MicroRNA-372 Is Associated with Poor Prognosis in

1 downloads 0 Views 778KB Size Report
Mar 26, 2012 - Shinya Yamashita a Hirofumi Yamamoto a Koshi Mimori b Naohiro Nishida b. Hidekazu Takahashi a Naotsugu Haraguchi c Fumiaki Tanaka b ...
Clinical Study Received: November 8, 2011 Accepted after revision: January 4, 2012 Published online: March 26, 2012

Oncology 2012;82:205–212 DOI: 10.1159/000336809

MicroRNA-372 Is Associated with Poor Prognosis in Colorectal Cancer Shinya Yamashita a Hirofumi Yamamoto a Koshi Mimori b Naohiro Nishida b Hidekazu Takahashi a Naotsugu Haraguchi c Fumiaki Tanaka b Kohei Shibata b Mitsugu Sekimoto a Hideshi Ishii c Yuichiro Doki a Masaki Mori a a

Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Osaka University, Suita, Division of Molecular and Surgical Oncology, Department of Molecular and Cellular Biology, Kyushu University, Medical Institute of Bioregulation, Beppu, and c Department of Frontier Science for Cancer and Chemotherapy, Graduate School of Medicine, Osaka University, Suita, Japan b

Key Words MicroRNA ⴢ Colon cancer ⴢ Liver metastasis ⴢ Prognosis ⴢ LATS2

Abstract Objective: MicroRNA-372 (miR-372) is reportedly shown to be an oncogene in human testicular germ cell tumors and gastric cancers, but its expression in colorectal cancer (CRC) is not yet determined. This study investigated the clinical significance of miR-372 expression in CRC. Methods: qRT-PCR was used to evaluate miR-372 in 144 CRC patients, and large tumor suppressor 2 (LATS2) expression was also examined as the likely target gene of miR-372. In vitro assays were performed to evaluate the biological function of miR-372. Results: Multivariate analysis indicated that high miR-372 expression was an independent prognostic factor (p = 0.006). High miR-372 expression was associated with synchronous liver metastasis (p = 0.035). We found an inverse relationship between miR-372 and LATS2 by qRT-PCR (p = 0.007) and immunohistochemistry (p = 0.042) using CRC tissue samples. Furthermore, pre-miR-372 led to a decrease in the LATS2 protein and an increase in proliferative activity of LoVo cells. We also found a significant association between low LATS2 expression and liver metastasis (p = 0.042). Conclusions: This

© 2012 S. Karger AG, Basel 0030–2414/12/0824–0205$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ocl

study suggested that miR-372 was a novel independent prognostic factor in CRC. Our data suggest that LATS2 may serve as one of the target genes of miR-372 in clinical CRC tissues. Copyright © 2012 S. Karger AG, Basel

Introduction

Colorectal cancer (CRC) is the second most common cause of cancer mortality in developed countries and has a high mortality rate [1]. In Japan, CRC is the third leading cause of cancer death [2]. Although monoclonal antibodies, including bevacizumab, an inhibitor of vascular endothelial growth factor, and cetuximab, an epidermal growth factor receptor inhibitor [3, 4], are currently feasible as novel molecular-based therapies, many patients with CRC still die from disease recurrence, mainly due to liver metastasis. Therefore, further elucidation of the molecular mechanisms of CRC is essential for developing novel therapeutic strategies. MicroRNAs (miRNAs) constitute a class of small (19– 25 nucleotide) non-coding RNAs that function as posttranscriptional gene regulators and work as a novel class of global gene regulators by binding to partially compleHirofumi Yamamoto, MD, PhD Yamadaoka 2-2 Suita, Osaka 565-0871 (Japan) Tel. +81 6 6879 3251 E-Mail hyamamoto @ gesurg.med.osaka-u.ac.jp

mentary sequences in 3ⴕ untranslated regions of downstream target mRNAs [5]. Alterations in miRNA expression are involved in the initiation, progression, and metastasis of human cancers [6, 7]. Moreover, miRNA is considered superior to mRNA as a molecular marker because of its small size, stability, and resistance to RNase degradation [8]. In CRC, miR-135a, miR-135b, and miR675 are considered as oncogenic miRNAs [9, 10], while miR-126 and miR-145 are reported to be tumor suppressors [11]. Moreover the expression of miR-21 and miR-155 were reported as biomarkers of poor prognosis [12]. Previous studies showed that miR-372 enhances cell proliferation, stimulates cell cycle progression, or decreases apoptosis in testicular germ cell tumors [13] and in gastric cell tumors [14]. By contrast, miR-372 suppresses cell growth in human cervical cancer [15]. However, to our knowledge, the role of miR-372 in CRC has not been analyzed. The current study aimed to assess a prognostic value of miR-372 in CRC. Toward this end, we used qRT-PCR to examine miR-372 expression in 144 CRC samples. We also examined expression of the large tumor suppressor 2 (LATS2) gene as a possible target of miR-372 in clinical CRC samples. To our knowledge, this is the first report on the clinical significance of miR-372 in human CRC.

