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 λZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using BamH 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 receptorα (FRα) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRα gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72 h.

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 FRα gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Key Words: $[Keywords]

INTRODUCTION

Gastric cancer is common in China[1-38]. Garlic has been used for over three thousand years in traditional Chinese medicine and is widely used in preventing manifold diseases all over the world. Epidemiological studies indicated that garlic in diet had remarkable antithrombotic, hypolipidemic, hypocholesterolemic and antineoplastic effects[39-41]. Both Chinese and Italian scholars reported protective efficacies of garlic in cases of human gastric cancer (HGC)[40]. Allitridi is an important constituent of garlic oil mainly containing diallyl trisulfide (DATS) and diallyl disulfide (DADS), which are widely used in cancer chemoprevention and anti-cardiovascular disease research[42]. Our data also indicated that Allitridi could arrest HGC cell line BGC823 in G1 phase and induce morphological changes of such cells (data not shown). Recent studies suggested that garlic may exert its chemopreventive effects by modulating lipid peroxidation, enhancing the levels of glutathione (GSH), glutathione peroxidase (GPx), glutathione S-transferase (GST) and NAD (P)H: quinone oxidoreductase (NQO)[43,44]. However, a definitive molecular mechanism of anticarcinogenic activities of garlic has not been established.

The cDNA Representational Difference Analysis (cDNA RDA) method with high specificity is widely used in identifying differentially expressed genes in different developmental stages and cell cycle phases or in following the progression of expression in proceeding with a particular event, for example, stimulation of cells with a growth factor[45-47]. Enough double stranded cDNA will be available in cDNA libraries for subtractive cloning. In order to detect the feasibility of cDNA RDA based on cDNA libraries and to identify Allitridi inducible expression genes at the same time, Allitridi-treated and parental BGC823 cell cDNA libraries were constructed respectively by using λZAP II vector. We further took advantage of restriction site harbored in the vector and replaced four-cutting restriction endonuclease Dpn II used in classical cDNA RDA with six-cutting enzyme BamH I to select representations and to perform cDNA RDA following the classical procedure.

MATERIALS AND METHODS

Cell culture and cDNA library construction

The HGC cell line BGC823 was grown at 50 mL•L⁻¹ CO₂ in DMEM medium supplemented with 50 mL•L⁻¹ fetal calf serum. Allitridi used in our experiments contained 97.98% DATS and 0.85% DADS. BGC823 cells were incubated in medium containing 25 mg•L⁻¹ Allitridi for 72 h. Total RNA isolated from parental and Allitridi-treated BGC823 cells was separated by using guanidinium thiocyanate acid-phenol-chloroform method, followed by purification of poly (A)+ mRNA with the Poly (A)+ Quik mRNA Isolation Kit (Stratagene). 5 μg mRNA were collected and λZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene) was used to construct two cDNA libraries which were Allitridi-treated and parental cell cDNA libraries respectively.

Establishment of cDNA RDA based on cDNA libraries

By using Allitridi-treated BGC823 (Alli823) library DNA as tester and parental cell (BGC823) library DNA as driver, we performed cDNA RDA following the protocol supplied by classical cDNA RDA. Library DNAs (40 μg) of the two cDNA libraries were prepared respectively and digested with BamH I, phenol extracted, ethanol precipitated and resuspended in 20 μL 1 × TE. 12 μL (about 24 μg) digested DNAs were then conjugated to 18 μL R-Bam-24 (10 μmol•L⁻¹) and R-Bam-12 (10 μmol•L⁻¹) adapters. PCRs were set up to generate the initial representations by using R-Bam-24 as a primer. Afterward the R-adapters were removed from the representation of BGC823 with BamH I to form the driver. Representation of Alli823 was also digested with BamH I and the product was purified using Qiaex resin (Qiagen). This formed the tester, of which 2 μg was conjugated to the J-Bam-12/24 adapters. For the first subtractive hybridization, 0.4 μg J-conjugated tester was mixed with 24 μg driver, and the mixture was allowed to anneal 20 h at 67 °C. The annealing products were used as templates and 25 cycles of PCR were performed to generate the first difference product (DP1). J-adapters on DP1 were changed for N-Bam-12/24 adapters. The ratio for subtractive hybridization was changed for 50 ng∶40 μg and the processes were repeated to generate the second difference product (DP2). To generate the third difference product (DP3), 400 pg J-conjugated DP2 was mixed with 40 μg driver and the process repeated. Sequences of oligonucleotides used in cDNA RDA were as follows: R-Bam-24 5'-AGCACTCTCCAGCCTCTCACCGAG-3'; R-Bam-12 5'-GATCCTCGGTGA-3'; J-Bam-24 5'-ACCGACGTCGACTATCCATGAACG-3'; J-Bam-12 5'-GATCCGTTCATG-3'; N-Bam-24 5'-AGGCAACTGTGCTATCCGAGGGAG-3'; N-Bam-12 5'-GATCCTCCCTCG-3'. The 5’end of 12-mer adapters was dephosphorylated. cDNA RDA based on cDNA libraries is showed schematically in Figure 1.

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

Northern blot hybridization

Total RNA extracted from parental and Allitridi-treated BGC823 cells for 72 h (10 μg for each sample) were fractionated on formaldehyde agarose gel and transferred onto Nitrocellulose filters (S&S Company), then cross-linked by using a UV Stratalinker. Probes (DP3s) were labeled by random primer extension and hybridization carried out in 1 mmol•L⁻¹ EDTA, 0.25 mmol•L⁻¹ Na₂HPO₄ and 70 g•L⁻¹ SDS solution for 16 h at 60 °C. Following hybridization, the filters were washed twice in 1 mmol•L⁻¹ EDTA, 40 mmol•L⁻¹ Na₂HPO₄ and 50 g•L⁻¹ SDS for 25 min at 60 °C and twice in 1 mmol•L⁻¹ EDTA, 40 mmol•L⁻¹Na₂HPO₄ and 10 g•L⁻¹ SDS for 25 min at 60 °C again. Filters were exposed to a phosphor screen for 48 h and analyzed.

