Composite

Part:BBa_K2624005

Designed by: Shuyao Zhou, Huandi Xu, Junqing Zhang, Yali Wu   Group: iGEM18_CPU_CHINA   (2018-08-17)


hulc-pri-miRNA(MAP4K4)

This composite consists of two parts. One is the core promoter of the gene highly up-regulated in liver cancer (HULC), the other designed to encode a primary microRNA analogue targeting MAP4K4. This expression unit is up-regulated in hepatocellular carcinoma and liver cancer cell lines at transcription level, hence constitutive knock-down of MAP4K4 if such cells are transfected with.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 130


Usage

The 2018 CPU_CHINA project is a gene therapy strategy based on conditional RNA interference. The effect silencer RNA (microRNA) comes from this part. With nothing standing in the pri-miRNA’s maturing way, it becomes a miRNA and knock down the expression of MAP4K4. In order to specifically target hepatocellular carcinoma, we found this promoter so that only in liver cancer cells will the composite takes great effect and damage the cancer cells itself.

Biology

A novel mRNA-like long ncRNA, highly up-regulated in liver cancer (HULC) is one of the most up-regulated genes in hepatocellular carcinoma. Researchers found that such a regulation is at transcriptional level. There’s one cAMP response element binding protein (CREB) binding site within the Hulc proximal promoter region that can specifically bind phospho-CREB transcription factors through the PKA pathway. Moreover, phospho-CREB is able to “open” and maintain the local chromatin structure across the Hulc promoter. MicroRNA (miRNA) act as post-transcriptional gene suppressors by base-pairing with their target mRNAs and inducing either translational repression or mRNA destabilization. Their genes are transcribed as primary miRNAs (pri-miRNAs) by RNA polymerase II (Pol II) in the nucleus. The long pri-miRNAs are cleaved by Microprocessor, which includes Drosha and DiGeorge syndrome critical region 8 (DGCRB), to produce 60-70-nucleotide precursor miRNAs (pre-miRNAs). The pre-miRNAs are then exported and further processed by Dicer to produce the mature miRNAs. According to the “base-anchor” model of Han et al., DGCR8 anchors to the basal ssRNA and directs Drosha cleavage 11 bp up the stem of a given pri-miRNA.

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