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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (1) : 52-60     DOI: 10.1007/s11684-016-0433-3
RESEARCH ARTICLE |
Protein phosphatase magnesium-dependent 1δ is a novel tumor marker and target in hepatocellular carcinoma
Zhi Xu1,*(),Chunxiang Cao1,Haiyan Xia1,Shujing Shi1,Lingzhi Hong1,Xiaowei Wei1,Dongying Gu1,Jianmin Bian2,Zijun Liu2,Wenbin Huang3,Yixin Zhang4,Song He5,Nikki Pui-Yue Lee6,Jinfei Chen1,*()
1. Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China
2. Department of General Surgery, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China
3. Department of Pathology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China
4. Department of General Surgery, Nantong Tumor Hospital, Nantong 226361, China
5. Department of Pathology, Nantong Tumor Hospital, Nantong 226361, China
6. Department of Surgery, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
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Abstract  

Hepatocellular carcinoma (HCC) is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 1δ (PPM1D) was highly expressed in the majority of HCC cases (approximately 59%) and significantly associated with high serum α-fetoprotein (AFP) level (P= 0.044). Kaplan-Meier and Cox regression data indicated that PPM1D overexpression was an independent predictor of HCC-specific overall survival (HR, 2.799; 95% CI, 1.346–5.818, P = 0.006). Overexpressing PPM1D promoted cell viability and invasion, whereas RNA interference-mediated knockdown of PPM1D inhibited proliferation, invasion, and migration of cultured HCC cells. In addition, PPM1D suppression by small interfering RNA decreased the tumorigenicity of HCC cells in vivo. Overall, results suggest that PPM1D is a potential prognostic marker and therapeutic target for HCC.

Keywords PPM1D      hepatocellular carcinoma      prognosis      target therapy     
Corresponding Authors: Zhi Xu,Jinfei Chen   
Just Accepted Date: 30 December 2015   Online First Date: 25 January 2016    Issue Date: 31 March 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0433-3     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I1/52
Clinicopathological features Total patients PPM1D expression P value
N = 81 (%) Low (n = 33) High (n = 48)
Age
Year [(mean±SD)] 48.9±9.9 48.8±9.5 49.0±9.8 0.938
Gender
Male 70 (86.4) 28 38
Female 16 (13.6) 5 10 0.574
Tumor sizea
≤5 cm 49 (60.5) 23 26
>5 cm 32 (39.5) 10 22 0.175
Hepatitis
Negative 19 (23.5) 8 11
HBV 62 (76.5) 25 37 1.000
Tumor nodules
Single 59 (72.8) 24 35
Multiple 22 (27.2) 9 13 1.000
Capsule
No 53 (65.4) 23 30
Yes 27 (34.6) 9 18 0.472
Vascular invasionb
No 67 (82.7) 28 39
Yes 14 (17.3) 5 9 0.771
Tumor stage (AJCC 7th)
Stage I 12 (14.8) 6 6
Stage II 33 (40.7) 14 19
Stage III 36 (44.4) 13 23 0.676
Tumor differentiation
Well to moderately 43 (53.1) 18 25
Poorly 38 (46.9) 15 23 1.000
Serum AFP level
≤400 ng/ml 58 (71.6) 28 30
>400 ng/ml 23 (29.4) 5 18 0.044c
Tab.1  Table 1 Clinical correlations of PPM1D in HCC
Fig.1  Overexpression of PPM1D in HCC was correlated with poor overall post-operative survival. (A) Representative photomicrographs of PPM1D-stained HCC tissue are shown. Grade 2 staining is shown on the left, and grade 4 staining is shown on the right. Scale bar: 20 μm. (B) PPM1D overexpression in HCC was associated with poor OS in 81 patients as assessed based on PPM1D immunoreactivity.
