Part:BBa_K3170997

HRE(Hypoxia response elements)

In solid tuumors, rapid proliferation of tumor cells leads to increased oxygen consumption of tumor cells, which in turn leads to hypoxia microenviroment. Hypoxia is one of the basic characteristics of tumor microenvironment, playing an important role in the proliferation, invasion and metastasis of tumor cells. In our experiments, we transfect 293T cell with HRE-miniCMV and when it senses the condition of hypoxia in tumor microenvironment, HIF-1(hypoxia inducible factor 1) is produced, and it can bind to HRE(hypoxia response elements), then activating the minimized CMV promoter. Through this mechanism, we can switch the interest gene specifically in tumor microenvironment. In order to enhance its induction effect,we designed six HRE repeats components.After pHRE, miniCMV was added to transcribe the downstream genes.

• HIF-1 is a transcription factor widely distributed in mammals and humans. It is a heterodimeric nuclear transcription factor composed of two subunits, HIF-1α and HIF-lß, belonging to the bHLH-PAS family, including HIF. -1α is a major oxygen-regulating subunit and a functional subunit, which regulates the expression of various target genes such as glucose transporter, VEGF, erythropoietin, etc. The HIF-ß subunit is an arylhydrocarbon receptor. Nuclear translocator (ARNT), stable expression in cells. Anoxic microenvironment can induce the increase of HIF-1α expression level, form a stable dimer with HIF-lß subunit, and bind to target gene hypoxia with the help of coactivators (such as CBP, P300, etc.) The component (hypoxia-responsive elemene, HRE) up-regulates the expression of the target gene. The amino acid 401-603 region of the HIF-1α subunit is an oxygen-depengent degradation domain (ODD). Under normoxic conditions, HIF-1α protein is ubiquitinated by VHL (von hippel lindau) white-mediated by ODD region and rapidly degraded by protease (half-life <5 min). As the oxygen concentration decreases, the degradation process of HIF-1α protein is inhibited and the protein level increases exponentially. Studies have shown that HIF-1α is highly expressed in various tumor tissues such as breast cancer, prostate cancer and lung cancer. HIF-1α plays a central role in the process of tumor hypoxia, which plays an important role in maintaining energy metabolism of tumor cells, stimulating neovascularization, and promoting tumor cell proliferation and metastasis.

Experimental Results

• We tested the induced expression of pHRE with CopGFP. CoCl(II) is used to produce anoxic conditions. Figure 1~4 shows the effect of pHRE on HEK 293T cells. The experimental results show that the fluorescence intensity of CopGFP increases with the increase of CoCl(II) concentration. At a concentration of 500 uM, the fluorescence intensity decreased significantly. This is the result of inhibition of cell viability by high concentrations of CoCl(II). A similar effect was observed in SW1990 cells. In addition, the fluorescence intensity of SW1990 is usually lower than that of HEK293T, which may be due to the poor expression efficiency of SW1990 as a differentiated cell.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 5
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]