Breast cancer cell line MCF7 and cell line 293T used for lentivirus packaging were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA), trypsin and fetal bovine serum DMEM were purchased from Gibco (Grand Island, NY, USA), dimethyl sulfoxide was from Sigma (St. Louis, MO, USA), CCK-8 kit was from Dojindo (Kumamoto, Japan), RNA extraction kit and TRIzol were obtained from Takara (Otsu, Japan), and the rabbit anti-human p-p44/42MAPK, p-p38 MAPK, p-AKT, cyclin A2, CDK2 and mouse anti-human GAPDH monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

MCF7 cells were cultured in DMEM medium containing 10% fetal bovine serum and the medium was placed in an incubator containing 5% CO2 at 37°C. Cells were digested by 0.25% trypsin, followed by passage; and cells in logarithmic phase were used for the experiment.

According to DNA sequence of PTENP1 in National Center for Biotechnology Information (NCBI), the target gene PTENP1-3′UTR was amplified with DNA in human skin tissue as the template. Gene primer sequences were as follows: sense strand: 5′-AGTCACCTGTTAAGAAAATGAGAAGACAAA-3′, antisense strand: 5′-CTGTCCCTTATCAGATACATGACTTTCAA-3′. The recombinants expressing PTENP1 were constructed by LV003-GFP vector, followed by sequencing and identification.

Four kinds of plasmids (pspax2, pMD2G, pLVX-IRES-ZsGreen1 and pLV003-PTENP1) in lentiviral packaging system were prepared; ZsGreen1 expression cassette on the plasmid could express GFP; 293T cells were transfected with the plasmids containing the target gene and the four kinds of plasmids in lentiviral packaging system for virus packaging, and the virus supernatant was collected; the supernatant was centrifuged in a 40 ml ultra-speed centrifuge tube at 80,000 × g for 2 h at 4°C; the in a 40 ml ultracentrifuge tube. The virus precipitation was resuspended using iced PBS, dissolved at 4°C overnight and stored at −80°C.

When the cell fusion of MCF7 reached 60–70%, GFP-PTENP1 and LV003-GFP were added on the cell surface at the multiplicity of infection (MOI)=10, respectively. After 8 h, the normal DMEM medium containing 10% fetal calf serum was used. The experimental group (MCF7 cells transfected with LV003-GFP-PTENP1) and control group (MCF7 cells transfected with LV003-GFP) were set up. Puromycin (1 µl/ml) was added for screening after green fluorescence was observed under the fluorescence microscope, fluorescence photos were taken after 3 days, and the expression of PTENP1 was detected by real-time fluorescent quantitative PCR.

Cells taken from the two groups were inoculated onto the 96-well plates (2×10³/well) with 5 repetition wells in each group. CCK-8 solution was added with 10 µl/well at 12, 24, 48 and 72 h, respectively, and the cells were incubated in an incubator for 2 h. A450 was measured with the microplate reader, and the growth curve was drawn according to A450s.

Cells taken from experimental group and control group were digested to prepare the single-cell suspension, and the cell density was adjusted to 2×10³/ml, and 100 µl cell suspension was taken and inoculated onto the 6-well plate, and then 2 ml medium was added. The cell suspension was cultured in an incubator for 1–2 weeks and was seeded when the clone formation was visible, followed by Giemsa staining; and the number of clones was counted.

The above-mentioned cells were inoculated onto the 6-well plate, and when the cell fusion reached 100%, a straight line was drawn in the middle of each well with 1-ml spearhead, and the photos of cells were taken after 24 h; the area change before and after the scratch was calculated. Cell migration rate (%) = (area in scratch - area after 24 h/area in scratch) × 100%.

Cells in logarithmic phase were taken from experimental group and control group; the total protein of each group was extracted and quantified according to the procedures in protein extraction kit. The protein was transferred to polyvinylidene chloride membrane by wet transfer method after acrylamide gel electrophoresis. The membrane was sealed by skimmed milk for 1 h. And then primary rabbit monoclonal p-AKT antibody (dilution, 1:500; cat. no. ab81283); rabbit polyclonal t-AKT antibody (dilution, 1:500; cat. no. ab38449); rabbit monoclonal p-P44/42MAPK antibody (dilution, 1:500; cat. no. ab53277); rabbit monoclonal t-P44/42 MAPK antibody (dilution, 1:500; cat. no. ab50011); rabbit polyclonal p-p38 MAPK antibody (dilution, 1:500; cat. no. ab47363); rabbit polyclonal t-p38 MAPK antibody (dilution, 1:500; cat. no. ab197348); rabbit monoclonal cyclinA2 antibody (dilution, 1:500; cat. no. ab32386); rabbit monoclonal CDK2 antibody (dilution, 1:500; cat. no. ab32147); rabbit polyclonal GAPDH antibody (dilution, 1:500; cat. no. ab37168) were added. All the antibodies were all purchased from Abcam (Cambridge, MA, USA). Then the protein was incubated overnight at 4°C. After the protein was washed with TBST three times, secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2,000; cat. no. ab6721) was added for incubation for 1 h, followed by development via enhanced chemiluminescence (ECL).

SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for data analysis. The t-test was used for the comparison of protein expression levels and absorbance values between the two groups. P<0.05 was considered to indicate a statistically significant difference.

The growth curve of breast cancer MCF7 cells is shown in Fig. 1. A450 of cells in experimental group at 48 h and 72 h was 1.4±0.3 and 2.3±0.47, respectively, which were significantly lower than those in control group (3.2±0.39, 3.4±0.58) (P<0.05). Colony-forming assay is shown in Fig. 2, and the results showed that the number of cell clones in experimental group was 48±13, which was significantly lower than that in control group (159±16) (P<0.01).

Scratch assay showed that the cell migration rate in experimental group was 22.8±3.3%, which was significantly lower than that in control group 61.8±5.2% (p<0.01) (Fig. 3).

Cyclin A2 and CDK2 are the important proteins that promote cells to transform from S phase to G2 phase. Western blot detection showed that the expression levels of cyclin A2 and CDK2 in experimental group were significantly lower than those in control group (Fig. 4).

Western blot detection showed that the expression of p-AKT protein, an important factor in AKT signaling pathway, in experimental group was significantly decreased compared with that in control group. In addition, the protein expression levels of p-p38 MAPK and p-p44/42 MAPK, important factors in MAPK signaling pathway, in experimental group were significantly decreased compared with those in control group (Fig. 5).