Clinicopathological features Univariate analysis Multivariate analysis
Median OS (month) P value HR P value HR 95% CI
Age NA 0.509 1.010 0.450 1.012 0.981–1.044
Gender 0.536 0.505
Male 44.0 1 1
Female 64.0 0.774 0.728 0.286–1.852
Tumor size <0.001b 0.146
≤5 cm 63.8a 1 1
>5 cm 20.0 2.786 3.459 0.648–18.468
Tumor differentiation 0.029b 0.111
Well to moderately 61.7a 1 1
Poorly 26.0 1.986 1.756 0.879–3.509
Tumor staging (AJCC) 0.008b 0.194
Stage I 68.4a 1 1
Stage II 36.0 0.111 2.341 0.091 2.675 0.854–8.384
Stage III 20.0 0.002b 3.010 0.865 0.865 0.144–5.105
PPM1D expression 0.013b 0.006b
Low 66.0a 1 1
High 32.0 2.350 2.799 1.346–5.818
Tab.2  Cox regression analysis of OS (n = 81)
Fig.2  In vitro functional assays for examining the tumorigenic properties of PPM1D in HCC cells. (A) Western blot analysis showed the overexpression of PPM1D in HepG2 and Huh7 cells through transient transfection of PPM1D plasmid (left panel). Semi-quantitative analysis showed a twofold overexpression of PPM1D in the transfected cells than that in cells transfected with the vector (right panel). (B) When assessed by MTT assay, the ectopic expression of PPM1D in HepG2 and Huh7 cells significantly enhanced cell growth from day 3 to 5 compared with the vector controls (HepG2-Vec, Huh7-Vec). All experiments were conducted in triplicate in two independent experiments. (C) In the Matrigel invasion assay, penetrated cells were stained with 0.1% crystal violet. Representative images of invaded cells on the membrane are shown. Huh7 cells overexpressing PPM1D (Huh7-PPM1D) showed enhanced ability to penetrate the Matrigel membrane after 36 h of incubation (left panel). Histograms in the right panel showed the mean number of invaded cells. Scale bar: 80 μm. Original magnification: 100×. Data are expressed as mean±2×SEM. *P<0.05; ** P<0.01.
Fig.3  In vitro functional assays for examining the effects of PPM1D inhibition on HCC cells. (A) Reduced expression of PPM1D was observed after transient transfection of PPM1D siRNA (siPPM1D-1 and siPPM1D-2) in Hep3B and MHCC97L cells. Successful reductions in PPM1D protein and transcript levels were confirmed by immunoblotting (left panel) and quantitative PCR assay (right). Data are expressed as mean±SD. (B) Suppression of PPM1D in Hep3B and MHCC97L cells significantly impaired cell growth rate compared with that in the scrambled control (Hep3B-siCon, MHCC97L-siCon) from day 3 to 5. All experiments were conducted in triplicate in two independent experiments. Data are expressed as mean±2×SEM. **P<0.01. (C) Wound healing assay showed that Hep3B-PPM1D-1 and MHCC97L-PPM1D-1 cells exhibited lower closure rate of artificially created wounds after 48 h than the scrambled siRNA-transfected control cells. Original magnification: 200×. (D) Matrigel invasion assay indicated that Hep3B-PPM1D-1 and MHCC97L-PPM1D-1 cells showed reduced ability to penetrate the Matrigel membrane after 36 h of incubation. Representative images of invaded cells on the membrane are shown. Histograms in the right panel show the mean number of invaded cells. Scale bar: 80 μm. Original magnification: 100×. All experiments were conducted in triplicate in two independent experiments. *P<0.05, **P<0.01.
Fig.4  Inhibition of PPM1D reduced the sizes of tumor xenografts in mice. (A) Photo shows HCC tumor xenograft-bearing mice from two experimental groups. (B) Tumor growth curves of MHCC97L-PPM1D-2 and MHCC97L-Vec up to 27 days after subcutaneous inoculation of tumor cells. Data are expressed as mean tumor size±SD. (n = 6 per group) at each time point. *P<0.05.
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