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Efficient inhibition of human glioma development by RNA interference-mediated silencing of PAK5.

Gu X, Wang C, Wang X, Ma G, Li Y, Cui L, Chen Y, Zhao B, Li K - Int. J. Biol. Sci. (2015)

Bottom Line: Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients.Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin.In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Neurology, Guangdong Medical College, Zhanjiang, China. ; 2. Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China.

ABSTRACT
Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment.

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Lentivirus-mediated RNAi significantly inhibits PAK5 expression in human glioma cells. (A) The lentiviral transduction efficiency in U87 cells was estimated 3 days after infection at an MOI of 5. Light micrograph (upper); fluorescent micrograph (lower) (× 400). (B) Total RNA of U87 cells was extracted at 4 days after infection, and relative PAK5 mRNA expression was determined by qRT-PCR. GAPDH was used as an internal standard. The data represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01. (C-E) Western blot analysis of PAK5 protein expression levels of Scr-shRNA-infected and PAK5-shRNA-infected cells in U87 (C), SHG-44 (D) and CHG-5 (E) cells.
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Figure 2: Lentivirus-mediated RNAi significantly inhibits PAK5 expression in human glioma cells. (A) The lentiviral transduction efficiency in U87 cells was estimated 3 days after infection at an MOI of 5. Light micrograph (upper); fluorescent micrograph (lower) (× 400). (B) Total RNA of U87 cells was extracted at 4 days after infection, and relative PAK5 mRNA expression was determined by qRT-PCR. GAPDH was used as an internal standard. The data represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01. (C-E) Western blot analysis of PAK5 protein expression levels of Scr-shRNA-infected and PAK5-shRNA-infected cells in U87 (C), SHG-44 (D) and CHG-5 (E) cells.

Mentions: While normal glial cells do not express PAK5 5, our results indicated that glioma cells express high levels of PAK5. This difference between normal glial cells and glioma cells suggests that PAK5 expression might affect glioma development. To investigate the role of PAK5 in glioma tumorigenesis, we used lentivirus-mediated RNAi to silence PAK5 expression in a human glioma cell line. Lentiviral vectors expressing PAK5-specific shRNA and Scr-shRNA were used to infect U87 cells. After three days, high-efficiency infection was observed (Fig. 2A). To determine the effectiveness of the inhibition of PAK5 expression by the PAK5-shRNA-expressing lentiviral vectors, we detected PAK5 mRNA and protein levels in infected cells by real time-PCR and western blot. Both PAK5-shRNAs significantly reduced PAK5 mRNA expression (Fig. 2B). In addition, PAK5 protein levels were also prominently decreased in the two PAK5-shRNA-infected groups compared with the Scr-shRNA infected group (Fig. 2C). In addition, SHG-44 and CHG-5 cells also showed the similar results (Fig. 2D and E). Therefore, both types of PAK5-shRNA efficiently downregulated PAK5 expression in human glioma cells.


Efficient inhibition of human glioma development by RNA interference-mediated silencing of PAK5.

Gu X, Wang C, Wang X, Ma G, Li Y, Cui L, Chen Y, Zhao B, Li K - Int. J. Biol. Sci. (2015)

Lentivirus-mediated RNAi significantly inhibits PAK5 expression in human glioma cells. (A) The lentiviral transduction efficiency in U87 cells was estimated 3 days after infection at an MOI of 5. Light micrograph (upper); fluorescent micrograph (lower) (× 400). (B) Total RNA of U87 cells was extracted at 4 days after infection, and relative PAK5 mRNA expression was determined by qRT-PCR. GAPDH was used as an internal standard. The data represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01. (C-E) Western blot analysis of PAK5 protein expression levels of Scr-shRNA-infected and PAK5-shRNA-infected cells in U87 (C), SHG-44 (D) and CHG-5 (E) cells.
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Related In: Results  -  Collection

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Figure 2: Lentivirus-mediated RNAi significantly inhibits PAK5 expression in human glioma cells. (A) The lentiviral transduction efficiency in U87 cells was estimated 3 days after infection at an MOI of 5. Light micrograph (upper); fluorescent micrograph (lower) (× 400). (B) Total RNA of U87 cells was extracted at 4 days after infection, and relative PAK5 mRNA expression was determined by qRT-PCR. GAPDH was used as an internal standard. The data represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01. (C-E) Western blot analysis of PAK5 protein expression levels of Scr-shRNA-infected and PAK5-shRNA-infected cells in U87 (C), SHG-44 (D) and CHG-5 (E) cells.
Mentions: While normal glial cells do not express PAK5 5, our results indicated that glioma cells express high levels of PAK5. This difference between normal glial cells and glioma cells suggests that PAK5 expression might affect glioma development. To investigate the role of PAK5 in glioma tumorigenesis, we used lentivirus-mediated RNAi to silence PAK5 expression in a human glioma cell line. Lentiviral vectors expressing PAK5-specific shRNA and Scr-shRNA were used to infect U87 cells. After three days, high-efficiency infection was observed (Fig. 2A). To determine the effectiveness of the inhibition of PAK5 expression by the PAK5-shRNA-expressing lentiviral vectors, we detected PAK5 mRNA and protein levels in infected cells by real time-PCR and western blot. Both PAK5-shRNAs significantly reduced PAK5 mRNA expression (Fig. 2B). In addition, PAK5 protein levels were also prominently decreased in the two PAK5-shRNA-infected groups compared with the Scr-shRNA infected group (Fig. 2C). In addition, SHG-44 and CHG-5 cells also showed the similar results (Fig. 2D and E). Therefore, both types of PAK5-shRNA efficiently downregulated PAK5 expression in human glioma cells.

Bottom Line: Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients.Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin.In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Neurology, Guangdong Medical College, Zhanjiang, China. ; 2. Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China.

ABSTRACT
Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment.

Show MeSH
Related in: MedlinePlus