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Expression of Ribophorine II Is a Promising Prognostic Factor in Human Gastric Adenocarcinoma

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Expression of Ribophorine II Is a Promising Prognostic Factor in Human Gastric Adenocarcinoma 448 INTERNATIONAL JOURNAL OF ONCOLOGY 50: 448-456, 2017 Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma DAISUKE Fujimoto, TAKANORI GOI and YASUO HIRONO Department of Surgery 1, Faculty of Medicine, University of Fukui, Fukui 910-1193, Japan Received October 17, 2016; Accepted December 19, 2016 DOI: 10.3892/ijo.2016.3822 Abstract. The increased invasiveness of gastric adenocarci- Introduction noma is important for progression and metastasis. In recent molecular biological studies, ribophorine II (RPN2) induced Gastric adenocarcinoma has one of the highest prevalence epithelial-mesenchymal transition and metastatic activity. and mortality rates among malignant tumors (1). Global However, no studies have evaluated the relationship between incidence of primary tumor locations and the histological RPN2 expression, ability of cancer to invade/metastasis, and types are constantly changing: in United States and in patient prognosis in gastric adenocarcinoma. Therefore, we Western Europe the incidence of Barrett's type carcinoma have examined these factors. Immunohistochemical staining and gastric cardia adenocarcinoma is increasing (2), while was performed to detect RPN2 and p53 in the primary there has been a reduction of incidence of distal gastric lesion and adjacent normal gastric mucosa of 242 gastric adenocarcinoma since the 1970s (3). Although gastric adeno- adenocarcinoma patients who underwent resection surgery. carcinoma mortality has been reduced, it remains a disease We conducted clinicopathologic examinations and analyzed with poor prognosis and high mortality, second only to lung patient prognoses with the Kaplan-Meier method. Further, tumor in the world. The prognosis of gastric adenocarcinoma multivariate analysis was conducted using a Cox hazard depends on stage and signature metastasis type of peritoneal model. Also, we analyzed the ability of invasion under inhib- dissemination have poor prognosis and when the disease is ited RPN2 expression in vitro. RPN2 expression was observed confined to the stomach mucosa, 5-year survival is ~95%, in 119 of 242 cases of gastric adenocarcinoma patients. RPN2 while the reported 5-year survival rate for advanced gastric expression was associated with a higher incidence of depth adenocarcinoma with peritoneal dissemination varies from of wall invasion, lymph node metastasis, lymphatic invasion, 10 to 20% (3). venous invasion, peritoneal dissemination, histopathological Peritoneal dissemination of gastric adenocarcinoma is stage, and p53 expression. In stage II and III curative resec- particularly frequent, driving the need for therapies that tion cases, where recurrence is the most serious problem, minimize the cancer spread. One model for the mechanism cases that expressed RPN2 had a significantly lower 5-year of peritoneal metastasis of gastric adenocarcinoma involves survival rate and higher recurrence rate compared to the cases growth of tumor at the primary lesion, invasion into serous with no RPN2 expression. In the multivariate analysis for surface, expose abdominal cavity, dissociation of cancer cells, prognosis, RPN2 expression was found to be an independent and implantation and growth on the peritoneum. Various factor. Also, gastric adenocarcinoma cell, had mutant-type genetic alterations have been studied, and involvement of a p53, reduced the ability of invasion by knockout of RPN2 number of genes has been reported. expression in vitro. RPN2 expression correlates with gastric MicroRNA (miRNA) is a small non-coding RNA that adenocarcinoma cell invasion and shows promise as a new functions in RNAs silencing and post-transcriptional regu- prognostic factor in human gastric adenocarcinoma. lation of gene expression (4-6). These are the fundamental gene regulators that control proliferation, differentiation, and apoptosis during development. They target a large number of mRNAs and induce mRNA degradation or inhibition of translation by targeting the 30-untranslated regions (5). Some miRNAs had controlled the peritoneal dissemina- Correspondence to: Dr Daisuke Fujimoto, Department of tion of gastric adenocarcinoma (7-9). miR-27 promoted Surgery 1, Faculty of Medicine, University of Fukui, 23-3 Matsuoka epithelial-mesenchymal transition (EMT) and metastasis Shimoaiduki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan of human gastric cancer cells (10). EMT is a portmanteau E-mail: [email protected] concept that can be applied to the metastatic behavior of carcinoma cells at a number of junctures, and has been Key words: ribophorine II, gastric adenocarcinoma, prognostic factor, associated with peritoneal dissemination