Decreased expression of SPRR3 in Chinese human oesophageal cancer
Bao-Sheng Chen,
Ming-Rong Wang1,
Yan Cai,
Xin Xu,
Zhi-Xiong Xu,
Ya-Ling Han and
Min Wu
National Laboratory of Molecular Oncology, Cancer Institute, CAMS, PUMC, Beijing 100021, China
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Abstract
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cDNA fragments that were differentially expressed between human oesophageal carcinomas and matched normal adjacent mucosa were isolated using an improved mRNA differential display technique. One of them was identified as the 3'-untranslated region of SPRR3 and was homologous to the esophagin cDNA. Northern blot, dot blot and reverse transcriptionpolymerase chain reaction (RTPCR) analyses revealed that SPRR3 expression was lost in three cell lines of oesophageal carcinoma and was dramatically decreased in 54 out of 57 primary oesophageal carcinomas compared with adjacent normal mucosa. Esophagin has been shown to be down-regulated in western oesophageal carcinomas. The data suggest that esophagin is probably the protein product of the gene SPRR3 and that altered mRNA expression of SPRR3/esophagin is a frequent event in the development of Chinese oesophageal cancer.
Abbreviations: RTPCR, reverse transcriptionpolymerase chain reaction; SPRR, small proline-rich protein.
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Introduction
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The molecular basis underlying the transformation of normal oesophageal mucosa to squamous carcinoma of the oesophagus is not well understood. Genetic changes that have been identified in oesophageal cancer include point mutations of the genes encoding p53 and p16, amplification of the Int-2, Hst-1, cyclin D and epidermal growth factor receptor (EGFR) genes and loss of heterozygosity of retinoblastoma protein (Rb) (1). Abraham et al. (2) recently reported a novel gene (named esophagin) that was expressed in normal squamous mucosa and undetectable in oesophageal tumors. They showed that this gene was associated with the differentiation and the morphogenesis of oesophageal epithelia. In the present study, we have isolated and characterized cDNA fragments differentially expressed between oesophageal carcinomas and adjacent normal mucosa using the mRNA differential display technique. One of the cDNA fragments was identical to that for SPRR3, a small proline-rich protein. We found that esophagin also shared great homology to SPRR3. SPRR3 is one of the cornified envelope precursor proteins whose expression is restricted to terminally differentiating squamous cells, preferentially located at the cell periphery. SPRR3 is abundant in oral and oesophageal epithelium (3).
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Materials and methods
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Sample collection
Fifty-seven pairs of oesophageal carcinomas and matched adjacent normal mucosa were obtained at surgery. Eleven pairs were obtained from a high-incidence area (Anyang, Henan Province, China), and the others from a low-incidence area (Beijing, China). The samples were frozen in liquid nitrogen until DNA and RNA could be purified.
Cell lines and cell culture
Three oesophageal carcinoma cell lines, EC8712 and EC9706 and EC109, were established in our laboratory (4,5). These cell lines were maintained in M199 medium with 15% fetal bovine serum and cultured at 37°C, 5% CO2.
RNA extraction and cDNA synthesis
Tumour samples were grossly dissected to trim away non-tumour tissues. Matched adjacent normal mucosa was separated from underlying muscle layers. Total RNAs were prepared using Trizol reagent (Gibco) according to the manufacturer's instructions. Fifty micrograms of total RNAs from tumour tissues and adjacent normal epithelia were treated with DNase I for 3040 min at 37°C, extracted with phenol/isopropanol, precipitated in 3 M sodium acetate (pH 5.2) overnight at 4°C, and dissolved in DEPC-treated water. Five micrograms of each RNA was reverse transcribed with 200 units of Superscript II reverse transcriptase (Gibco) in the presence of 50 µM 3'-anchored oligo(dT) primer in 20 µl reverse transciptionpolymerase chain reaction (RTPCR) buffer [200 mM TrisHCl pH 8.4, 500 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM dNTP], at 42°C for 50 min, followed by heating to 70°C for 15 min to inactivate reverse transcriptase.
mRNA differential display
mRNA differential display was performed with a modification of the procedure described by Liang and Pardee (6). In brief, PCR amplification was done using 1 µl of the cDNA, primed with mixed anchored primers (GT15N, where N = A, C and G, equal number of moles) and 10-mer arbitrary primers (Operon). The cycle parameters were 94°C for 4 min, 39°C for 4 min, 72°C for 2 min, and then 35 cycles of 95°C for 20 s, 39°C for 2 min, 72°C for 1 min. A final extension was carried out at 72°C for 5 min. The resultant products were electrophoresed on an 8% polyacrylamide gel and stained with silver. Identified bands were excised from the gel, and the cDNA was recovered by incubation at 37°C in 0.1x TE overnight.
