Silencing of Xeroderma Pigmentosum Group D Gene Promotes Hepatoma Cell Growth by Reducing P53 Expression

Abstract

BACKGROUND This study investigated the effect of xeroderma pigmentosum group D (XPD) silencing on the growth of hepatoma cells and assessed the mechanisms. MATERIAL AND METHODS XPD gene was silenced by siRNA in hepatoma cells. The experiments were randomly divided into a control group, a liposome control group, a negative control (NC) group, an XPD siRNA group, and an XPD siRNA + P53 inhibitor group. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was used to detect cell viability 24 h after gene silencing and treatments. Terminal deoxynucleotidyl transferases (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect apoptosis. Invasive ability was detected by Transwell assay. Additionally, the expression of mouse double-minute 2 homolog (Mdm2), mouse double-minute 4 homolog (Mdm4), CyclinD1, P21, Bax, P53, C-sis, and Bcl-2 was detected by real-time polymerase chain reaction and Western blotting. RESULTS Compared with the NC group, XPD siRNA significantly reduced XPD expression at both mRNA and protein levels. XPD siRNA significantly promoted cell proliferation, reduced apoptosis, and promoted cell invasive ability. Expression of CyclinD1, Bcl-2, and C-sis increased significantly after XPD silencing, while the expression of P21, Mdm2, Mdm4, Bax, and P53 significantly decreased (vs. NC, P<0.05). Importantly, P53 inhibitor (1 μM bpV) further enhanced the effect of XPD silencing (vs. XPD silencing, P<0.05). CONCLUSIONS Our data revealed that XPD silencing promoted growth of hepatoma cells by reducing P53 expression.

Figure 1

XPD siRNA reduced XPD expression in hepatoma cells. (A) Representative blots; (B) Quantification data the protein expression; (C) Relative mRNA expression of XPD after siRNA treatment. Compared with control, * P<0.05.

Figure 2

XPD siRNA promoted cell viability of hepatoma cells. Compared with NC, * P<0.05; Compared with XPD siRNA, ^# P<0.05.

Figure 3

XPD siRNA reduced apoptosis of hepatoma cells. (A) Representative images of TUNEL assay. Scale bar: 100 μm; (B) Representative images of flow cytometry; (C) Quantification data of apoptosis. Compared with NC, * P<0.05; Compared with XPD siRNA, ^# P<0.05.

Figure 4

XPD siRNA promotes cell invasion of hepatoma cells. Magnification: ×200. Compared with NC, * P<0.05; Compared with XPD siRNA, ^# P<0.05.

Figure 5

XPD siRNA promotes Cyclin D1, Bcl-2, C-sis and reduces P21, Mdm2, Mdm4, Bax, and P53 expression. (A) Relative expression of CyclinD1, P21, Mdm2, and Mdm4 at mRNA level; (B) Relative expression of Bax, Bcl-2, C-sis, and P53; (C) Representative blots of the related proteins; (D) Quantification data of protein expression. Compared with NC, * P<0.05; Compared with XPD siRNA, ^# P<0.05.