Human colon cancer cell lines, Caco-2, HT-29 and LoVo, were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI-1640 (Sigma, St. Louis, MO, USA) and antibiotics, supplemented with 10% fetal bovine serum and 100 U/ml of penicillin and streptomycin at 37°C in a humidified 5% CO₂ atmosphere and stored at −20°C.

By standard methods, genomic DNA was isolated by digestion with Proteinase K followed by phenol:chloroform (1:1) extraction and ethanol precipitation from Caco-2, HT-29 and LoVo cell lines. Total-RNA extraction was performed using TRIzol reagent (Invitrogen Life Technologies, Groningen, The Netherlands) according to the manufacturer’s instructions. To avoid the genomic DNA contamination, RNA samples were treated with DNase I (RNase-free) (Takara Bio, Inc., Shiga, Japan) for 20 min at 37°C. The enzyme was heat inactivated and the RNA was ethanol precipitated following phenol chloroform extraction. Following precipitation, the RNA was resuspended in 10 μl of diethylpyrocarbonate (DEPC)-treated water and stored at −80°C.

RNA (5 μg) was used as template in the first strand complementary DNA (cDNA) synthesis in a 20 μl reaction volume, which consisted of oligo-dT primer (0.2 μg/reaction), dNTP (0.5 mM of each), avian myeloblastosis virus reverse transcriptase (AMV RT; Promega Corporation, Madison, WI, USA) (20 U/reaction), RNasin (Takara Bio) (20 U/reaction). The reaction was incubated at 42°C for 60 min, followed by heating at 99°C for 5 min. Reactions in which pure water replaced the RNA were used as RT-negative controls.

All primers and amplification conditions used in this study are listed in Table I. Single round β-actin amplification was used to demonstrate RNA integrity and the RT performance. The reaction mixture consisted of the template (50–100 μg of cDNA), dNTP (0.2 mM of each), Taq DNA polymerase (Promega Corporation) (0.625 U/reaction) and a selected primer pair (20 pmol/primer/reaction) in a total volume of 25 μl. The final products were analyzed by electrophoresis on 2% agarose gels containing ethidium bromide.

Genomic DNA (2 μg) was denatured with 0.3 M NaOH for 10 min at 37°C and mixed with 30 μl of 10 mM hydroquinone (Sigma), 510 μl of 3.0 M NaHSO₃ (pH 5.0; Sigma), covered with paraffin oil, then deaminated in the dark for 16 h at 50°C. To use, 10 mM hydroquinone and 3.0 M NaHSO₃ (pH 5.0) were freshly prepared and mixed. Bisulfite-treated DNA was purified using a Wizard DNA Clean-Up System (Promega Corporation). Subsequently, purified DNA samples were desulfonated with 0.3 M NaOH for 5 min at room temperature, neutralized with ammonium acetate, ethanol precipitated and resuspended in 20 μl Tris-EDTA buffer.

To determine whether the loss of DLC-1 expression was associated with promoter hypermethylation, we used MSP to detect the methylation status of the 5′CpG island. Bisulfite-treated DNA was amplified by PCR with methylation status-specific primer pairs. The primer sequences for the MSP and amplification conditions used in this study are listed in Table I. The sodium bisulfite reaction converts unmethylated cytosine in DNA to uracil while leaving the methylcytosine unchanged, so that methylated and unmethylated alleles can be distinguished by MSP. Bisulfite-treated DNA (8 μl) was subjected to PCR using bisulfite treated DNA PCR primer (the primer can amplify both the sequences methylated and unmethylated) (Table I), a 292 bp fragment, particularly for the upstream region of the basic promoter of DLC-1 was amplified and PCR product was sequenced.

In order to construct two plasmids, two pairs of shRNA sequence were designed, one is according to the DLC-1 sequence in the GenBank (AF035119), as pGCsiDLC-1, another sequence of shRNA has no homology with human sequence, as a control group. Each pair contained a unique 19-nt double-stranded sequence that is separated by a loop of 9-nt sequence (ttcaagaga). Following purification and restriction digestion, the oligonucleotides were ligated into plasmid pGCsi with polymerase III U6 promoter, purchased from GeneChem Inc, China. After amplifying the plasmids into E.coli, the selected clones with the shRNA insert were selected and purified with plasmid purification kit (Promega Corporation). The oligonucleotide sequences of siRNA are listed in Table II.

To generate DLC-1 transfected DLC-1 cells, 3 μg of plasmid DNA were transfected in 1×10⁵ cells in a 60-mm dish using Lipofectamine^® 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Selection for transfected cells was carried out in a medium containing 400 μg/ml G418 (Geneticin; Life Technologies, Inc., USA).

