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Xiaopeng Li, MD1, Xin Chen, MD2, Jue Jiang, MD1, Shuanying Yang, MD3, and Ya Gao, MD4. Abstract. Aims: Nuclear receptor binding protein 2 is ubiquitously ...
Original Article

NRBP2 Overexpression Inhibits Cell Proliferation and Migration and Increases Cisplatin Sensitivity in Intrahepatic Cholangiocarcinoma

Technology in Cancer Research & Treatment 2017, Vol. 16(6) 1235–1243 ª The Author(s) 2017 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1533034617747174 journals.sagepub.com/home/tct

Xiaopeng Li, MD1, Xin Chen, MD2, Jue Jiang, MD1, Shuanying Yang, MD3, and Ya Gao, MD4

Abstract Aims: Nuclear receptor binding protein 2 is ubiquitously expressed in all tissues in humans. However, few studies have reported the function of nuclear receptor binding protein 2 in human cancers. Methods: Immunohistochemistry and Reverse Transcription-PCR (RT-PCR) were used to detect nuclear receptor binding protein 2 expression in intrahepatic cholangiocarcinoma tissues. Cell Counting Kit-8 assay, flow cytometry, Transwell assay, wound healing assay, and Western blotting were used for the functional study of nuclear receptor binding protein 2. All statistical analyses were performed using SPSS 19.0. Results: Survival analysis showed that high expression of nuclear receptor binding protein 2 led to better prognosis. Overexpressed nuclear receptor binding protein 2 can inhibit the proliferation rate of cholangiocarcinoma cells while having a slight effect on cell apoptosis. Gain-of-function experiments showed that overexpressed nuclear receptor binding protein 2 could lead to G1 phase arrest in RBE and CCLP cell lines. Furthermore, Transwell assay showed that overexpressed nuclear receptor binding protein 2 could inhibit the migration ability of RBE and CCLP cell lines. Western blot analysis showed that E-cadherin was upregulated, while N-cadherin and vimentin were downregulated. In addition, we observed that overexpressed nuclear receptor binding protein 2 can also increase the cisplatin sensitivity of cholangiocarcinoma cells by regulating the Mammalian Target of Rapamycin (mTOR) pathway. Conclusions: Our study observed that nuclear receptor binding protein 2 played a tumor suppressive role in intrahepatic cholangiocarcinoma, which may be attributable to the induction of G1 phase arrest and inhibition of progression of epithelial–mesenchymal transition, and overexpression of nuclear receptor binding protein 2 leads to improved efficiency of cisplatin treatment. Keywords nuclear receptor binding protein 2 (NRBP2), intrahepatic cholangiocarcinoma (ICC), cisplatin sensitivity, epithelial-mesenchymal transition (EMT), G1 arrest Abbreviations CCA, cholangiocarcinoma; CCK-8, Cell Counting Kit-8; cDNA, complementary DNA; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; mRNA, messenger RNA; NRBP 1, nuclear receptor binding protein 1; NRBP 2, nuclear receptor binding protein 2. Received: April 25, 2017; Revised: October 27, 2017; Accepted: November 3, 2017. 1

Introduction Human cholangiocarcinoma (CCA) is a highly malignant, generally fatal neoplasm and primarily occurs in Chile, South Korea, and Northeast Thailand, while a rare cancer (incidence of less than 6 cases per 100 000) in Western countries.1 Although we have several methods for CCA diagnosis, including imaging studies, cytology and/or histology, and serum tumor markers, patients diagnosed in the advanced stage have few options for surgery.2 Furthermore, those patients who are eligible for radical operations only survive 1 to 3 years.3 Therefore, it is necessary to

Department of Ultrasonography, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 2 Department of Radiology, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 3 Department of Respiratory Medicine, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 4 Department of Pediatric Surgery, The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China Corresponding Author: Ya Gao, MD, Department of Pediatric Surgery, The Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xiwu road, Xi’an 710004, China. Email: [email protected]

Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Technology in Cancer Research & Treatment 16(6)

understand the molecular mechanism underlying tumor progression, by which tumor cells gain the properties of proliferation, drug resistance, invasiveness, and metastasis. Nuclear receptor binding protein 2 (NRBP2) is a gene with unknown function that shows 59% amino acid similarity to the putative adaptor protein, nuclear receptor binding protein 1 (NRBP1).4,5 A recent study revealed that NRBP2 was associated with neuronal differentiation and affects neuronal cell survival.6 Zhang and his colleagues determined that NRBP2 overexpression can enhance sorafenib sensitivity of the hepatocellular carcinoma (HCC) cell line by inhibiting annexin A2 (ANXA2) expression and activity of the AKT/PKB (Protein Kinase B) signaling pathway.7 However, the function of NRBP2 in CCA is unclear, especially in intrahepatic cholangiocarcinoma (ICC). In this study, we aimed to preliminarily analyze the role of NRBP2 in CCA cells and the relationship between the expression of NRBP2 and the prognosis of patients with ICC.

