Cancer Letters

Cancer Letters

Volume 200, Issue 1, 8 October 2003, Pages 69-76
Cancer Letters

Overexpression of Reg IV in colorectal adenoma

https://doi.org/10.1016/S0304-3835(03)00460-9Get rights and content

Abstract

Identification of molecular markers associated with colorectal adenoma may uncover critical events involved in the initiation and progression of colorectal cancer. Our previous studies, mainly based on suppression subtractive hybridization, have identified Reg IV as a strong candidate for a gene that is highly expressed in colorectal adenoma when compared to normal mucosa. In this study, we sought to determine the mRNA expression of Reg IV in colorectal adenoma, in comparison with normal colorectal mucosa and carcinoma in multiple samples. Semi-quantitative RT-PCR was performed in 12 colorectal adenomas and 10 concurrent carcinomas. Reg IV mRNA level was higher in all adenomas (12/12) (p=0.001) and in 9/10 concurrent colorectal carcinoma (p=0.021) when compared to paired normal colorectal mucosa. Northern blot analysis further confirmed these results. In situ hybridization with digoxigenin (DIG)-labeled cRNA was performed in 32 colorectal adenomas with varying degree of dysplasia. Compared with paired normal tissues, Reg IV was overexpressed in 74% (14/19) adenomas with mild or moderate dysplasia and 100% (13/13) cases of adenoma with severe dysplasia. In addition, higher levels of Reg IV mRNA was consistently scored in regions with more severe dysplasia within the same adenoma sample displaying varying degree of dysplasia. The strongest staining was seen within carcinomoutous areas of the 12 adenoma cases (p=0.002). Our results support that overexpression of Reg IV may be an early event in colorectal carcinogenesis. Detection of Reg IV overexpression may be useful in the early diagnosis of carcinomatous transformation of adenoma.

Introduction

Colorectal cancer is one of the most common cancers and is the second leading cause of cancer death for men and women [1], [2]. The mortality from colorectal cancer has changed very little over the past 50 years, which emphasizes the need for therapies based on an improved understanding of the molecular changes that underlie colorectal cancer [3]. As an initial step to understand the gene expression differences between normal colorectal mucosa and adenoma, we have created a profile of genes with altered mRNA levels in primary colorectal adenoma by suppression subtractive hybridization (SSH) [4]. A subtracted cDNA library (A-N library) was constructed from extracts of normal mucosa and adenoma tissue from a single female patient, who suffered from adenocarcinoma with villous adenoma displaying mild-to-moderate dysplasia. Reverse Northern blot showed 75% clones of the A-N library (adenoma and paired normal mucosa) were up-regulated in adenoma compared with paired normal mucosa. A large scale sequencing analysis identified Reg IV in 13 of the 109 clones derived from the library [5]. Previous studies have identified Reg IV from an inflammatory bowel disease library and mRNA expression of Reg IV was significantly up-regulated upon mucosa injury from active Crohn's disease or ulcerative colitis [6]. Its role in colorectal adenoma, however, has not been reported.

Other members of regenerating gene family members, Reg Iα and Reg Iβ, have been indicated to play important roles in colorectal carcinogenesis [7]. Overexpression of Reg I is a good marker for digestive tract mucosa at risk for development of cancer. Reg I plays a role in the early stages of carcinogenesis, probably via a cell-proliferative effect. Because Reg IV showed homology to Reg I, we hypothesize that it might have a similar role in colorectal carcinogenesis. There have been no reports on the involvement of Reg family in adenoma so far. We firstly reported its higher expression in colorectal adenomas. We were interested in the expression pattern of Reg IV in transition from normal mucosa to adenoma and eventually to adenocarcinoma.

In this study, we determined the expression level of Reg IV in colorectal adenoma and carcinoma as compared to that of paired normal tissues. Our results show that Reg IV is overexpressed in human colorectal adenoma and carcinoma, especially in adenoma with carcinomoutous transformation.

Section snippets

Tissue collection and processing

Normal colon tissue samples as well as adenoma (n=12) and concurrent carcinoma samples (n=10) were obtained from patients undergoing surgery for their colorectal tumors. Normal tissue was taken from the same patient at a distance of at least 10 cm from the tumor site. Tissue samples were frozen in liquid nitrogen immediately on surgical removal and stored at −80 °C until ready for total RNA extraction.

Fresh tissues of 32 colorectal adenomas were obtained by endoscopy and biopsy for in situ

Identification of Reg IV overexpression in colorectal adenoma and adenocarcinoma using RT-PCR

By semi-quantitative RT-PCR with these primers, Reg IV was shown up-regulated in 12/12 adenomas and 9/10 carcinomas when compared to paired normal mucosa (Fig. 1). The ratios of intensities of Reg IV/ G3PDEH or β-actin were calculated (Fig. 2). There are significant differences of Reg IV mRNA between normal mucosa and either adenoma (p=0.001) or carcinoma (p=0.021). There is no significant difference of Reg IV mRNA between adenoma and its concurrent carcinoma (p=0.075) (Table 3).

Northern blot analysis

Northern blot

Discussion

Most colorectal cancers are thought to develop through the ‘adenoma-to-carcinoma sequence’ model [10], in which adenomas are recognized as precursor lesions of the vast majority of colorectal cancers. Identification of the adenoma–carcinoma sequence with its corresponding molecular genetic alterations will significantly increase our understanding of the pathogenesis of colorectal carcinoma. However, since most of adenomas are very small and the fresh tissues of adenoma are difficult to be

Acknowledgements

This study was supported by a grant from National Natural Science Foundation of People's Republic of China (No. 30200333). We thank Jun Luo, PhD, Johns Hopkins University, and Dr Welch, Loma Linda University, who are very nice to provide help to edit this manuscript.

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