(B) The graph compared the nine TFs based on the database.
Mostly, the expression of genes in vivo (get from patients’ tissue) is dramatically different with cell lines, although which are derived from patients. But on the phase of experiments, we need to imitate the process that could happen when our system get into body. In this case, we are supposed to test the expressions of the above nine TFs in breast cancer cell lines to see whether it can be used in our project.
Firstly, the table below (Table1) indicates detailed information about five cell lines we used, related with breast and different types of breast cancer. In length, HBL-100 is a spontaneously immortalized cell line derived from breast milk from a lactating, young, healthy woman[2]; MDA-MB-231 represents the basal B TNBC and often be used to identify genes and pathways that regulate metastasis to different sites[3-6], etc.
So, we extracted the total RNA from the five cell lines mentioned above and did the real-time RCR to examined the relative mRNA expression (compared to HBL-100, the normal breast cells), which shows the specific&high expression of ESR1 and GATA3 in TNBC cell lines (Figure1).
Figure2: The graph shows the expression of nine TFs screened form database in five breast-related cell lines.
The other significant factor we need to confirm is the miRNA targeting specific binding sites, which directly connected to the efficiency of our designed system. We define the miRNA down-regulated in TNBC cell lines by scanning the existed literature (Table2).
Table2: This table shows 14 miRNAs we screened from literatures that down-regulated in TNBC cell lines, compared to normal breast cells.
In order to confirm the results we get form online resources, we detected the true expression level in the cells lines we interested (Figure2), which demonstrates that the miR-141&miR148b is actually up-regulated in MDA-MB-231, while miR-146&miR-409-3p is on the contrary.
Figure3:The real-time PCR results shows the expression level in MDA-MB-231 and MCF-7(use as an positive control) compared to HBL-100. (A) The down-regulated miRNAs in MDA-MB-231; (B) The up-regulated miRNAs in MDA-MB-231.
Module1
Specific function of synthetic promoter in TNBC cell line.
We combined the synthetic promoter designed by GATA3 binding sites with lateADE promoter, followed by GAD gene , which can drive the promoter G8p. We use the G8p-luciferase and G8p-mKate plasmid respectively to verify the function (Figure3).
Figure4:(A) The luciferase assay compared the efficiency of Module1 between the TNBC cell line MDA-MB-231 and normal breast cell line HBL-100. (B) The fluorescence image shows that in MDA-MB-231 the efficiency is significantly high than the HBL-100.
Module2
High efficiency of synthetic sponge inhibiting the endogenous miRNA.
Based on the sequence of mature miRNA, we used the online software miRNAsong to design and modify the structure of miRNA141-sponge and miRNA-148b-sponge.
When sponges driven by the synthetic promoter s(ESR1)p, they are supposed to inhibit the endogenous miRNA specifically, so that the miRNA will not disrupt the function of Module1 and Module3. Figure5shows that our design of miRNA148b-sponge actually play a role in down-regulating the level of miRNA-148b, which can be used in our system.
Figure5:(A)The pattern diagram shows the structure of working Module2 plasmid, which we used in experiments of what B shows. (B) The qPCR results demonstrate that miR141-sponge has no function in inhibiting miRNA while miRNA-148b-sponge does.
Module1&2&3
Specific damaging effect of the whole system towards TNBC cells.
We transfected the TNBC cell line 231 and normal cell line HBL-100 with the whole system, in which the module3 integrate ycD as the killer part. We use DAPI to stain the nuclei that shows the survival of those cells 2 days and 3 days after adding the 5-FC.
Figure6:The presented images of MDA-MB-231 and HBL-100 several days after transfection and 5-FC treatment.
References
[1] Bell, D., Berchuck, A., Birrer, M., Chien, J., Cramer, D.W., Dao, F., Dhir, R., DiSaia, P., Gabra, H., Glenn, P., et al.; Cancer Genome Atlas Research Network (2011). Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615.
[2] Gaffney EV. A cell line (HBL-100) established from human breast milk. Cell Tissue Res. 1982;227:563–568.
[3] Bos PD, Zhang XH, Nadal C, et al. Genes that mediate breast cancer metastasis to the brain.Nature. 2009; 459:1005–1009.
[4] Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell. 2003; 3:537–549.
[5] Minn AJ, Gupta GP, Siegel PM, et al. Genes that mediate breast cancer metastasis to lung. Nature.2005; 436:518–524.
[6] Minn AJ, Kang Y, Serganova I, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. 2005; 115:44–55.