Bombyx mori Dihydroorotate Dehydrogenase: Knockdown Inhibits Cell Growth and Proliferation via Inducing Cell Cycle...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the de novo pyrimidine biosynthesis pathway.
• DHODH catalyzes the oxidation of dihydroorotate to orotate, essential for nucleotide synthesis.

Purpose of the Study:

• To clone and characterize the dihydroorotate dehydrogenase (dhod) gene from the silkworm, Bombyx mori (B. mori).
• To investigate the role of BmDHODH in silkworm development, tissue expression, cell growth, proliferation, and DNA replication.

Main Methods:

• Cloning and sequencing of the B. mori dhod cDNA.
• Analysis of the BmDHODH protein structure, identifying key domains.
• Gene expression analysis across different developmental stages and tissues.
• RNA interference (RNAi) for gene knockdown and cell proliferation assays.
• Treatment with a DHODH inhibitor (leflunomide) to assess effects on cell growth and DNA replication.

Main Results:

• The full-length B. mori dhod cDNA is 1339 bp, encoding a 390 amino acid protein (BmDHODH) with DHO_dh and transmembrane domains.
• BmDHODH is expressed throughout silkworm development and in various tissues.
• Knockdown of BmDHODH or treatment with leflunomide inhibited cell growth and proliferation, causing G2/M phase cell cycle arrest.
• Leflunomide treatment decreased cyclin B and cdk2 levels and partially inhibited endomitotic DNA replication in silk gland cells.

Conclusions:

• Downregulation of BmDHODH significantly inhibits cell growth and proliferation in silkworm cells.
• BmDHODH plays a critical role in regulating endomitotic DNA replication within silkworm silk gland cells.