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Desmoglein-2 Overexpression Predicts Poor Prognosis in Hepatocellular Carcinoma Patients

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Desmoglein-2 Overexpression Predicts Poor Prognosis in Hepatocellular Carcinoma Patients European Review for Medical and Pharmacological Sciences 2018; 22: 5481-5489 Desmoglein-2 overexpression predicts poor prognosis in hepatocellular carcinoma patients C.- P. H A N1.2, Y.-H. YU1, A.-G. WANG2, Y. TIAN3, H.-T. ZHANG2, Z.-M. ZHENG2, Y.-S. LIU2 1Department of Radiation Oncology, Shandong Cancer Hospital and Institute Affiliated to Shandong University, Jinan, P.R. China 2Department of Oncology, Qianfoshan Hospital Affiliated to Shandong University, Jinan, P.R. China 3Department of Radiation Oncology, Qianfoshan Hospital Affiliated to Shandong University, Jinan, P.R. China Introduction Abstract. – OBJECTIVE: Desmoglein-2 (Dsg2) plays a crucial role in the assembly and adhesion of desmosomes. The absent or aber- Hepatocellular carcinoma (HCC) is one of rant expression of Dsg2 was reported to be as- the most common cancer types, with an in- sociated with the progression of varies human creasing incidence and mortality worldwide1-3. In cancers. However, the expression of Dsg2 in he- 2006, 466,100 new diagnosed HCC patients and patocellular carcinoma (HCC) and its associa- 422,100 HCC-related death were estimated for tion with tumor prognosis is still unknown. The 4 aim of this study was to evaluate the expression 2015 in China . Liver transplantation and surgical level of Dsg2 in HCC and of the correlation be- resection were still the most effective treatment tween Dsg2 expression and clinicopathological methods for HCC5,6. However, the overall sur- variables. vival of HCC patients remains poor because the PATIENTS AND METHODS: A total of 104 pa- vast majority of patients were diagnosed at an tients diagnosed with HCC were enrolled in this advanced stages7. Therefore, efforts to elucidate study. Real time-quantitative PCR (RT-qPCR) and Western blot were performed to determine the molecular mechanisms underlying the pro- the expression level of Dsg2 in HCC tumor tis- gression of HCC are urgently needed. sues and matched noncancerous tissues. Cell Desmosomes are multi-protein complexes and proliferation and cell cycle were measured by also a type of cell-cell anchoring junction in cell counting kit-8 (CCK-8) assay and flow-cy- tissues8-10. The transmembrane protein core of tometry assay, respectively. RESULTS: the desmosome is constituted by desmosomal Our results revealed that Dsg2 ex- 11,12 pression was significantly higher in HCC tu- cadherins, desmogleins, and desmocollins . mor tissues than in matched noncancerous tis- In humans, four desmoglein isoforms and three sues (p < 0.01), positively correlated with tumor desmocollin isoforms have been identified13. size (p = 0.035) and tumor stage (p = 0.021). Uni- A study14 revealed the palmitoylation of Des- variate and multivariate analyses demonstrat- moglein-2 (Dsg2) plays a vital role in the as- ed Dsg2 expression was an independent prog- sembly of desmomosome. Also, researches15-17 nostic factor for overall survival. Meanwhile, we demonstrated that altering junction stability or found knockdown the expression of Dsg2 using small interfering RNA (siRNA) could efficiently assembly state can lead to inherited disorders, impaired HCC cell proliferation rate and cell cy- blistering diseases, and cancer. Dsg2 is a cell cle progression (p < 0.05). surface expressed adhesion protein and belongs CONCLUSIONS: Taken together, our results to the cadherin family18. However, to date, the suggest that increased Dsg2 expression was role of Dsg2 in cancer is still uncertain. Emerg- associated with tumor progression in HCC and ing studies indicated Dsg2 expression was may function as a promising biomarker for unfa- 19 vorable prognosis of HCC. downregulated in gastric cancer , prostate can- cer20, melanoma21, pancreatic cancer22, and colon Key Words: cancer23, which showed Dsg2 functions as a tu- Desmoglein-2, Hepatocellular carcinoma, Progno- mor suppressor. In contrast, other investigations sis, Biomarker. reported that Dsg2 was overexpressed in skin Corresponding Author: Yonghua Yu, MD; e-mail: [email protected]. 5481 C.-P. Han, Y.-H. Yu, A.-G. Wang, Y. Tian, H.-T. Zhang, Z.-M. Zheng, Y.-S. Liu SCC and basal cell carcinoma24 and non-small nitrogen after surgical resection. The clinicopath- cell lung cancer25, while implied the oncogenic ological variables including age, gender, HBsAg, function of Dsg2 protein. However, its expres- smoking status, tumor size, and tumor stage col- sion pattern and clinical significance in hepa- lected from patients were summarized in Table tocellular carcinoma (HCC) remains unknown. I. Overall survival (OS) was defined as the time In this work, we measured Dsg2 expression in that elapsed between the date of surgery and the HCC tumor tissues and adjacent noncancerous patient’s death from HCC. tissues using quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) and Western blot. Cell Lines and Cultures Also, we investigated the