Yonsei Med J.  2014 Mar;55(2):324-330.

Small Double-Stranded RNA Mediates the Anti-Cancer Effects of p21(WAF1/ClP1) Transcriptional Activation in a Human Glioma Cell Line

• Dong Z
• Dang Y
• Chen Y

Abstract

PURPOSE
This study was conducted to investigate the small double-stranded RNA (dsRNA) mediated anti-tumor effects of p21(WAF1/ClP1) (p21) transcriptional activation in vitro in the human glioma SHG-44 cell line.
MATERIALS AND METHODS
Human glioma SHG-44 cells were transfected with dsRNA using LipofectAMINE 2000 transfection reagent. Real-time PCR and Western blot analysis were conducted to detect p21 and survivin mRNA and protein levels, respectively. Cell proliferation was examined by MTT assay. Cell cycle distribution and apoptosis were detected by flow-cytometric analysis.
RESULTS
We found that dsRNA targeting p21 promoter (dsP21) significantly induced the expression of p21 at transcription and protein levels, and reduced the expression of survivin. AS well, dsP21 transcription significantly inhibited human glioma SHG-44 cell proliferation. Analysis of cell cycle distribution revealed that dsP21 transfection increased accumulation of cells in the G0/G1 phase and reduced accumulation of cells in the S phase. Further analysis revealed that dsP21 transcription led to an increase in both early and late stages of apoptosis in human glioma SHG-44 cells.
CONCLUSION
In the present study, P21 activation by RNA-induced gene activation (RNAa) induced anti-tumor activity in vitro in a human glioma SHG-44 cell line. The results suggested that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.

Keyword

p21; glioma; apoptosis; cell cycle

MeSH Terms

Apoptosis
Blotting, Western
Cell Cycle
Cell Line*
Cell Proliferation
Genes, Tumor Suppressor
Glioma*
Humans*
Methods
Real-Time Polymerase Chain Reaction
RNA, Double-Stranded*
RNA, Messenger
S Phase
Transcriptional Activation*
Transfection
RNA, Double-Stranded
RNA, Messenger

Figure

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  Fig. 1 The transfection efficiency of human glioma SHG-44 cells. The transfection efficiency was 57.2% (magnification×10). (A) Inverted phase contrast microscope Bright field 10×. (B) Inverted phase contrast microscope Darkfield 10×.

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  Fig. 2 dsP21 induces p21 expression in glioma SHG-44 cells. Induction of p21 mRNA expression was detected by real-time PCR, p21 mRNA expression levels were normalized to mock and calculated by 2-ΔΔCt, and the results were presented as means±SD of three independent experiments. Compared to mock, there was a two-fold induction in dsP21 mRNA expression (mock vs. dsControl: p=0.400, p>0.05; mock vs. dsP21, p=0.001, p<0.05; dsControl vs. dsP21, p=0.000, p<0.05) (A). In addition, p21 protein expression levels were detected by Western blot analysis. β-actin levels were detected as an endogenous control. p21 protein expression levels were normalized to β-actin and presented as the means±SD of three independent experiments (p<0.05, mock: 0.426±0.010, dsControl: 0.400±0.005 vs. dsP21: 0.954±0.030) (B and C). *Compared to Mock and dsControl, p<0.05.

• 

  Fig. 3 Upregulation of p21 leads to an inhibition of SHG-44 cell proliferation in a time-dependent manner. The spectrometric absorbance at 490 nm of dsP21 transfected glioma cells significantly decreased to 0.134±0.035, 0.138±0.038, 0.150±0.05, 0.178±0.03 at 3, 4, 5, and 6 days, compared to mock and dsControl (p<0.05, mock: 0.225±0.063, 0.367±0.057, 0.375±0.049, 0.609±0.051; dsControl: 0.233±0.061, 0.336±0.044, 0.393±0.066, 0.627±0.066). (A) Cell growth curve. (B) Histogram. *Compared to Mock and dsControl, p<0.05.

• 

  Fig. 4 dsP21 treatment induced apoptosis of human glioma SHG-44 cells. The early and the late apoptosis rates of dsP21 transfected cells increased to 8.14% and 50.0%, compared to the mock and dsControl treatments (early apoptosis rate, p<0.05, 8.14% vs. 2.11%, 2.37%), (late apoptosis rate, p<0.05, 50.0% vs. 11.7%, 13.9%), respectively. Meanwhile, there were no obvious differences in apoptosis rates between the latter two types of cells (the early apoptosis rate, p>0.05, 2.11% vs. 2.37%; the late apoptosis rate, p>0.05, 11.7% vs. 11.9%). (A) Apoptosis detection. (B) Histogram. *Compared to Mock and dsControl, p<0.05. 1, necrosis cells; 2, late apoptotic cells; 3, live cells; 4, early apoptotic cells.

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  Fig. 5 Effect of dsP21 treatment on cell cycle distributions of glioma SHG-44 cells. The percentage of cells in the G0/G1 phase significantly increased to 72.29% in the dsP21-transfected cells, compared to those in the mock and dsControl cells (p<0.05, 72.29% vs. 67.52%, 64.88%), while there were no obvious differences between the latter two types of cells (p>0.05, 67.52%, 64.88%). Furthermore, a decrease in the S-phase fraction was seen in the dsP21-transfected cells, compared to those in the mock and dsControl cells (p<0.05, 10.26% vs. 14.83%, 19.67%). No change was seen in the G2/M phase cell populations (p>0.05, 17.45% vs. 17.65%, 15.45%). (A) Cell cycle analysis. (B) Histogram. *Compared to Mock and dsControl, p<0.05.

• 

  Fig. 6 dsP21 transfection is associated with a decrease in survivin expression in human glioma SHG-44 cells. Induction of survivin mRNA expression was detected by real-time PCR; survivin mRNA expression levels were normalized to mock and calculated by 2-ΔΔCt, and the results were presented as means±SD of three independent experiments. Compared to mock, a two-fold reduction in survivin mRNA expression occurred in dsP21 transfected cells (mock vs. dsControl: p=0.562, p>0.05; mock vs. dsP21, p=0.000, p<0.05; dsControl vs. dsP21, p=0.007, p<0.05) (A). In addition, survivin protein expression levels were detected by Western blot analysis. β-actin levels were detected as an endogenous control. Survivin protein expression levels were normalized to β-actin and presented as the means±SD of three independent experiments (p<0.05, mock: 0.562±0.051, dsControl: 0.543±0.012 vs. dsP21: 0.193±0.009) (B and C).

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