BT474 [ERBB2(+), TACC1(−)] and MCF-7 [ERBB2(−), TACC1(+)] (22) cancer cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (fetal bovine serum) (v/v) and penicillin (100 U/ml)/streptomycin (100 mg/ml) at 37°C in 5% CO₂, 95% air.

We designed and cloned an shRNA template into a lentiviral vector. A self-inactivating lentiviral vector containing a GFP reporter and a U6 promoter upstream of the cloning restriction sites (HpaI and XhoI) was used. The introduction of oligonucleotides encoding shRNAs between these restriction sites enables the production of the shRNA in vitro. Four coding regions corresponding to targeted human ERBB2 starting at different positions were used. We constructed four shRNA-ERBB2 lentiviral vectors, namely pLL2G-shERBB2-1, pLL2G-shERBB2-2, pLL2G-shERBB2-3 and pLL2G-shERBB2-4, respectively (Table I). Briefly, oligonucleotides were annealed, digested and inserted between the HpaI and XhoI restriction sites of the plasmid vector. Some mutations were introduced in the sense sequence of the hairpin structure to facilitate sequence and avoid destruction by bacteria during amplification in the bacterial host. Correct insertions of shRNA cassettes were confirmed by restriction mapping and direct DNA sequencing. We constructed a vector, pLL2G-shERBB2 (Fig. 1), co-infected with recombinant lentiviral vectors into 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). To detect the interference effects of the target, the expression of ErbB-2 mRNA and protein was determined using real-time PCR and western blotting, respectively. Recombinant and control lentiviral vectors were produced by co-transfecting 293T cells with the lentiviral expression plasmid and packaging plasmids (pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5). Infectious lentiviral vectors were harvested at 48 h post-infection, centrifuged to remove cell debris, and filtered through 0.45-μm cellulose acetate filters. The infectious titer was determined by hole-by-dilution titer assay. The virus titers produced were ~4.9×10⁷ TU/ml.

Cells grown in 6-well culture plates (500,000 cells/well) were treated as required. When the cells were ~80% confluent in complete RPMI-1640 medium, they were infected with the lentiviral constructs. BT474 cells infected with lentiviral-mediated Lenti-shERBB2-eGFP were used as the study cells; cells infected with the negative shRNA (Lenti-sh-control) were used as the negative controls, and cells without infection were considered as the blank controls.

Total RNA was isolated using RNAiso (Takara, Japan). The concentration and purity of RNA were determined using a spectrophotometer. Reverse transcriptase was used to create cDNA for further analyses. Reverse transcriptase and quantitative real-time polymerase chain reaction (RT-PCR) assays were carried out using SYBR Premix Ex Taq II (Perfect Real-Time) (Takara) and real-time PCR amplification equipment. Reverse transcriptase and real-time PCR was carried out according to the instructions provided in the kit. The PCR primers (synthesized by Sangon Biotech, Shanghai, China) used to detect ErbB-2, TACC1 and GAPDH were as follows: ErbB-2, sense strand 5′-CATGTCATCGTCCTCCAGCAG-3′, and antisense strand 5′-TTGACTCTGAATGTCGGCCAA-3′, with a product length of 161 bp; TACC1, sense strand 5′-TGGTCAGAAATCAGCTGGTGC-3′, and antisense strand 5′-GCACAGTGGACCCGTCTTCTT-3′, with a product length of 230 bp; GAPDH, sense strand 5′-CTGCACCACCAACTGCTTAG-3′, and antisense strand 5′-TGAAGTCAGAGGAGACCACC-3′, with a product length of 135 bp. The real-time PCR procedure consisted of two steps: one cycle at 95°C for 10 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 20 sec. The expression of ErbB-2 was determined by normalization of the threshold cycle of these genes to that of the control housekeeping gene (GAPDH). Data were analyzed using the comparative ΔΔCT method.

Whole-cell proteins in various breast cancer cell lines were isolated. The lysates were centrifuged, and the supernatant was collected and stored at −80°C according to the manufacturer’s instructions. Total protein (10 μl) was loaded per well, separated by 10–15% SDS-PAGE, and transferred to polyvinylidene difluoride membranes at 60 V for 1 h at 4°C. The membranes were blocked and incubated with primary antibodies (Bioworld Co., USA; diluted 1:1,000 in TBS-A). The membranes were rinsed thrice with 0.1% Tween 20-PBS for 30 min. The secondary antibodies (Abcam Co., Cambridge, UK; diluted 1:1,200 in TBS-A) were used with Peroxidase-conjugated Affinipure goat anti-mouse IgG (1:8,000) and Peroxidase-conjugated Affinipure goat anti-rabbit IgG (1:8,000) for 1 h at room temperature. The blotted membranes were washed three times with 0.1% Tween 20-PBS for 15 min and three times with PBS for 15 min. The immunoblots were detected using an electrochemiluminescence kit and exposed to the Vilber Fusion FX5 automatic gel imaging analysis system (Vilber, Marne La Vallée, France).

