J Vet Sci.  2016 Jun;17(2):171-178. 10.4142/jvs.2016.17.2.171.

Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells

• Wan X
• Yang R
• Liu G
• Zhu M
• Zhang T
• Liu L
• Wu R

• KMID: 2413166
• DOI: http://doi.org/10.4142/jvs.2016.17.2.171

Abstract

Cellular prion protein (PrP(C)) is ubiquitously expressed in the cytomembrane of a considerable number of eukaryotic cells. Although several studies have investigated the functions of PrP(C) in cell proliferation, cell apoptosis, and tumorigenesis of mammals, the correlated functions of chicken PrP(C) (chPrP(C)) remain unknown. In this study, stable chPrP(C)-downregulated Marek's disease (MD) virus-transformed avian T cells (MSB1-SiRNA-3) were established by introducing short interfering RNA (SiRNA) targeting chicken prion protein genes. We found that downregulation of chPrP(C) inhibits proliferation, invasion, and migration, and induces G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells compared with Marek's disease virus-transformed avian T cells (MSB1) and negative control cells. To the best of our knowledge, the present study provides the first evidence supporting the positive correlation between the expression level of chPrP(C) and the proliferation, migration, and invasion ability of MSB1 cells, but appears to protect MSB1 cells from apoptosis, which suggests it functions in the formation and development of MD tumors. This evidence may contribute to future research into the specific molecular mechanisms of chPrP(C) in the formation and development of MD tumors.

Keyword

Marek's disease virus-transformed avian T cells; apoptosis; chicken prion protein; invasion; short interfering RNA

MeSH Terms

Animals
*Apoptosis/genetics
Cell Cycle
Cell Line
Cell Movement
Cell Proliferation/genetics
Cell Transformation, Viral
*Chickens
*Down-Regulation
Herpesvirus 2, Gallid/*physiology
Marek Disease/virology
Poultry Diseases/*virology
Prion Proteins/*genetics/metabolism
RNA, Small Interfering/genetics/metabolism
T-Lymphocytes/*physiology/virology
Prion Proteins
RNA, Small Interfering

Figure

• 

  Fig. 1 Relative expression of chicken cellular prion protein (chPrPC) mRNA and protein. (A) quantitative real-time PCR (qRT-PCR) was performed to detect chPrPC mRNA levels of in Marek's disease virus-transformed avian T cell line (MSB1), MSB1-SiRNA-3, and MSB1-SiRNA-NC cells from three separate experiments. β-actin mRNA was amplified as the endogenous control. (B) Western blot analysis. Lane 1, MSB1; Lane 2, MSB1-SiRNA-NC; Lane 3, MSB1-SiRNA-3. β-actin was used as a control. (C) Analysis of band intensities to quantify chPrPC protein expression using the Image-Pro-Plus software. **p < 0.01 compared with the MSB1 and MSB1-SiRNA-NC cells.

• 

  Fig. 2 Downregulation of chPrPC inhibited proliferation and clone formation of MSB1-SiRNA-3 cells. (A) Clones formed in soft agarose stained with 0.1% crystal violet. (B) Calculated numbers of clones from three separate experiments. (C) Cell proliferation of MSB1, MSB1-SiRNA-3, and MSB1-SiRNA-NC cells was measured by cell counting kit (CCK)-8 assay. *p < 0.05 compared with the MSB1 and MSB1-SiRNA-NC cells.

• 

  Fig. 3 Downregulation of chPrPC induced G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells. (A) The cell apoptosis distribution of MSB1, MSB1-SiRNA-3 and MSB1-SiRNA-NC cells. FITC-labeled annexin V-positive cells (upper and lower right) were considered apoptotic. (B) Cell cycle analysis of MSB1, MSB1-SiRNA-3, and MSB1-SiRNA-NC cells by flow cytometry. The population of MSB1-SiRNA-3 cells increased significantly at the G1 phase compared with the MSB1 and MSB1-SiRNA-NC cells (p < 0.05). Data are representative of three independent experiments.

• 

  Fig. 4 Downregulation of chPrPC inhibited MSB1-SiRNA-3 cells migration and invasion. (A) cell migration through the polycarbonate membrane to the lower chambers. (B) The relative number of migrated cells was determined by CCK-8 assay. (C) Cell invasion through the membrane of Matrigel to the lower chambers. (D) The relative number of invading cells was determined by CCK-8 assay. *p < 0.05 compared with the MSB1 and MSB1-SiRNA-NC cells. 200× (A and C).

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