Cloning and DNA sequencing of differential display cDNA
PCR-amplified cDNA were cloned into the pGEM-T easy vector (Promega). Plasmid DNA was prepared by using Wizard Miniprep Purification System (Promega). The sequence reactions were performed by TaKaRa Corp. (Dalian City, China).
Northern blot analysis
Total RNA was denatured in the presence of 50% (v/v) formamide and 2.2 M formaldehyde and subjected to electrophoresis on 1.2% agaroseformaldehyde gels. Following electrophoresis, the RNA was transferred to nylon membranes (Bio-Rad) by capillary transfer in 10x SSC. Northern blots were prehybridized for 1 h in 1 M EDTA, 0.25 M Na2HPO4 (pH 7.2) and 7% sodium dodecyl sulfate (SDS) at 65°C. The probe consisted of a 303 bp fragment synthesized using the Prime-a-Gene labelling system (Promega) in the presence of [32P]dATP and [32P]dCTP. Hybridization was carried out in hybridization solution at 65°C for 1624 h and blots were washed first for 20 min in 1 mM EDTA, 40 mM Na2HPO4, 5% SDS at 65°C, and then in 1 mM EDTA, 40 mM Na2HPO4, 1% SDS. The membrane was exposed to X-ray film at 70°C for 16 h. To ensure equality of loading of the RNA samples, blots were also probed with a random-primed human ß-actin cDNA.
Dot blot analysis
Total RNA (10 µg) with formaldehyde and 20x SSC was denatured at 68°C for 15 min then transferred to nylon membrane (Bio-Rad). Hybridization and washings were performed as described for northern blotting.
RTPCR detection
Primers were designed based on the sequence of SPRR3. The upstream primer was 5'-CTTCTCTGCACAGCAGGTCC-3' and the downstream primer 5'-AGCAATTTAATGAGGGAAGAGC-3'. Five micrograms of total RNA was used to synthesize the first strand of cDNA using Superscript II (Gibco). The PCR reaction was carried out with 1 unit Taq polymerase in 15 µl PCR buffer including 0.5 µl cDNA template, 200 mM TrisHCl pH 8.4, 500 mM KCl, 1.5 mM MgCl2, 200 mM dNTPs and 0.5 µM primer. After a 4 min denaturation at 94°C, PCR was performed for 25 cycles (each consisting of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min) followed by a 72°C elongation step for 6 min. Five microlitres of each PCR product was electrophoresed on a 1.5% agarose gel.
Forty-three pairs of surgical samples and three oesophageal carcinoma cell lines (EC9706, EC8712 and EC109) were assayed. We amplified GAPDH as a control with the same templates. The upstream primer of GAPDH was 5'-GACCACAGTCCATGCCATC-3' and the downstream primer 5'-ACCAGGAAATGAGCTTGACA-3'. The PCR products of SPRR3 and GAPDH are 860 and 416 bp, respectively.
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Results
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Identification of the SPRR3 gene by mRNA differential display
With the arbitrary primer OPA and GT15N, several differentially expressed bands were displayed in all three pairs of samples (Figure 1
). One of them was predominantly expressed in normal oesophageal mucosa but was undetectable in tumour tissues. This cDNA was recovered from silver-stained gel, reamplified and cloned into the pGEM-T easy vector. Sequencing analysis showed that the cloned cDNA fragment (318 bp) was identical to the SPRR3 gene and homologous to the esophagin cDNA.

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Fig. 1. mRNA differential display of oesophageal carcinomas (T) and adjacent normal mucosa (N) with the arbitrary primer OPA1 and the anchored GT15N. Arrow indicates differentially expressed bands. M, Marker ( X174/HaeIII).
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Differential expression of SPRR3: confirmation by northern and dot blotting
We performed northern blot and dot blot analyses to verify that the SPRR3 gene was indeed down-regulated in oesophageal cancer. Northern blotting showed that SPRR3 expression was decreased in six oesophageal cancer tissues compared with the matched adjacent normal squamous epithelia. Dot blotting revealed that SPRR3 was down-regulated in 10/10 oesophageal cancer tissues (Figure 2
).