The drug sensitivity assay was carried out using the mitochondrial reduction activity assay. According to the protocol of the CCK-8 assay kit (Dojindo, Kumamoto, Japan), cells grown in 96-well culture plates (8,000 cells/well) were treated as required. The experimental group consisted of cells infected with the lentivirus and the control group consisted of non-infected cells. After a 24-h culture, docetaxel (Aventis Pharma, Guildford, UK) was added at the following concentrations: 0.01, 0.05, 0.25, 0.5, 0.75, 1, 5 and 25 μg/ml. Next, cells in each well were incubated with 10 μl of CCK-8 at 37°C for 4 h. The optical density (OD) for each well was measured at 450 nm using a microplate reader (Bio-Rad Model 550; Bio-Rad Laboratories, Hercules, CA, USA). CCK-8 experiments were repeated three times on different days. The means and standard deviations of the optical density (OD) of the replicates were calculated for each well. The cell inhibitory rate was calculated according to the following equation: Cell inhibitory rate = [1 − (OD experiment − OD blank)/(OD control − OD blank)] × 100%. The 50% inhibition concentration (IC₅₀) of the drug was determined by chartography.

For all measurements as needed, the statistical significance between groups was assessed by one-way ANOVA based on the homogeneity of variance test (SPSS 13.0, SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Four plasmids containing shERBB2 (pLL2G-shERBB2) were coinfected into 293T cells, respectively. GFP expression in the 293T cells was observed under a fluorescence microscope 48 h after infection with pLL2G-shERBB2. According to the results of the western blotting assay, pLL2G-shERBB2-4 was the most effective lentiviral vector and, thus, was used in the subsequent research (Fig. 2).

BT474 cells were infected with the lentiviral vector encoding GFP, resulting in GFP expression in the majority of cultured cells. The infection efficiency was assayed by fluorescence microscopy after cells were infected by the lentivirus for 48 h. A robust infection efficiency of 79% was observed (Fig. 3). The fluorescence gradually weakened and the infected cells began to undergo apoptosis on the fifth day resulting in the gradual decline of the infection rate. BT474 cells that were infected with Lenti-shERBB2-eGFP and non-infected cells co-cultured showed no synergistic growth inhibition effect when treated using the same protocols. We observed that the proliferation of BT474 cells was significantly inhibited after infection with Lenti-shERBB2-eGFP when compared with the Lenti-shcontrol-eGFP and normal BT474 cells (P<0.05). Real-time PCR and western blot analysis showed that the mRNA and protein expression levels of ErbB-2 were lower in the cells infected with Lenti-shERBB2-eGFP than in the cells infected with the Lenti-shcontrol and the normal BT474 cells (P<0.05) (Figs. 4 and 5).

Following lentiviral infection of BT474 cells, the mRNA expression of ErbB-2 and TACC1 was examined by real-time PCR. A significant difference was noted between the silenced and control cells (P<0.05), a reduction in the expression of ErbB-2 was noted. The expression of ErbB-2 mRNA in the BT474 cells was successfully knocked down. On the other hand, the expression of ErbB-2 mRNA showed no significant difference between the blank control group and the negative control-shRNA group (P>0.05). As shown in Fig. 4B, the expression of TACC1 mRNA was affected following ErbB-2 knockdown, with a different expression level noted in the ErbB-2-silenced cells when compared to the control cells. The expression of TACC1 in the ErbB-2-positive cells (BT474) was expressed at a low level. However, after interference of the expression of ErbB-2, expression of TACC1 was upregulated. The result of real-time PCR showed that silencing of the ErbB-2 gene markedly increased the levels of TACC1 mRNA (P<0.05) (Fig. 4).

The protein expression of ErbB-2 and TACC1 was evaluated by western blotting. The gray scale of the stained area was measured under identical conditions. The average optical density for ErbB-2 protein expression in the ErbB2-shRNA-infected cells was lower when compared with the value in the blank control and the negative control-shRNA cells, respectively. This indicated marked reduction in ErbB-2 protein following ErbB2-shRNA infection; the observed differences were significant (P<0.05) (Fig. 5). Consistent with the above mRNA result, the average optical density for TACC1 protein expression in the BT474, MCF-7, Lenti-shcontrol and Lenti-shERBB-2 cells showed significant differences between the cell groups (P<0.05) (Fig. 5). TACC1 was overexpressed when the ErbB-2 protein was markedly inhibited by ErbB2-shRNA in BT474 cells. The results were in accordance with the mRNA levels (Fig. 4B).

Breast cancer cells that overexpress ErbB-2 are more resistant to chemotherapeutic agents such as paclitaxel (Taxol) and docetaxel (Taxotere) than cells that do not overexpress ErbB-2 (23). After inhibiting ErbB-2 expression using Lenti-shERBB2-eGFP infection, the IC₅₀ (index of chemotherapy sensitivity to docetaxel) of the Lenti-shERBB2-infected cells was 2.77±0.04 μg/ml. The IC₅₀ of the BT474 cells was 30.01±0.13 μg/ml, and this value for the MCF-7 cells was 1.15±0.03 μg/ml. That is, after ErbB-2 was inhibited, the breast cancer cell sensitivity to chemotherapy improved 10-fold. The result indicated that the supression of ErbB-2 expression inhibits the proliferation of BT474 cells and increases the cell sensitivity to docetaxel treatment; this difference was statistically significant (Fig. 6).