Introduction

Colorectal cancer is one of the most frequently diagnosed cancers worldwide, accounting for an estimated 9.4% of all malignancies [1]. In China, colorectal cancer remains the fifth most common cancer type and the fourth most common cause of cancer-related death [2]. Despite advances made in surgical techniques and chemotherapeutic options, the survival rate of colorectal cancer has not been substantially improved, and approximately 20% of advanced patients die of recurrence of the disease [3]. Colon carcinogenesis is a complex multistage process that involves a progressive disruption of homeostatic mechanisms controlling intestinal epithelial cell proliferation, differentiation, and programmed cell death [4]. Therefore, advances in treatment of this disease are likely to come from a better understanding of its pathogenesis and biological features.

The extracellular matrix plays a critical role in modulating epithelial cell morphology, growth, migration, and differentiation in a wide array of tissue types [5]. Biglycan is an extracellular matrix protein that belongs to the leucine-rich repeat (LRR) protein family. It is characterized by a 38-kDa core protein containing ten LRRs flanked by disulfide bond stabilized loops [6, 7]. Biglycan is found to be expressed both at the cell surface and in the pericellular space in various tissues of mainly mesenchymal origin [7]. A major physiological role for biglycan in bone formation was revealed using biglycan knockout (bgn) mice, which display a reduced bone mass and an osteoporosis-like phenotype [8]. Recently, overexpression of biglycan has been found in a variety of human epithelial tumors, such as liver [9], ovary [10], odontogenic [11], and colon [12], suggesting an important role of biglycan in cancer progression and pathogenesis. In addition, Weber et al. [13] found that biglycan is strongly overexpressed in pancreatic cancer tissues and inhibits the growth of pancreatic cancer and colon carcinoma cell lines by induction of p27, leading to cell cycle arrest in G1. More recently, Mikula et al. [12] identified biglycan as a novel biomarker for colon tumor by integrating microarray data with the results of a high-throughput proteomic workflow. Nonetheless, the expression and clinicopathological significance of biglycan in colorectal cancer need to be further elucidated.

The aim of this study is to examine biglycan mRNA expression in human colorectal cancer specimens (primary colorectal cancer tissues and corresponding adjacent normal tissues) using quantitative real-time RT-PCR and to elucidate the clinicopathological significance of biglycan expression in colorectal cancer.

Materials and methods

Patients and tissue samples

A total of 55 patients with colorectal cancers, who had undergone curative-intent surgery at Liaoning Cancer Hospital and Institute (Shenyang, China) between May 2009 and August 2010, were enrolled in this study. They were 39 men and 16 women, with a mean age of 59.1 years (range, 31–75 years). None of the patients had received radiotherapy or chemotherapy before surgical resection, and all the patients were treated with routine chemotherapy after the operation. The clinicopathological parameters of the patients were obtained by a medical history review and are summarized in Table 1. A total of 110 samples of colorectal cancer tissue and paired adjacent normal colorectal mucosa were collected, with each pair taken from the same patient. Surgically removed tumors and matched non-cancerous (normal) tissue samples were immediately frozen in liquid nitrogen until extraction of RNA. The histological types were assigned according to the criteria of the World Health Organization classification system by at least two expert pathologists independently in a double-blinded fashion. This study was performed following an Institutional Ethics Review Board approved protocol, and informed consent was obtained from all patients prior to surgery.

Table 1 Association between biglycan expression and clinicopathological factors of colorectal cancer patients
Full size table

Total RNA isolation and reverse transcription

Total RNA was extracted from tumor tissues and corresponding normal tissues using TRizol reagent (Life Technologies, Inc., Rockville, MD), according to the manufacturer’s instructions. The concentration and purity of the RNA in each sample were determined by measuring the absorbance at 260 and 280 nm. RNA integrity was confirmed by electrophoresis on a 1% agarose gel. Reverse transcription of RNA was carried out using the TIANscript RT kit (Tiangen Biotech Co., Ltd., Beijing, China). In brief, 1 μg of each total RNA sample was reverse transcribed using anchored oligo-dT primers and TIANScript M-MLV reverse transcriptase following the manufacturer’s recommended protocol and subsequently diluted with RNase-free ddH2O and stored at −20°C.

Quantitative real-time RT-PCR

Quantitative real-time RT-PCR was performed using SYBR Green (Tiagen) on an Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The primers for biglycan were as follows: forward, 5′-TCAACAACCCCGTGCCCTA-3′; reverse, 5′-TGCCTCTACTTTTTGTAGTTGCCA-3′. The reaction mixture consisted of 1 μl cDNA, 10 μl 1× SYBR Green (Tiagen), 200 nM of each primer, and distilled water to bring to the final volume of 20 μl. After an initial denaturation step of 10 s at 95°C, a three-step cycle procedure was used (denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and elongation at 72°C for 40 s) for 40 cycles. The specificity of the PCR was confirmed by examining the dissociation reaction plot subsequent to real-time RT-PCR. β-actin served as the constitutive control. PCR of each sample were conducted in triplicate.