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Fig. 2. Northern blot and dot blot analyses of SPRR3 in normal epithelia (N) and matched tumour tissues (T). Ethidium bromide-stained total RNA and ß-actin hybridization are shown as loading control for the northern blot and dot blot, respectively.
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Expression of SPRR3 was down-regulated in oesophageal cancer as judged by RTPCR analysis
Forty-three pairs of operative samples and three cell lines of oesophageal carcinoma were analysed. SPRR3 was expressed highly in all the normal tissues but was hardly detectable in the three oesophageal cancer cell lines and in 40 out of 43 tumour tissues, including four adenocarcinomas. In the remaining tumour tissues (three squamous cell carcinomas, all from male patients) SPRR3 expression was not lower than that in the corresponding normal mucosa (Figure 3
).

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Fig. 3. Representative RTPCR analysis of SPRR3. M, Marker ( X174/HaeIII); N, normal epithelia; T, tumor tissues; lane 1, EC9706; lane 2, EC8712.
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Taken together, the results of the northern blotting, dot blotting and RTPCR analyses showed that SPRR3 expression was lost in all three oesophageal carcinoma cell lines tested and dramatically decreased in 54 out of 57 primary oesophageal carcinomas compared with the matched normal tissues. There was no significant difference in gender, age, stage, grade and geographical origin.
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Discussion
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In order to elucidate the molecular mechanisms of oesophageal malignant alteration, we initiated a differential displayPCR study to identify genes differentially expressed between oesophageal squamous carcinomas and normal oesophageal squamous epithelia. One of the cDNA fragments was found to be the 3'-untranslated region of SPRR3, a gene encoding a small proline-rich protein (SPRR). SPRR genes are clustered within a 300 kb DNA segment on chromosome 1q21. They encode a class of polypeptides that are strongly induced during differentiation of human epidermal keratinocytes in vitro and in vivo. This cluster contains approximately two SPRR1 genes, eight SPRR2 genes and a single SPRR3 gene. Because of their highly inducible nature during squamous differentiation and because of the presence of a region homologous to loricrin and involucrin, SPRRs are regarded as precursor proteins of the cornified envelope and as markers of squamous differentiation (79). In 1996, Abraham et al. (2) reported a novel gene named esophagin. They found that this gene was undetectable in oesophageal tumours and displayed varying degrees of reduced expression in dysplastic cells. Esophagin had significant homology to SPRR1 and SPRR2 at the protein level and was localized in the upper and middle layers of normal epithelia. The authors postulated that esophagin expression was strongly associated with oesophageal epithelial differentiation (2).
The cDNA sequence of esophagin was not deposited in GenBank. We compared the cDNA sequence of SPRR3 with that of esophagin described by Abraham et al., and found that they were 99% identical. Because the northern blot analysis did not display two separate hybridization bands, we think that esophagin is the protein product of the SPRR3 gene.
Most of the oesophageal cancers found in China are squamous cell carcinomas. We have analysed 57 pairs of samples, only four of which were adenocarcinomas. Expression of SPRR3 was reduced in 94.7% (54/57) of the carcinomas compared with their adjacent normal mucosa. Among the tumours tested, 21 were diagnosed as stage IIa, six as stage IIb and 30 as stage III; 16, 28 and 13 tumors were classified as grades G1, G2 and G3, respectively. Our results show that loss of SPRR3 expression was common in the three stages and the three grades of oesophageal cancers. There was no significant difference in the rate of SPRR3 down-regulation in the high-incidence area and the low-incidence area. No age- or gender-related differences in expression of SPRR3 were found.
SPRR3/esophagin mRNA was barely detectable in any of the Chinese oesophageal cancer samples examined, as has been seen in western oesophageal carcinomas. The data indicate that down-regulation of SPRR3/esophagin is a frequent event in the development of oesophageal squamous cancer.
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Notes
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1 To whom correspondence should be addressed 
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Acknowledgments
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This work was supported by grants from the Chinese Hi-Tech R&D Program (Z19-02-01-03), Chinese National Ninth 5-year Medical Strategic Science and Technology Plan (96-906-01-22) and State Key Basic Research Grant of China (G1988051205).
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Received February 8, 2000;
revised July 7, 2000;
accepted August 17, 2000.