of gastric adeno- CRISPR/Cas9, immunohistochemistry carcinoma (11). Various genetic alterations associated with EMT have been studied, and involvement of a number of genes has been reported. Ribopholin II (RPN2) is one of the Fujimoto et al: RPN2 is A promising prognostic factor 449 genes that promote EMT through the stabilization of mutant nobenzidinetetrahydrochloride for 5 min and the reaction was p53 (12), and there is a possibility that miRNA is controlling stopped in TBS. Finally, the slides were lightly counterstained RPN2. However, in gastric adenocarcinoma, no report exists with hematoxylin. The expression was interpreted as positive on the relation between RPN2 and metastasis and prognosis of when the protein was expressed in >20% of the cancer cells gastric adenocarcinoma. using ImageJ software (http://rsb.info.nih.gov/ij/). This study was conducted to compare the expression of RPN2 with the invasiveness of the primary lesion in human Statistical analysis. Statistically significant differences in gastric adenocarcinoma, and the clinicopathological features clinicopathological findings were assessed by cross-tabulation, and patient prognosis, as reported below. and statistical evaluations were determined by the χ2 test using SPSS software (IBM SPSS Statistics, IBM Corp., USA). Materials and methods Patient survival was calculated using the Kaplan-Meier technique. The outcomes from different groups of patients Patients and samples. Surgical specimens and adjacent normal were compared by log-rank test using SPSS software. The Cox gastric tissues were obtained from 242 patients with sporadic proportional hazards model was used in multivariate regres- primary gastric adenocarcinoma, and surgically resected in sion analysis of survival data using SPSS software. P-values the Department of First Surgery, University of Fukui, Japan, <0.05 were considered statistically significant. between 2002 and 2010. As histopathological findings varied within the same tumors, the diagnosis was based upon the Cell culture and RT-PCR analysis. Human gastric cancer cell dominant pattern evaluated by two pathologists. All samples lines MKN45, MKN74, NUGC3, NUGC4, SNU5, KATO III were fixed in 10% paraformaldehyde (pH 6.8) for 24 h, and and TMK1 (which were obtained from JCRB Cell Bank) embedded in paraffin. were maintained in RPMI-1640 supplemented with 10% FBS The eligibility criteria were as follows: i) histopatho- (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin at logically confirmed primary gastric adenocarcinoma, 37˚C with 5% CO2 incubation. RNA was isolated from the ii) resection of gastric adenocarcinoma with extended (D1+ above cells using Isogen (Wako, Japan), according to the or D2) lymph node dissection (13), iii) pathological diag- manufacturer's instructions. cDNA was synthesized from 2 µg nosis for the classification used the 7th edition UICC TNM of total RNA by reverse transcription, using High-Capacity classification, iv) histological curative resection (stage I-III), cDNA Reverse Transcription kits (Applied Biosystems). The v) an Eastern Cooperative Oncology Group performance resulting cDNA was used for the subsequent PCR assays. status of 0 or 1, vi) no chemotherapy or radiotherapy before RPN2 was amplified by using the primers with following surgical resection, vii) patients with stage II/III received sequences: forward, 5'-GCCAGGAAGTGGTGTTTGTT-3'; S-1 after surgical resection, viii) patients with stage IV and reverse, 5'-ACAGAGCGAAGAGCAGAAGC-3'. received S-1 + cisplatin after surgical resection, ix) patients with stage I received no chemotherapy after surgical resec- Cell transfection. MKN74 and KATO III cells were seeded tion, and x) all patients were followed up for recurrence onto 6-well plates (Corning) 24 h before transfection. Cells were at regular intervals for five years, underwent chest X-ray, transfected using Lipofectamine 3000 (Life Technologies) at computed tomography, and testing of gastrointestinal fiber. 80-90% confluency according to the manufacturer's instruc- All the patients provided written informed consent before tions. For each well of a 6-well plate, a total of 1 µg of sgRPN2 the samples were collected. (single-guided RNA to RPN2) + pGuide-it-ZsGreen1 plasmid or empty plasmid was used. The CRISPR/Cas9 system used Ethical approval. We attest that the research was performed in this study was Guide-it CRISPR/Cas9 systems (Clonetch). in accordance with the humane and ethical rules for human We designed sequences targeting the RPN2 locus (Fig. 4A). experimentation that are stated in the Helsinki Declaration The plasmid enhanced green fluorescent protein (eGFP) and of 1964 and latest version. The procedures of our study received was used as a fluorescent marker to sort transfected cells. ethical approval with Institutional Committee Responsible for Forty-eight hours posttransfection,
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