Cloning of novel LERGU mRNAs in GPR30 3′ untranslated region and detection of 2 bp‐deletion polymorphism in gastric cancer

Abstract

The improved IGCR (In-Gel Competitive Reassociation) method was applied to the analysis of human gastric cancer genomic DNA to identify its alterations, and it appeared that the IGCR library contained a fragment of 3′ ′ ′ ′-untranslated region (3′ ′ ′ ′ UTR) of G-protein coupled receptor 30 (GPR30) mRNA. When we searched genomic DNA pairs of gastric cancer patients with this IGCR clone, we found the deletion polymorphism with or without 2 bp (Cytosine and Thymine; CT). We confirmed the existence of a novel mRNA in GPR30 3′ ′ ′ ′UTR by northern blotting, cloned this novel mRNA and named it Leucine Rich Protein in GPR30 3′ ′ ′ ′UTR (LERGU). The EST database search gave one alternative splicing form in this 3′ ′ ′ ′ UTR, which was named as LERGU-1. A novel alternative splicing form of this mRNA was also identified from the stomach total RNA, which was named LERGU-2. The LERGU mRNA was also detected in eight gastric cancer cell lines, but GPR30 mRNA scarcely existed. Furthermore, we detected the 2 bp-deletion form in genomic DNAs and mRNAs derived from gastric cancers, but not in other type cancers. Since the 2 bp-deletion position on LERGU corresponds to its alternative splicing site, this deletion may produce a frame-shifted protein. G astric cancer is the most frequent malignancy of the gastrointestinal tract in Japanese and certain South-east Asian populations, and one of the leading causes of cancer mortality in the world. Genetic aberrations associated with gastric cancers have been reported in the gene encoding Ecadherin, p53, the transforming growth factor-β receptor and so on, but it is believed that a much greater number of genetic changes could account for the phenomena.(1-4)The original In-Gel Competitive Reassociation (IGCR) technique is one of the DNA subtractive hybridization techniques, which consists of the size separation of double strand DNA fragments by electrophoresis, the denaturation in gel with an alkaline buffer and the renaturation by exchanging the buffer. (5)(6)(7)(8)(9)(10)(11) This technique had achieved the sufficient enrichment of the DNA sequences only in the target to detect small aberrations between two DNA populations such as recombination junctions in extrachromosomal DNA, (9) restriction fragment length polymorphism in the genome, (10) and methylation sites in the genome.(6) We have improved the IGCR technique to overcome several disadvantages such as time-consuming and complex process with poor reproducibility. In the denaturing step, we employed a heat denature method instead of the alkaline denature protocol. In addition, to reduce the genomic complexity and prevent the amplification of undesirable polymerase chain reaction (PCR) bands, smaller fragments were cut off with DNA binding resins in the purification of Mse I cut fragment mixture, and a 'competitor' was introduced to the IGCR technique. Our improved IGCR method efficiently detected Epstein-Barr virus (EBV) genomic DNA fragments, which were contained only in a tester DNA of gastric cancer. (12) G protein coupled rece...

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