Part:BBa_K3170512

ALKBH5 alkB homolog 5

RNA demethylase

• ALKBH5 is an mRNA m6A demethylase, a nuclear speckle body located at the site of nuclear mRNA cleavage. ALKBH5 is a dioxygenase that uses alpha-ketoglutaric acid and oxygen as a reaction matrix to mediate demethylation. At least 8 Alkb homologs (human AlkB homologs) have been identified in the human genome as ALKBH1-8 (or ABH1-8). The human ALKBH5 gene is located in the 17p11.2 region of the chromosome and encodes a protein of 394 amino acid residues with a molecular weight of approximately 43 kDa. ALKBH5 has a stronger RNA binding ability.
• Alkbh5 has Fe2+ and a-KG-dependent free-chain enzyme activities. It is detected by isotopic labeling that Alkbh5 can oxidize a-KG to oxidize and produce C02 in the presence of a-KG oxidase cofactor Fe2+ and ascorbic acid. In the absence of Fe2+ or ascorbic acid, the activity of the decarboxylase of Alkbh5 was decreased, while in the presence of the inhibitor PDCA, no product was detected, indicating that Alkbh5 is an active a-KG oxygenase. In 2011, Thalhammer et al. found that ALKBH5 is a downstream target gene of Hypoxia-Inducible Factor 1α (HIF-1α) (Thalhammer et al., 2011). Hypoxia-inducible factor (HIF-1) is a transcription factor that is active under hypoxia and is a heterodimer composed of two subunits, HIF-1α and HIF-1ß. Thalhammer et al. exposed three different cell lines (MCF7, U20S, IMR32) to a hypoxic environment and detected changes in the transcriptional level of the AlkB family protein gene by real-time quantitative PCR. The transcription levels of Alkbh5 in the three cells after hypoxia treatment were significantly increased (more than two times higher), while the mRNA contents of other AlkB protein genes did not change significantly. The researchers further demonstrated that the cells treated with DMOG (dimethyl oxalylglycine, inhibiting the degradation of HIF-1a) and the results of hypoxia treatment, the mRNA content and protein expression of A] kbh5 increased significantly; meanwhile, under normal oxygen conditions, By inducing the expression of HIF-1α protein regulated by doxycycline, it was found that with the increase of HIF-1α expression, the intracellular lung lung Alkbh5 mRNA content also increased significantly, which proved that the transcription of Alkbh5 was indeed under hypoxic conditions. HIF-1α factor regulated.

Experimental Results

• In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and performed a scratch test to detect the migration and crawling ability of pancreatic cancer cells.
• A and D are blank control groups. B and E overexpress the ALKBH5 cell scratch assay; panels C and F are knockdown ALKBH5 cell scratch assays.
• The results showed that the migration ability of the siALKBH5 (knockdown expression) group was stronger than that of the Ctrl group (blank control group), p<0.01. The migration ability of the ALKBH5-WT (overexpression) group was higher than that of the Ctrl group (blank control group).

• In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and Transwell to detect the invasive ability of pancreatic cancer cells.
• Ctrl is the blank control group, ALKBH5-WT is the ALKBH5 overexpression group, and siALKBH5 is the knockdown expression group.
• The results showed that the invasive ability of ALKBH5-WT group was weaker than that of Ctrl group, p<0.01; the invasive ability of siALKBH5 group was stronger than that of Ctrl group, p>0.01.

• Western blot analysis showed that the expression levels of β-catenin and p-GSK-3β in Wnt/β-catenin signaling pathway were up-regulated in the pancreatic cancer cell SW1990 compared with the control group (3xFlag-Vector). Significantly decreased, the total GSK-3β protein expression was unchanged, this result indicates that up-regulation of ALKBH5 can enhance the activity of GSK-3β and promote the phosphorylation of β-catenin, resulting in a decrease in intracellular β-catenin and inhibition of Wnt/ --catenin signaling pathway.

Usage and Biology

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1013
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 255