Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells - PubMed (original) (raw)

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

Yong Li et al. World J Gastroenterol. 2002 Apr.

Abstract

Aim: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells.

Methods: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products.

Results: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h.

Conclusion: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

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Figures

Figure 1

Figure 1

Schematic diagram of preparing representations for cDNA RDA based on cDNA libraries. Using _Bam_H I alone (A) or _Bam_H I and Xho I together (B) to digest library DNAs. Black boxes represent insert cDNAs of cDNA libraries. White boxes represent two arms of cDNA library vector (λZAP II vector). “B” epresents _Bam_H I and “X” Xho I.

Figure 2

Figure 2

Agarose gel electrophoresis of digestion products of Alli823 (lanes 1, 3, 5) and BGC823 (lanes 2, 4, 6) cDNA library DNAs digested with _Bam_H I at 37 °C for 2 (lanes 1, 2), 4 (lanes 3, 4), and 12 h (lanes 5, 6) respectively. The digestion products of Alli823 (lane 7) and BGC823 (lane 8) library DNAs digested with _Bam_H I and Xho I together (A fragment, 850 bp in size, appeared in the digestion products). λPhage/_Hin_d III size marker (lane M).

Figure 3

Figure 3

Agarose gel electrophoresis of amplicons derived from different enzyme digestions. A: The amplicons obtained by using _Bam_H I to digest Alli823 (lane 1) and BGC823 (lane 2) cDNA library DNAs respectively; B: The amplicons obtained by using _Bam_H I and Xho I together to digest Alli823 (lane 1) and BGC823 (lane 2) library DNAs respectively. 1 kb size marker (lane M).

Figure 4

Figure 4

A: Agarose gel electrophoresis of reamplified difference products SH1-4 (lanes 1-4 respectively). PCR product of GAPDH (400 bp in size) used as quantity control (lane G); B: Slot blots analysis showing differentially expressed cDNAs. Reverse transcription products (first stranded cDNA) of total RNA of BGC823 and Alli823 cells used as probe respectively; C: The degree to which each cDNA was up-regulated.

Figure 5

Figure 5

Sequences of two novel ESTs (SH1 and SH4) isolated by cDNA RDA based on cDNA libraries.

Figure 6

Figure 6

Northern blotting result showing the expression level of calcyclin gene in human gastric cancer cell lines BGC823, MGC803, PAMC82, SGC7901 and MKN45 (lanes 1-5) respectively.The arrow showing the novel transcript derived from the recombinant gene merged from calcyclin gene and HNRPA0 gene.

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