ManTM MicroRNA Reverse Transcription kit (Applied Biosystems). Reverse transcription conditions were as described previously [18]. miRNA expression was determined as an expression value relative to that of RNU6B (control) and was analyzed using the 2–⌬⌬Ct method [19]. Evaluation of LATS2 mRNA Expression in Clinical Samples Frozen tissue specimens were homogenized, and total RNA was extracted using the modified acid-guanidine-phenol-chloroform method described previously [20, 21]. Total RNA (8.0 ␮g) was reverse transcribed to cDNA with M-MLV RT (Invitrogen, Carlsbad, Calif., USA). The PCR primer sequences for LATS2 mRNA were as follows: LATS2-F, 5ⴕ-AAGAGCTACTCGCCATACGCCTTT-3ⴕ; LATS2-R, 5ⴕ-AGCTTTGGCCATTTCTTGCTCCAG-3ⴕ. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the GAPDH primers were as follows: GAPDH-F, 5ⴕ-TTGGTATCGTGGAAGGACTCTA-3ⴕand GAPDH-R 5ⴕ-TGTCATATTTGGCAGGTT-3ⴕ. Real-time monitoring of PCR was performed with the LightCycler system (Roche Applied Science, Indianapolis, Ind., USA) and SYBR Green I dye (Roche Diagnostics, Tokyo, Japan). Monitoring was performed according to the manufacturer’s instructions as described previously [22]. In brief, a master mixture was prepared on ice that contained 1 ␮l of cDNA, 2 ␮l of DNA Master SYBR Green I mix, 50 ng of primers, and 24 ␮l of 25 mmol/l MgCl2. The final volume was adjusted to 20 ␮l with water. qRT-PCR was performed as described previously [23] with the following cycling conditions: initial denaturation at 95 ° C for 10 min, followed by 45 cycles of 95 ° C for 10 s, annealing at 60 ° C for 10 s, and extension at 72 ° C for 10 s. After amplification, PCR products were subjected to a temperature gradient from 65 to 95 at 0.2 ° C increase per second under continuous fluorescence monitoring to produce a melting curve of the products. The concentrations of unknown samples were calculated by plotting their crossing points against the standard curve and dividing by the GAPDH content.  

 

 

 

 

 

 

 

 

Materials and Methods Clinical Tissue Samples A total of 144 patients analyzed in this study underwent resection of the primary CRC at Kyushu University Hospital at Beppu or at its affiliated hospitals between 1992 and 2000. Resected tissue samples were immediately cut and embedded in Tissue-Tek OCT (optimum cutting temperature) medium (Sakura, Tokyo, Japan), frozen in liquid nitrogen, and stored at –80 ° C until RNA extraction. Clinicopathological factors and clinical stage were classified using the criteria of the International Union against Cancer [16]. All sample data, including age, sex, tumor size and depth, lymphnode metastasis, vascular invasion, distant metastasis, clinical stage, and histologic grade, were obtained from the clinical and pathological records and are summarized in table 1. The mean follow-up period was 39.6 months. In this study, none of the patients had preoperative chemotherapy or irradiation. After surgery, patients with stage III/IV tumors were basically treated with 5-fluorouracil-based chemotherapy. The Human Ethics Review Committee of Osaka University and Kyushu University approved the use of the resected samples. REMARK criteria for tumor marker studies were used for the preparation of this article [17].  

 

Evaluation of miR-372 Expression in Clinical Samples For qRT-PCR analysis of miR-372, cDNA was synthesized from 10 ng of total RNA using TaqManTM MicroRNA hsa-miR-372 specific primers (Applied Biosystems, Tokyo, Japan) and the Taq-

206

Oncology 2012;82:205–212

 

Immunohistochemistry Immunostaining of the LATS2 protein was performed on formalin-fixed, paraffin-embedded sections, as we previously described [24]. Heat antigen retrieval was performed in 10 mM of citrate buffer, pH 6.0, at 95 ° C for 40 min. The rabbit polyclonal antibody against LATS2 (SH030422B; Abgent, Tokyo, Japan) was diluted at 1: 50 and incubated with the specimens overnight at 4 ° C. For negative controls, nonimmunized immunoglobulin G (Vector Laboratories, Burlingame, Calif., USA) was used as a substitute for the primary antibody.  

 

 

 

Cell Cultures The colon cancer LoVo cell line was maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS (Sigma-Aldrich Co., Saint Louis, Mo., USA), and 100 U/ml penicillin and 100 ␮g/ ml streptomycin, in a humidified atmosphere with 5% CO2 at 37 ° C. Cell count was assessed by a hemocytometer according to the manufacturer’s instruction.  

 

Transfection of miRNA-372 Precursor (Pre-miR-372) Cells were grown to 60–80% confluence and transfected with 5 nmol/l of pre-miR-372 or negative control oligonucleotides (Applied Biosystems) using siPORT NeoFX Transfection Agent (Ambion Inc., Austin, Tex., USA).

Yamashita et al.

Table 1. Patient and tumor characteristics according to miR-372 expression

Age, mean 8 SD Sex, male/female Histological grade, well or mod/por and others Size,