associations of Dsg2 Human normal liver cell line HL-7702 and expression with clinicopathological variables and HCC cell lines (QGY-7404 and Bel-7402) were overall survival data of patients with HCC. The obtained from the Institute of Biochemistry and effects of Dsg2 knockdown on HCC cell prolifer- Cell Biology of the Chinese Academy of Sciences ation were also investigated in vitro. (Shanghai, China). Cells were cultured in Ros- well Park Memorial Institute-1640 (RPMI-1640) medium (Invitrogen, Carlsbad, CA, USA) supple- Patients and Methods mented with 10% fetal bovine serum (FBS; Invi- trogen, Carlsbad, CA, USA) at 37°C, humidified Patients and Tissue Samples incubator containing 5% CO2. This investigation was approved by the Ethics Committee of Shandong Cancer Hospital and In- siRNA Transfection of Cell Lines stitute Affiliated to Shandong University. A total The siRNA sequences we used were the same of 104 patients who diagnosed as HCC and went as the previous study25. The detailed siRNA se- through surgical resection at Shandong Cancer quences were as follows: Dsg2: 5’-CCUCCAGU- Hospital and Institute Affiliated to Shandong GUUCUACCUAATT-3’ (sense) and 5’-UUAG- University between Mar 2009 and Mar 2011 GUAGAACACUGGAGGTT-3’ (antisense), neg- were enrolled in this study. The written informed ative control: 5’-UUCUCCGAACGUGUCAC- consent was obtained from all the patients. 104 GUTT-3’ (sense) and 5’-ACGUGACACGUUCG- pairs of HCC tumor tissues and adjacent normal GAGAATT-3’ (antisense). These double-strand- tissues were immediately snap-frozen in liquid ed siRNAs were synthesized by GenePharma Table I. Correlations of clinicopathological variables and Dsg2 expression. Dsg2 expression Variables N Low High p-value Age (years) 0.410 ≥ 50 51 19 32 < 50 53 17 36 Gender 0.728 Male 53 18 35 Female 51 18 33 HBsAg 0.087 Positive 54 16 38 Negative 50 20 30 Smoking status 0.277 Non-smoker 48 17 31 Smoker 56 19 37 Tumor size (cm) 0.035 ≥ 5 59 20 39 < 5 45 16 29 Tumor stage 0.021 I-II 43 15 28 III 61 21 40 Dsg2: Desmoglein-2; HCC: hepatocellular carcinoma; HBsAg: Hepatitis B surface antigen. 5482 Desmoglein-2 overexpression predicts poor prognosis in hepatocellular carcinoma patients (Shanghai, China) and transfected into HCC cell RT-qPCR Assay lines by Lipofectamine 2000 (Invitrogen, Carls- RT-qPCR was performed to measure the mR- bad, CA, USA) per the manufacturer’s recom- NA expression level of Dsg2 using the BeyoFast™ mendations. SYBR Green qPCR Mix Kit (Beyotime, Haimen, Jiangsu, China) at an ABI 7300 Real Time-PCR Cell Proliferation Assay System (Applied Biosystems, Life Technologies The rate of cell proliferation was determined GmbH, Darmstadt, Germany). The following by the Cell Counting Kit-8 (CCK-8) assay fol- PCR reaction was used: step 1: 95°C for 10 min, lowing the protocols provided by the supplier 1 cycle; step 2: 95°C for 15 sec, 60°C for 1 min, (Beyotime, Haimen, Jiangsu, China). Briefly, 40 cycles. The primers used in this study were the human normal liver cell line HL-7702, HCC cell same as the previous one21 and listed as follows: lines (QGY-7404 and Bel-7402), Dsg2 specific Dsg2: forward 5’-TGGACACCCAAACAGTG- siRNA (si-Dsg2) or negative control siRNA (NC GCCCT-3’, reverse 5’-CTCACTTTGTTGCAG- siRNA) transfected HCC cell lines were seeded CAGCACAC-3’; β-actin: forward 5’-GGCACCA- into 96-well plate at a density of 2×103 cells/ CACCTTCTACAATGA-3’, reverse 5’-TCTCCT- well and cultured in the aforementioned cul- TAATGTCACGCACGAT-3’. All samples were ture conditions. Subsequently, 20 µl of CCK-8 run in triplicates, and samples were normalized solution was added to the each well at indicated against an endogenous internal control, β-actin. time points (0 d, 1 d, 2 d, and 3 d) and incubated Levels of Dsg2 mRNA were quantified using −2 at 37°C for another 2 h. The absorbance was ΔΔCq method26. measured at 450 nm using an epoch microplate spectrophotometer (BioTek Instruments Inc., Western Blot Assay Winooski, VT, USA). Each sample was repeated Total protein was extracted from the tissues in triplicates. and cell lines using the RIPA buffer consisting of 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, Flow-Cytometry Assay 0.5% sodium deoxycholic acid, and 0.1% sodi- For cell cycle analysis, the cells cultured to um dodecyl sulfate as reported27. The protein logarithmic phase were harvested and washed sample was quantified using the Bradford Pro- with PBS. Then, the cells were treated with tein Concentration Determination Kit (Beyotime, trypsin and fixed using pre-cold 75% ethanol at Haimen, Jiangsu, China). The same amount of 4°C for 2 h. Following, the cells were stained protein sample was separated using a 10% sodium with propidium iodide (PI)/RNase A mixture dodecyl sulphate-polyacrylamide gel electropho- (Beyotime, Haimen, Jiangsu, China) at darkness resis (SDS-PAGE) gel and transferred to a poly- for 15 min at room temperature. Cell cycle was vinylidene difluoride membrane (PVDF, EMD analyzed using FACSCalibur™ flow cytometer Millipore Corporation, Billerica, MA, USA) at (BD Biosciences, Franklin Lakes, NJ, USA). 80 V for 2 h at 4°C.
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