Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells

Abstract

Aim: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC).

Methods: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using lambdaZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using Bam H I cutting-site and abundant cDNA messages provided by the libraries. Northern blot analysis was applied to identify the obtained difference products.

Results: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptor alpha (FRalpha) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRalpha gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h.

Conclusion: The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA. Up-regulation of FRalpha gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Figure 1

Schematic diagram of cDNA RDA based on cDNA libraries, illustrating the subtractive hybridization and amplification steps following the representation stage and preparation of amplicons. Gray boxes (24-mer, 12-mer) represent R (or J, N)-Bam adapters

Figure 2

Morphological changes of human gastric cancer cell line BGC823 after Allitridi treatment. A: The parental cells (used as control); B: Neurite-like structures outgrew from the cell bodies and formed interconnections after exposure to 25 mg•L⁻¹ Allitridi for 96 h (5 × 40).

Figure 3

Insert sequences released from randomly selected clones (Lanes 1-12) of parental cell (BGC823) cDNA library (A) and Allitridi-treated cell (Alli823) cDNA library (B) by digestion with EcoR I and Xho I. λPhage/Hind III size marker was indicated in Lane M.

Figure 4

Agarose gel electrophoresis of difference products obtained after the first, second and third round hybridizations. BamH I-digested library DNA extracted from Alli823 and BGC823 cDNA libraries respectively (Lanes 2, 3); Amplicons of cDNA population prepared from Allitridi-treated and parental groups (used as tester and driver respectively) (Lane 4, 5); First, second and third difference products respectively (Lanes 6, 7, 8); 1 kb size marker (Lane 1) and PUC18/Hae III DNA size marker (Lane 9).

Figure 5

Northern blot analysis of FRαgene and calcyclin gene expression in BGC823 cells. FRα mRNA (A) and calcyclin mRNA (B) level was elevated after exposure to 25 mg•L⁻¹ Allitridi for 72 h.