Prp19 Facilitated p21-Dependent Senescence of Hepatocellular Carcinoma Cells

Abstract

Introduction: Evidence suggests that the role of senescence in the development of cancer is context-dependent. An orthologue of human pre-mRNA processing factor 19 (Prp19) attenuates the senescence of human endothelial cells. Prp19 has been reported to be involved in the progression of hepatocellular carcinoma (HCC). This work aims to investigate the effect of Prp19 on the senescence of HCC.

Materials and methods: Senescence of L02 cells and HCC cells under different stimuli was detected through cell cycle analysis, SA-β-gal staining, and senescence associated secretory phenotype analysis. The relationship between Prp19 and senescence-related proteins was evaluated using real-time RT-PCR, western blot assay, and immunohistochemistry. Subcutaneous xenograft tumors in nude mice were used to evaluate the role of Prp19 on senescence in vivo. Data analysis was carried out using GraphPad Prism 6.

Results: Prp19 facilitated the senescence of L02 cells and HCC cells under different stresses. Prp19 positively modulated p21 expression in the mRNA level. Downregulation of Prp19 promoted the growth of subcutaneous xenograft tumors generated by HCC cell lines.

Conclusions: Prp19 may promote senescence of HCC cells via regulating p21 expression.

Figure 1

Prp19 facilitates the senescence of L02 cells. (a) Cell cycle distribution of L02 cells transfected with myc-Prp19. (b) The effects of Prp19 on senescence of L02 cells were assessed using SA-β-galactosidase staining with or without incubation of 10 μM cisplatin for 24 h. (c) The effects of Prp19 on H-ras-induced senescence of L02 cells were assessed using SA-β-galactosidase. Null vector, NV. The data are shown as the mean ± SEM. Similar results were observed in three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Black scale bar: 100 μm.

Figure 2

Inhibiting Prp19 represses senescence of HCC cells. (a) Prp19 expression was decreased by siRNA silencing in HCC cells. (b) Cell cycle distribution of HCC cells with Prp19 knockdown. (c) The effects of Prp19 on HCC cells senescence were assessed using SA-β-galactosidase. (d) The effects of Prp19 on HCC cells senescence were assessed using SA-β-galactosidase staining with or without incubation of 10 μM cisplatin for 24 h. (e) Relative IL6, IL1a, IL1b, IL8, and CXCL1 transcript levels in human HCC cell lines were determined by qRT-PCR. The levels of mRNA were normalized to ACTIN and presented relative to the control cells. Negative control, NC. The data are shown as the mean ± SEM. Similar results were observed in three independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01. Black scale bar: 100 μm.

Figure 3

Prp19 modulates p21 expression in L02 cells and HCC cells. (a) Western blot assay of p16, p21, and p53 expression in L02 cells overexpressing Prp19. (b) Western blot assay of p16, p21, and p53 expression in HCC cells with Prp19 knockdown. (c) Relative levels of Prp19 and p21 transcripts in human HCC cell lines were determined by qRT-PCR. Levels of mRNA were normalized to ACTIN and presented relative to the control cells. Negative control, NC. The data are shown as the mean ± SEM. ^∗∗∗p < 0.001.

Figure 4

Prp19 knockdown promotes tumor growth of HCC in vivo. (a) Prp19 protein expression of stable HCC cell lines misexpressing Prp19. (b) Images of the subcutaneous xenograft tumors generated by HCC cells misexpressing Prp19 in nude mice. The weights of the xenograft tumors are summarized in the lower panel. (c) Comparison of tumor size at the indicated time after implantation (n = 5). (d) Representative images of HE, IHC staining of Prp19 and p21, and SA-β-galactosidase staining in xenograft tumors (original magnification 200x). (e) Prp19 and p21 protein expression in xenograft tumors. (f) Statistically analysis of SA-β-galactosidase positive cells in xenograft tumors. Values are mean ± SEM, ^∗p < 0.05and ^∗∗p < 0.01. Black scale bar: 100 μm.