Difference between revisions of "Part:BBa K2624000"

Latest revision as of 17:29, 21 October 2019

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.

The 2019 Nanjing_NFLS project is Cancer Immunotherapy with Trojan Horse Antigen (CITHA). Neoantigen is an immunogenic peptide formed by mutations in tumor cells and it is an ideal target for cancer immunotherapy. However, natural neoantigen is highly heterogeneous and difficult to identify. Here, we designed an artificial neoantigen with high immunogenicity, which could allow tumors to be recognized and killed by the immune system. We named this artificial neoantigen as the Trojan horse antigen. In this project, we constructed a Trojan horse antigen expression system: pCDNA6.2-hTERT-HBsAg-EmGFP-miR-HBsAg and a specific activation system in tumor cells: PCDNA3.1(+) - Hulc-CeR-HBsAg. These two systems, which contain cancer-specific promoters and miRNA, form an AND gate for regulating the expression of the Trojan target antigen only in liver cancer cells, but not in normal cells. Then the human immune system will kill tumor cells by identifying Trojan horse antigen-specific.
The hTERT promoter is a tumor-specific promoter and is used as the promoter in HBsAg expression system. HTERT is used to prime the expression of HBsAg (Trojan Horse) in the project. With this promoter, it’s more likely that normal cells would not express the protein contents that come after it. Only tumor cells express HBsAg, causing immune response.

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

Figure 1. Activity of hTERT promoter in different cell lines.

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.

Table1 Relative promoter activity of HULC and SV40 promoters
Vector HepG2 293T
PGL3-Basic 22.02 1.32
PGL3-hulc 237.38 130.39
PGL3-Control 2782.88 3318.18
The data is the mean of the relative Luc activity RL/RL (%) measured.
The 2019 Nanjing_NFLS used dual-luciferase reporter gene assay to test activity of hTERT promoter in both HEK293T and HepG2 cells.The PGL3-Basic is the negative control group while the PGL3-Control is the positive control group. From the table shown above, the PGL3-Basic group without any promoter showed a low relative Luc activity, indicating that the entire detection system exhibited no interference. The PGL3-Control group with SV40 promoter has good priming performance in both engineered HEK293T cell line and tumor cell HepG2 cells: maintains a good value of 3000% of relative Luc activity. However, it does not have a tumor-specific selection ability. The hTERT promoter works perfectly. In terms of specificity, its relative Luc activity was 597.56% in tumor cells, apparent compared to 2.30% in 293T cells, which showed perfect specific priming ability. In addition, the hTERT promoter has higher activity than Hulc. In HepG2 cells, the activity of the hTERT promoter is equivalent to 21% of SV40. Therefore, we successfully characterize hTERT promoter and verified its effectiveness in our project. It does work great as a tumor-specific promoter.

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.