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A novel microRNA mmu-miR-466h affects apoptosis regulation in mammalian cells

Aliaksandr Druz1, Chia Chu, Brian Majors

  • 1Biotechnology Core Laboratory National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike Bldg 14A Rm 176, Bethesda, Maryland 20892, USA.

Biotechnology and Bioengineering
|February 22, 2011
PubMed

Insights

Nutrient depletion up-regulates microRNA (miR)-466h in Chinese hamster ovary cells, promoting apoptosis by inhibiting anti-apoptotic genes. Inhibiting miR-466h reversed these effects, confirming its pro-apoptotic role.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Apoptosis is a critical cellular process regulated by complex molecular pathways.
  • MicroRNAs (miRs) are small non-coding RNAs that play significant roles in gene regulation, including apoptosis.
  • Understanding miR expression changes during apoptosis is crucial for deciphering cellular responses to stress.

Purpose of the Study:

  • To investigate the changes in microRNA expression in Chinese hamster ovary (CHO) cells undergoing apoptosis induced by nutrient depletion.
  • To identify specific miRs involved in the apoptotic process and elucidate their functional roles.
  • To determine the targets of the identified pro-apoptotic miR and its mechanism of action.

Main Methods:

  • Chinese hamster ovary (CHO) cells were cultured in nutrient-depleted media to induce apoptosis.
  • Cell viability and Caspase-3/7 activation were measured to confirm apoptosis onset.
  • Microarray analysis was performed to compare miR expression profiles between cells in fresh and depleted media.
  • Quantitative real-time PCR (qRT-PCR) was used to validate miR expression levels.
  • Bioinformatics tools (miRecords, DAVID NCBI) were employed to predict and identify miR targets.
  • mRNA levels of potential target genes were analyzed using qRT-PCR.

Main Results:

  • Nutrient depletion induced apoptosis in CHO cells, evidenced by reduced cell viability and increased Caspase-3/7 activity.
  • Microarray analysis revealed significant up-regulation of the mouse miR-297-669 cluster, particularly mmu-miR-466h, in nutrient-depleted cells.
  • qRT-PCR confirmed the overexpression of mmu-miR-466h under nutrient-depleted conditions.
  • Bioinformatic analysis predicted 8708 targets for mmu-miR-466h, narrowed down to 38 anti-apoptotic genes.
  • Experimental validation showed that mmu-miR-466h down-regulates the mRNA levels of anti-apoptotic genes, including bcl2l2, dad1, birc6, stat5a, and smo.
  • Inhibition of mmu-miR-466h led to increased expression of these anti-apoptotic genes, enhanced cell viability, and reduced Caspase-3/7 activation.

Conclusions:

  • The up-regulation of mmu-miR-466h in response to nutrient depletion plays a pro-apoptotic role in mammalian cells.
  • mmu-miR-466h induces apoptosis by simultaneously inhibiting multiple anti-apoptotic genes.
  • This study highlights the significant involvement of mmu-miR-466h in modulating the apoptotic pathway under stress conditions.

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