Asian Spine J.  2017 Oct;11(5):686-693. 10.4184/asj.2017.11.5.686.

Small Interfering RNA–Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

Affiliations
  • 1Department of Orthopaedic Surgery, Uijeongbu St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. spinepjb@catholic.ac.kr

Abstract

STUDY DESIGN: In vitro cell culture model. PURPOSE: To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. OVERVIEW OF LITERATURE: Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear.
METHODS
Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls.
RESULTS
Serum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both).
CONCLUSIONS
The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Keyword

Disc cells; Fas; Apoptosis; Cell proliferation; Small interfering RNA

MeSH Terms

Animals
Apoptosis*
Cell Culture Techniques
Cell Proliferation
Densitometry
Down-Regulation
Gene Expression
In Vitro Techniques
Intervertebral Disc Degeneration
Intervertebral Disc*
Oligonucleotides
Rats*
RNA Interference
RNA, Messenger
RNA, Small Interfering
Transfection
Oligonucleotides
RNA, Messenger
RNA, Small Interfering
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