Experiments were conducted in three groups, the pGCsiDLC-1, the mock and the control group. Following transfection for 24 and 48 h, LoVo cells (10×10⁶) were harvested and lysed in 60 μl cell lysis reagent, containing 50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 100 μg/ml phenylmethylsulfonyl fluoride (PMSF) and 1% TritonX-100. Equal amounts of total protein were separated by 5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF). After blocking in a 5% non-fat dry milk solution in washing buffer containing 10 mmol/l Tris (pH 7.6), 150 mmol/l NaCl and 0.05% Tween-20, membranes were incubated for 1 h at room temperature with human monoclonal anti-DLC-1 (1:200; clone 3; BD Biosciences Pharmingen, San Diego, CA, USA) as primary antibodies, washed 3 times, incubated again with bovine anti-mouse IgG (1:2,500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as second antibody. β-tublin (1:300) staining served as the internal standard for all membranes.

The transfected cells were plated in 96-well microtiter plates at a density of 1×10⁴ cells/well. Cells were further cultured for 24, 36 and 48 h respectively, after which the medium was replaced with 100 μl of fresh serum-free medium containing 50 μg MTT. Four hours later, the color reaction was quantified with an ELISA plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. The experiment was repeated three times independently.

The cells stably transfected with DLC-1 and control cells were seeded into six-well plates at a density of 1×10⁴ cells/well, the culture medium contained 500 μg/ml G418 (Geneticin; Life Technologies, Inc). After culturing for two weeks, the G418-resistant colonies were washed twice with phosphate-buffered saline (PBS), stained with Giemsa, and the colony formation efficiency was tested. For soft agar colony assays, the pGCsil-DLC-1, the mock and the control group were mixed with RPMI-1640 complete medium containing 0.4% agar and placed over 0.6% of basal agar in six-well plates. Cells were grown for two weeks and colonies were visualized microscopically and photographed.

RT-PCR was performed in Caco-2, LoVo and HT-29 cells to examine the expression of DLC-1 in colon cancer cell lines. To validate the PCR results, the same cDNA was amplified for β-actin. All the cell lines were positive for β-actin, which indicates both RNA preparation and cDNA synthesis were successful. Caco-2 and LoVo colon cancer cell lines were all positive for DLC-1 mRNA and a band of 262 bp, but low levels or absence of DLC-1 mRNA were observed in HT-29 cells (Fig. 1). In order to detect the protein expression of DLC-1 in these cells, western blot analysis was performed and the results were in agreement with the results obtained from RT-PCR. Loss of DLC-1 protein expression was detected in HT-29, Caco-2 and LoVo revealed a fragment of 123 kDa (Fig. 2).

To determine whether the loss of DLC-1 expression was mediated by promoter hypermethylation in human colon cancer cell lines, MSP was performed on Caco-2, LoVo and HT-29 cells. Representative results are shown in Fig. 2. All cells exhibited the bands that correspond to either unmethylated (178 bp) or methylated (172 bp) CpG island. PCR with methylated primers showed that HT-29 has methylated forms of DLC-1, which showed no expression or low levels of DLC-1 expression. On the other hand, the unmethylated band was observed in Caco-2 and LoVo cells (Fig. 3). To further confirm the methylation status as well as to determine whether the loss of DLC-1 expression was mediated by promoter hypermethylation in colon cancer cell lines, a 292 bp fragment of the DLC-1 promoter region was sequenced on LoVo and HT-29 cells. In agreement with the results obtained from MSP study, 6 CpG dinucleotides in LoVo cell were unmethylated, whereas HT-29 cells showed extensive hypermethylation at these CpG dinucleotides (Fig. 4).

The knockdown efficiencies of DLC-1 specific shRNAs in LoVo cells were analyzed by semiquantitative RT-PCR. Relative DLC-1 mRNA levels were normalized by internal control β-actin after transfection. Compared with the mock and the control group, the DLC-1 mRNA expression was reduced in the pGCsiDLC-1 LoVo cells, but it was not different in the mock and the control group (Fig. 5). The expression of DLC-1 protein can reflect post-transcription level of the DLC-1 gene. The knockdown efficiencies of DLC-1 protein in LoVo cells were analyzed by western blot analysis. β-tubulin protein expression was used to normalize the expression of DLC-1. The results showed DLC-1 protein expression to be downregulated following transfection, particularly after 48 h (Fig. 6).

To determine whether decreased DLC-1 expression may change the rate of LoVo cell proliferation, DLC-1 RNAi recombinant vector was transfected into LoVo cells to downregulate DLC-1 expression and MTT assay was carried out to test cell proliferation. Fig. 7 shows that after the cells were transfected with pGCsiDLC-1, the proliferation rate of the LoVo cells increased significantly at 48 h compared to the control and the mock group (P<0.01). No significant difference of growth inhibition was observed between the mock and the control group. To further assess the time of DLC-1 inhibitory effects, we tested the effect of DLC-1 on colony formation of LoVo cells; cells transfected with pGCsiDLC-1 and control vector were cultured in G418 for two weeks and the colony number was counted on Giemsa-stained dishes. A significant increase both in colony number and size of G418-resistant colonies was observed in transfection LoVo cell lines compared to the control group (Fig. 8A) and for soft agar assays, cells transfected with pGCsiDLC-1 formed colonies more quickly than the mock and the control group (Fig. 8B).