Protein phosphatase magnesium-dependent 1δ (PPM1D) mRNA expression is a prognosis marker for hepatocellular carcinoma

Abstract

Background: Protein phosphatase magnesium-dependent 1δ (PPM1D) is an oncogene, overexpressed in many solid tumors, including ovarian cancer and breast cancer. The current study examined the expression and the prognostic value of PPM1D mRNA in human hepatocellular carcinoma (HCC).

Methods: Total RNA was extracted from 86 HCC and paired non-cancerous liver tissues. PPM1D mRNA expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR). Immunohistochemistry assay was used to verify the expression of ppm1d protein in the HCC and non-cancerous liver tissues. HCC patients were grouped according to PPM1D mRNA expression with the average PPM1D mRNA level in non-cancerous liver tissue samples as the cut-off. Correlations between clinicopathologic variables, overall survival and PPM1D mRNA expression were analyzed.

Findings: PPM1D mRNA was significantly higher in HCC than in the paired non-cancerous tissue (p<0.01). This was confirmed by ppm1d staining. 56 patients were classified as high expression group and the other 30 patients were categorized as low expression group. There were significant differences between the two groups in term of alpha-fetoprotein (α-FP) level (p<0.01), tumor size (p<0.01), TNM stage (p<0.01), recurrence incidence (p<0.01) and family history of liver cancer (p<0.01). The current study failed to find significant differences between the two groups in the following clinical characteristics: age, gender, portal vein invasion, lymphnode metastasis, hepatitis B virus (HBV) infection and alcohol intake. Survival time of high expression group was significantly shorter than that of low expression group (median survival, 13 months and 32 months, respectively, p<0.01).

Conclusion: Up-regulation of PPM1D mRNA was associated with progressive pathological feature and poor prognosis in HCC patients. PPM1D mRNA may serve as a prognostic marker in HCC.

Figure 1. PPM1D mRNA expression in HCC and matched non-cancerous liver tissues.

(A) PPM1D mRNA expression was examined in 86 pairs of HCC and matched non-cancerous liver tissues with qPCR assay. Data were presented as the abundance relative to β-ACTIN mRNA. PPM1D mRNA in HCC tissues was significantly higher than in the non-cancerous liver tissues (p<0.01). (B) Representative image of 2% agarose gel electrophoresis of qPCR products, the size of PPM1D mRNA product was 237bp. M, marker; C, negative control; T, HCC sample; N, non-cancerous liver sample.

Figure 2. Immunohistochemical analysis of ppm1d in HCC and non-cancerous liver tissues.

(A) Representative images of ppm1d staining in HCC and non-cancerous liver tissues. The positive cells were stained dark brown. (a) Negative ppm1d staining in normal liver tissue. (b) Negative staining of HCC tissue without ppm1d primary antibody. (c) Ppm1d-positive non-cancerous liver tissue. (d) High expression of ppm1d in HCC tissue. Magnification, ×200; Scale bar, 100 µm. (B) The distribution of difference of ppm1d staining in HCC and non-cancerous liver tissues (ΔIRS = IRS_HCC-IRS_{non-cancerous}). p<0.01. IRS, immunoreactivity score.

Figure 3. Kaplan-Meier survival analysis stratified by PPM1D mRNA expression.

Overall survival was compared between patients with PPM1D mRNA high expression versus low expression with the Kaplan-Meier survival curve. The overall survival in subjects with high PPM1D mRNA expression was significantly lower than in patients with low expression (p<0.01, log-rank test).