Construction of NRBP2 Overexpression and mTOR Suppression in CCA Cell Line

Materials and Methods

Immunohistochemistry

Patients and Cancer Tissue Sample Collection

The paraffin slides were deparaffinized and placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 minutes. Next, antigen retrieval was performed by heating the slides in citrate buffer (pH 6.2) in the microwave oven for 20 minutes and washed in PBS (phosphate buffer saline) 3 times before staining with immunoperoxidase. Slides were incubated in a humidified chamber with blocking agent (5% FBS) for 60 minutes at room temperature. Next, slides were incubated overnight with anti-NRBP2 antibody (ab172866; 1:100 dilution; Abcam Biotechnology, Cambridge, United Kingdom) in 5% FBS overnight at 4 C. After PBS wash, they were incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin for 40 minutes at room temperature. The results were visualized by reaction with diaminobenzidine (3,30-diaminobenzidine tetrahydrochloride) and counterstained with hematoxylin. For each sample, we imaged with a high-power objective (20) under the same conditions. Next, the tissues were scored according to the intensity of the dye color and the number of positive cells. The intensity of the dye color was graded as 0 (no color), 1 (light yellow), 2 (light brown), or 3 (brown), and the number of positive cells was graded as 0 (75%). The 2 grades were multiplied together. Next, we assigned the score to 2 levels: 55 Gender Male Female Size of tumor, cm 5 >5 Number of tumor Single Multiple Vessel invasion Negative Positive Grade Well þ Moderate Poor TNM stage I-II III-IV

All Patients, N ¼ 29 Lowa Higha P Valuea 12 17

7 11

5 6

.728

10 19

6 12

4 7

.868

9 20

3 15

6 5

.032

22 7

12 6

10 1

.139

24 5

16 2

8 3

.264

8 21

2 16

6 5

.011

19 10

11 7

8 3

.523

Abbreviation: NRBP, nuclear receptor binding protein; TNM, Tumor Node Metastasis. a The median expression level was used as the cutoff. Low expression of NRBP2 in 17 patients was classified as values below the 50th percentile. High NRBP2 expression in 11 patients was classified as values at or above the 50th percentile. For analysis of correlation between NRBP2 expression and clinical features, w2 tests were used. Results were considered statistically significant at P < .05.

Results Nuclear Receptor Binding Protein 2 Was Downregulated in Human ICC Tissues To investigate the function of NRBP2 in the progression of ICC, the NRBP2 expression level was detected by immunohistochemistry (IHC) in 29 paired ICC tissues and adjacent neighbor tissues. The results showed that NRBP2 was primarily located in the cytoplasm and downregulated in ICC tissues, and 24 paired ICC tissues had lower expression than adjacent noncancer tissues. Only one paired was upregulated, while the others had no change (Figure 1A, B). Next, we used RT-PCR to detect the messenger RNA (mRNA) level of NRBP2 in another 24 paired ICC tissues and adjacent neighbor tissues, and the results were consistent with the IHC results (Figure 1C).

Figure 2. Results of the Kaplan-Meier survival analysis between the NRBP2 low-expression group and the NRBP2 high-expression group in 29 patients with ICC. NRBP2 indicates nuclear receptor binding protein 2.

Overexpression of NRBP2 Was Associated With Better Prognosis of Patients With ICC To further study the relationship between expression of NRBP2, clinicopathological features, and patient prognosis,

Li et al

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Figure 3. Overexpressed NRBP2 in CCA cells was associated with a decrease in the cell proliferation rate. A and B, Results for efficiency of NRBP2 transfection in RBE and CCLP cell lines; GAPDH was used as internal control. C, The CCK-8 results between NRBP2 overexpression and negative control. ***P < .001. D, Apoptosis results by flow cytometry are shown. Cell percentage of each apoptosis phase was analyzed, and the expression of pro-caspase-3 and cle-caspase-3 was detected. CCA indicates cholangiocarcinoma; CCK-8, Cell Counting Kit-8; NC, negative control; NRBP2, nuclear receptor binding protein 2; NRBP2-OE, NRBP2 overexpression.

we divided these 29 patients into 2 groups according to NRBP2 expression: those with IHC score 6, the high expression group, and those with IHC score