Biglycan expression scores

The relative biglycan mRNA expression was calculated by the 2−ΔCt method (ΔCt = Ct of Biglycan − Ct of β-actin) [14]. The biglycan expression score (fold change) in each tissue was defined as the ratio of relative biglycan mRNA expression in tumor tissue to that in corresponding normal tissues. The up-regulation of biglycan was considered to be positive only when the biglycan expression score was greater than 1.7, as described elsewhere [15–17].

Statistical analysis

All statistical analyses were performed using the SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). The correlation between biglycan expression and clinicopathological factors were analyzed by Chi-square test. Values of P < 0.05 (confidence level >95%) were considered statistically significant.

Results

Up-regulation of biglycan in colorectal cancer tissues

Biglycan mRNA expression was detectable in all the primary colorectal cancer tissues and adjacent normal colorectal mucosa. Notably, the relative expression of biglycan in colorectal cancer tissues was markedly higher than that in the corresponding normal tissues (P = 0.0264, Fig. 1). Biglycan up-regulation was positive in 61.8% (34/55) of colorectal cancer patients. Thus, the up-regulated biglycan expression might be associated with the progression of colorectal cancer.

Fig. 1
figure 1

Biglycan mRNA expression in colorectal cancer tissues and paired adjacent normal colorectal mucosa examined by quantitative real-time RT-PCR and normalized to β-actin. Bars represent the means of biglycan relative expression in cancer tissues and normal tissues, respectively

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Correlation of biglycan expression with clinical parameters

The relationship between the up-regulation of biglycan and clinicopathological variables are provided in Table 1. It was found that the up-regulation of biglycan was significantly correlated with poor tumor differentiation (P = 0.009), lymph node metastasis (P = 0.041), and distant metastasis (P = 0.036). However, there was no significant correlation between biglycan up-regulation and other clinicopathological factors including gender, age, tumor location, tumor size, TNM stage, and Dukes’ classification (P = 0.586, 0.345, 0.701, 0.431, 0.117, and 0.200, respectively). These findings suggest that up-regulation of biglycan may be a potential marker for the malignancy of colorectal cancer.

Discussion

Current clinical practice in colorectal cancer screening has contributed to a reduction in mortality; however, the cure rate for advanced colorectal cancer remains low and the morbidity remains high. In spite of several clinicopathological factors such as carcinoembryonic antigen (CEA) and CA19-9 have been used as serum markers for colorectal cancer, their sensitivities varied in different studies and their clinical usefulness remains controversial [18–20]. Therefore, it is necessary to explore novel markers for estimating the progression of colorectal cancer when designing individualized treatment procedures, which will be helpful to improve the success of treatment.

Recently, a number of microarray-based investigations have demonstrated that biglycan is frequently overexpressed in human cancer tissues and is associated with cancer progression [9, 10, 12, 21]. In particular, using proteomic and microarray technologies, Mikula et al. [12] identified an up-regulation of biglycan in colon adenocarcinoma samples at both protein and mRNA levels compared to normal colon mucosa. These novel findings suggest that overexpression of biglycan is a frequent event in the tumorigenesis of many types of cancer. To our knowledge, however, the clinicopathological significance of biglycan in colorectal cancer has not yet been determined. In the current study, we showed that up-regulation of biglycan occurred in more than half (61.8%, 34/55) of the colorectal cancer specimens, and the relative level of biglycan mRNA expression in primary colorectal cancer tissues was significantly higher than that in corresponding non-tumor colon tissues, suggesting that up-regulation of biglycan may be an important event in the carcinogenesis of colorectal cancer. Furthermore, the most important finding of the present study is the evidence that the up-regulation of biglycan was significantly associated with poor tumor differentiation, lymph node metastasis, and distant metastasis, but not with other factors of colorectal cancer patients including gender, age, tumor location, tumor size, TNM stage, and Dukes’ classification. These results agree with the fact that biglycan, a transforming growth factor β (TGF-β)-binding protein, is able to increase the probability of an interaction of TGF-β with its specific surface receptors, thereby contributing to the early progression of carcinomatous tumors [13]. Notably, several recent studies indicated a possible link between biglycan expression and malignant behavior of cancer cells. For example, Fang et al. [22] demonstrated that elevated expression of biglycan may be involved in the stem cell function of colon carcinoma. Glycosaminoglycans, including biglycan, are thought to be promising therapeutic targets in cancer [23]. Our current findings may also support this notion, because the majority of colorectal cancer specimens exhibited biglycan overexpression, and up-regulation of biglycan was significantly associated with poor tumor differentiation, lymph node metastasis, and distant metastasis. However, due to the limited number of patients in this study, further investigation of a larger patient population is necessary to confirm its clinical significance in colorectal cancer. In addition, the mechanisms leading to biglycan overexpression in colorectal cancer await further investigations. A limitation of our biglycan mRNA results is the lack of corresponding protein levels, and whether there is a coincidence between the expression of biglycan mRNA and protein in colorectal cancer is still unknown. Thus, immunohistochemistry and Western blotting studies are needed to evaluate the protein level of biglycan in human tumors.

In conclusion, we have demonstrated that the overexpression of biglycan gene possessed clinicopathological importance in colorectal cancer and might be a useful molecular marker for the malignancy of colorectal cancer. Further studies are needed to clarify the mechanisms by which biglycan is involved in the progression of colorectal cancer and its exact roles in colon carcinogenesis by functional analysis.