Effect of RNA Interference-Mediated Suppression of p75 on the Viability of Rat Notochordal Cells

Abstract

Study design: In vitro cell culture model.

Purpose: To investigate the effects of RNA interference (RNAi) on p75 expression and viability of rat notochordal cells treated with serum deprivation.

Overview of literature: RNAi enables the inhibition of specific genes by sequence-specific gene silencing using a double-stranded RNA.

Methods: Notochordal cells were isolated, cultured, and placed in 10% (control) or 0% (apoptosis-promoting) fetal bovine serum (FBS) for 48 hours. The expression of p75, apoptosis, and cell proliferation were determined. To suppress p75 expression, a small interfering RNA (siRNA) was synthesized against p75 (p75 siRNA) and transfected into cells. The suppression of p75 mRNA expression was investigated using the reverse transcription-polymerase chain reaction. The degree of p75 suppression was semiquantitatively analyzed using densitometry. The effect of p75 siRNA on apoptosis and proliferation of cells was determined. Solutions of an unrelated siRNA and transfection agent alone served as controls.

Results: Serum deprivation significantly increased apoptosis by 40.3%, decreased proliferation of notochordal cells by 45.3% (both, p<0.001), and upregulated p75 expression. The p75 siRNA suppressed p75 expression in cells cultured in 0% FBS. The rate of suppression by p75 siRNA of p75 mRNA was 72.9% (p<0.001). Suppression of p75 expression by p75 siRNA inhibited apoptosis by 7% and increased proliferation by 14% in cells cultured in 0% FBS (both, p<0.05).

Conclusions: siRNA-mediated suppression of p75 inhibited apoptosis and increased proliferation of notochordal cells under conditions of serum deprivation, suggesting that RNAi might serve as a novel therapeutic approach for disc degeneration caused by insufficient viability of disc cells through the suppression of the expression of harmful genes.

Keywords: Notochordal cells; RNA interference; Viability; p75.

Fig. 1. (A) TUNEL and flow cytometry results are displayed (×400). (B) Notochordal cells incubated in 0% FBS for 48 hours displayed a significantly greater rate of apoptotic cell death by 40.3% compared with cells incubated in 10% FBS (5.9±1.1% vs. 46.2%±3.5%, p<0.001). TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; FBS, fetal bovine serum; PI, propidium iodide. ^***p<0.001.

Fig. 2. MTS proliferation assay showing that notochordal cells incubated in 0% FBS for 48 hours displayed reduced proliferation ratios by 45.3% compared with those incubated in 10% FBS (1.111±0.24 vs. 0.608±0.12, p<0.001). MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; FBS, fetal bovine serum. ^***p<0.001.

Fig. 3. Western blot analysis demonstrating that p75 expression was upregulated in cells incubated in 0% FBS compared with those cultured in 10% FBS. FBS, fetal bovine serum.

Fig. 4. (A) Reverse transcription-polymerase chain reaction results. (B) p75 siRNA significantly suppressed p75 mRNA levels in notochordal cells cultured in 0% FBS. The rate of suppression of p75 mRNA by siRNA was 72.9% (p<0.001). siRNA, small interfering RNA; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^***p<0.001.

Fig. 5. TUNEL demonstrating that p75 siRNA significantly decreased apoptotic death of notochordal cells cultured in 0% FBS. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; siRNA, small interfering RNA; FBS, fetal bovine serum. (×400).

Fig. 6. (A) Flow cytometry results. (B) p75 siRNA significantly inhibited apoptosis by 7% in notochordal cells cultured in 0% FBS (46.2%±3.5% vs. 36.9%±2.7%, p<0.05). siRNA, small interfering RNA; FBS, fetal bovine serum; PI, propidium iodide. ^*p<0.05.

Fig. 7. MTS proliferation assay showing that p75 siRNA increased proliferation by 14% in notochordal cells cultured in 0% FBS (0.608±0.12 vs. 0.693±0.16, p<0.05). MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; siRNA, small interfering RNA; FBS, fetal bovine serum. ^*p<0.05.