Abstract. Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its etiology and molecular ...
ONCOLOGY REPORTS 34: 610-616, 2015
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Adrenomedullin promotes intrahepatic cholangiocellular carcinoma metastasis and invasion by inducing epithelial-mesenchymal transition Chuang Zhou1*, Yan zheng2*, Lin Li3*, Wenlong Zhai1, Renfeng Li1, Zhiwen Liang1 and Longshuan Zhao1 1
Department of General Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052; 2 Institutes of Biomedical Science, Fudan University, Shanghai 200032; 3Department of Dermatology, Zhengzhou Children's Hospital, Zhengzhou, Henan 450053, P.R. China Received March 7, 2015; Accepted May 18, 2015 DOI: 10.3892/or.2015.4034
Abstract. Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its etiology and molecular pathogenesis remain largely unknown. The present study aimed to investigate the association between adrenomedullin (ADM) and epithelialmesenchymal transition (EMT) in ICC and to elucidate the underlying signaling pathway. We evaluated the clinical significance of ADM in 133 ICC patients using tissue microarray analysis of ICC tissues. We also investigated the mechanisms of ADM in ICC EMT-mediated metastasis in cholangiocarcinoma cell lines in vitro. The results revealed that ADM was upregulated in human ICC tissues (73/133) compared with that in healthy controls. ADM expression was positively correlated with shorter overall survival (P60 ≤60
Tissue microarray (TMA) and IHC. A tissue microarray was constructed as described previously (12). Samples were taken from each representative tumor tissue and from liver tissue adjacent to the tumor (within 10 mm) to construct TMA slides (in collaboration with the Shanghai Biochip Company Ltd., Shanghai, China). Duplicate tissue cylinders were obtained from intratumoral and peritumoral areas (a total of 4 punches for each patient). Tissues were incubated with primary rabbit antiADM monoclonal antibody (1:200; Abcam, Cambridge, MA, USA), according to previously described IHC protocols (13), using the EnVision Plus detection system (EnVision; Dako, Carpinteria, CA, USA). Reaction products were visualized by incubation with 3,3-diaminobenzidine. Semi-quantitative analysis of IHC staining was performed by two experienced pathologists in two sections of each specimen in 10 fields from each section (magnification, x200). Immunostaining scoring was based on the intensity of staining and the percentage of positively stained cells: negative (-), 0-5%; intermediate (+), >5-10%; moderate (++), >10-25%; strong (+++), >25%. ADM staining ≥5% was considered positive. Construction of recombinant plasmids and transfection. Full‑length human ADM cDNA was amplified by PCR and cloned into the pEGFP-N1 expression vector (Clontech, Palo Alto, CA, USA) to construct pEGFP-N1-ADM, and then transfected into HuCCT1 cells using Lipofectamine 2000
Recurrence Non-recurrence group A group B Demographics (n=54) (n=79) P-valueb
Differentiationa Ⅰ-Ⅱ Ⅲ-Ⅳ Encapsulation Complete None
CA19-9 (U/ml) ≤37 >37 Lymph node metastasis Presence Absence
Tumor size (cm) ≤5 >5 ADM Low ≤6 High
28 26
52 27
0.10606
27 27
31 48
0.21914
33 21
53 26
0.47882
4 50
10 69
0.33254
28 42
38 25
0.01929
11 49
13 66
0.77174
21 33
42 37
0.10539
23 31
50 29
0.01848
Tumor differentiation was assigned by Edmondson's grading system. P-value for the comparison of cohort B with cohort A; ADM, adrenomedullin; CA19-9, carbohydrate antigen 19-9; ICC, intrahepatic cholangiocellular carcinoma.
a
b
(Invitrogen) according to the manufacturer's instructions. Cells transfected with pEGFP-N1 were used as a negative control. Stable ADM-expressing clones were selected using geneticin (Roche Diagnostics, Indianapolis, IN, USA) at a concentration of 500 µg/ml. Establishment of A DM-knockdown cells. Lentivirus containing short hairpin RNAs targeting ADM was purchased from GeneCopoeia (Rockville, MD, USA) and transfected into HUH28 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells transfected with the empty vector were used as controls. Stable clones were selected using puromycin (final concentration, 2 µg/ml). In vitro cell behavior assay. Cellular proliferation was assayed in cells seeded at a density of 5x103 cells/well in 96-well plates. The proliferation of the transfected cells was measured
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ONCOLOGY REPORTS 34: 610-616, 2015
Figure 1. Adrenomedullin immunohistochemical staining of tumor tissue. The specimens from ICC patients were evaluated by immunohistochemistry using a manual quantitative-scoring method derived from staining intensity and extensity. Shown are representative samples with a high (A and B) and a low (C and D) score.
log-rank test. Independent prognostic factors were identified by multivariate survival analysis using a Cox proportional hazards model. The median value was used to determine the cut-off value for high vs. low expression of ADM. Statistical analyses were performed with SPSS 16.0 for Windows (SPSS software; SPSS Inc., Chicago, IL, USA). Statistical significance was accepted for P5 vs. ≤5 cm) Differentiation (Ⅰ-Ⅱ vs. Ⅲ-Ⅳ) CA19-9 (>37 vs. ≤37 U/ml) Encapsulation (complete vs. none) ADM (positive vs. negative)
Univariate Multivariate ------------------------------------------------------------------------- -------------------------------------------------------------------------Hazard ratio (95% CI) P-valuea Hazard ratio (95% CI) P-valuea 0.782 (0.430-1.423) 0.899 (0.472-1.713) 0.902 (0.493-1.650) 2.581 (1.216-5.481) 0.779 (0.409-1.480) 2.609 (0.921-7.338) 2.869 (1.453-5.665)
0.421 0.746 0.738 0.014 0.445 0.041 0.002
2.177 (1.016-4.665)
0.045
1.497 (0.503-4.456) 2.412 (1.207-4.823)
0.469 0.013
ADM, adrenomedullin; CA19-9, carbohydrate antigen 19-9.
determined by functional assays. The cell growth rates in the ADM-transfected cells were significantly higher than the rates in the control cells (P
