Part:BBa_K2624000
hTERT promoter
It's the core promoter of human telomerase reverse transcriptase (hTERT) gene. And it's cancer-specific. If you want to do something specifically to cancer cells, for them to express certain product, you may need it.
Usage
The 2018 CPU_CHINA project is an AND-gated system targeting hepatocellular carcinoma. HTERT is one of the most known cancer-specific promoters, suitable for many cancer cell-related scheme. With this promoter, it’s more likely that normal cells would not express the protein contents that come after it. Only tumor cells (malignant ones especially) express NS5B and ‘open’ the AND gate, releasing miRNA that damage themselves.
Triple-negative breast cancer refers to breast cancer in which the immunohistochemical examination results showed that the estrogen receptor (ER), progesterone receptor (PR) and the original oncogene her-2 were all negative.This type of breast cancer accounts for 10.0%~20.8% of all pathological types of breast cancer, with special biological behavior and clinicopathological characteristics, and poor prognosis compared with other types.MCF-7 cells are one kind of triple-negative breast cancer cells.It retains the properties of multiple differentiated mammary epithelium, including the ability to process estradiol through cytoplasmic estrogen receptors and the ability to form domes.
The 2019 CSU_CHINA project is an AND-gated system targeting triple-negative breast carcinoma.hTERT is one of the most known cancer-specific promoters, suitable for many cancer cell-related scheme.Some studies have shown that hTERT activation in MCF-7 is specific.So we tested the activation of this part in MCF-7 cells (ER+, PR+/-, HER2-), MDA-MB-231 cells(Triple negative breast cancer cells), and HBL-100 cells (normal breast cells) , tool cells 293T as control. Through our experiments, we found that hTERT is exactly specific in MCF-7, and little more efficient than the normal cells HBL-100 and 293T. What we did both helped us find a relative specific promoter to fight with triple-negative breast cancer cells but not damage normal cells also widened the scope of hTERT's action.
Biology
Telomerase activation maintains telomeres and is critical for human carcinogenesis. Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule. Transcriptional regulation of the enzyme part (hTERT) gene has been found to be the major mechanism for cancer-specific activation of telomerase. A number of factors have been identified to directly or indirectly regulate the hTERT promoter, which include famous cellular transcriptional activators such as c-Myc, Sp1, HIF-1, AP2, ER, as well as the repressors p53, WT1, and Menin, most of which comprise tumor suppressor gene products.
The chromatin structure via the DNA methylation or modulation of nucleosome histones is also important for regulation of the hTERT promoter. DNA unmethylation or histone methylation around the transcription start site of the hTERT promoter triggers the recruitment of histone acetyltransferase (HAT) activity, allowing hTERT transcription.
Results
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Figure1 (A-D) The 2019 CSU_CHINA iGEM team integrated the hTERT promoter into pGL4.22 plasmid which contains luciferase after the promoter, and tested the relative initiate efficiency of hTERT promoter using a strong promoter CMV as a positive control. The figure shows that in MCF-7 cells, hTERT has a relative strong efficiency compared to HBL-100 and 293T cells; while that is lower in MDA-MB-231 cells.
The 2019 CPU_CHINA iGEM team used dual luciferase reporter gene assay to test activity of hTERT promoter in both HEK293T and HepG2 cells. Plasmids containing renilla luciferase vector and PGL3-Basic or PGL3-Basic were separately transfected into cells. The results show that hTERT promoter has a better efficiency in HepG2 cells instead of HEK293T cells.
NJTech_China 2019's Characterization
To determine the transcriptional activity of hTERT promoter in different cell lines, NJTech_China transfected pGL3-Basic (negative control) and pGL3-htert vector (experimental group) into HepG2 and HEK293T cells. Renilla luciferase vector was used as reference gene. The bioluminescence was measured at emission wavelengths of 590 and 460nm. The results demonstrated that hTERT promoter has a relative strong efficiency in HepG2 cells rather than in HEK293T cells.
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