Sequencing and Database Searching

Final difference products were amplified and cloned into pGEM-T Easy Vector (Promega). Double stranded plasmid DNA was prepared using miniprep columns (Promega) and sequenced with Ultra VGI 1280 (applying User Manual version 3.0). Resulting sequences were compared to the GenBank database by using the BLAST program.

RESULTS

Parental and Allitridi-treated BGC823 cell cDNA libraries

Morphological changes were observed in BGC823 cells after exposure to 25 mg•L⁻¹Allitridi for 96 h. The cells after treatment dramatically changed to an elongated shape with filamentous protrusions. Neurite-like structures outgrew from the cell bodies and formed interconnections (Figure 2). Titer for Allitridi-treated BGC823 (Alli823) cDNA library was 1.1 × 10¹³ clones•L⁻¹, and titer for parental cell (BGC823) cDNA library 2.0 × 10¹³ clones•L⁻¹. The size of insert sequences for those two cDNA libraries ranged from several hundred base pairs to several kilobase pairs (kb), and the average length was about 2 kb (Figure 3).

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.

cDNA RDA based on two cDNA libraries

cDNA RDA based on cDNA libraries is an extension of classical cDNA RDA in application and follows the protocol of the latter. λZAP II vector used to construct cDNA libraries has three BamH I restriction sites with one site located very near the 5'-end of insert sequence. Thus BamH I adapters can be introduced into the 5'-end of insert sequence. The shortest fragment cleaved from library vector DNA is 6.4 kb in size, unable to compete with insert sequence during subsequent PCR amplification. Therefore, we could replace four-cutting restriction endonuclease Dpn II used in classical cDNA RDA with six-cutting enzyme BamH I to select representations and perform cDNA RDA. With same copies three distinct fragments appeared in the digestion products of library DNA (about 22.1, 11.3 and 6.4 kb in size respectively). We saw a stepwise reduction of complexity of the products in each successive difference product, until two clear bands with little background were visible by ethidium staining in the third difference products (DP3). The larger band named as DP3-1 is about 340 bp and the shorter one DP3-2 about 180 bp in size. The amplicons and difference products obtained after the first, second and third round hybridization-amplification steps are showed in Figure 4.

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).

Identification of differentially expressed genes in parental and Allitridi treated BGC823 cells

The third difference products, DP3-1 and DP3-2 were cloned into pGEM-T Easy Vector and resulting sequences were compared to the GenBank database using the BLAST program. Results showed that: DP3-1 had remarkable homology with FRα mRNA as high as 98%; DP3-2 sequence showed a remarkable homology of 97% with human calcyclin mRNA. Northern blot results using DP3-1 and DP3-2 as probes showed a 4-fold increase in FRα gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72 h (Figure 5).

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.

DISCUSSION

cDNA RDA based on cDNA libraries shares the chief merits of classical cDNA RDA, and has in addition several distinct advantages: Firstly, enough double stranded cDNAs needed for subtractive hybridization can be obtained from cDNA libraries and thus expand the application of cDNA libraries. Next, with same copies, three distinct fragments (about 22.1, 11.3 and 6.4 kb in size respectively) appear in the products of library DNA digestion with BamH I and act as good marks indicating the appropriate digestion degree for library DNA and thus increasing the specificity and reproducibility of cDNA RDA. Thirdly, by replacing four-cutting enzyme Dpn II with six-cutting enzyme BamH I to digest library DNA, longer difference products can be obtained; this will increase the specificity of subsequent Northern blot analysis, Database homology searching and library screening. Finally, through replacing or combining BamH I cutting site with other restriction sites (for example, EcoR I or Xho I) harbored in library vector, more difference products including low abundant messages can be obtained.

FRα functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate and 5-methyltetrahydrofolate to the cytoplasm of the cell[48]. Deficiency of folate has already been linked with increased incidence of neural tube defects and of cardiovascular disease through elevated plasma homocysteine levels[49]. Recent studies showed that folate deficiency may cause an imbalance in DNA precursors, uracil misincorporation into DNA, and chromosome breakage. By reducing intracellular S-adenosylmethionine (SAM), it can also cause global DNA hypomethylation, leading to inappropriate activation of proto-oncogenes and induction of malignant transformation[50-52]. Folate deficiency is reported to be associated with increased risk of colorectal carcinoma and breast cancer[53-55]. Our findings suggested that up-regulation of FRα gene expression by Allitridi may compensate for folate deficiency and correspond with the preventive efficacy of garlic against HGC as well as other human diseases. Calcyclin gene, as a member of the S100 family for generating small calcium-binding proteins, was reported to be a cell cycle-dependent gene and to be associated with cell differentiation[56-59]. Our results showed that calcyclin gene expression in BGC823 cells was up-regulated by Allitridi treatment before the neurite-like structures generated in the cells, and this would suggest the impact of garlic on regulating calcium metabolism and the involvement of calcyclin gene in HGC cell differentiation.

In conclusion, cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with the abundant cDNA messages in cDNA library, and thus expands the application of cDNA library and increases specificity of cDNA RDA. Our findings suggest that up-regulation of FRα gene and calcyclin gene expression induced by Allitridi may play an important role in HGC cell differentiation and may provide valuable molecular evidence for demonstrating the important preventive efficacy of garlic against HGC.