Contributed equally
We aimed to investigate the influence of long non-coding RNA (lncRNA) PTEN pseudogene-1 (PTENP1) on the proliferation, migration and cycle of breast cancer cells and its mechanism. Lentiviral vectors expressing PTENP1 were synthesized and breast cancer cells MCF7 were transfected with LV003-GFP-PTENP1 and LV003-GFP, respectively. The proliferation capacities of breast cancer cells were detected using CCK-8 assay, and the migration capacities of breast cancer cells were detected using scratch assay; flow cytometry was used to detect the cell cycles and Western blot was used to detect the expression levels of cyclin A2, CDK2, p-p44/42 MAPK, t-p44/42 MAPK, p-p38 MAPK, t-p38 MAPK, p-AKT, t-AKT in AKT and MAPK pathways. The absorbance values (A450) of cells in experimental group at 48 and 72 h were 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). The number of cell colonies in experimental group was (48±13), which was significantly lower than that in control group (159±16) (P<0.01). 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). Western blot detection showed that the expression levels of cyclin A2, CDK2, p-AKT, p-p44/42 MAPK and p-p38 MAPK in experimental group were significantly decreased compared with those in control group. LncRNA PTENP1 can inhibit the proliferation and migration of breast cancer cells via the AKT and MAPK signaling pathways.
Breast cancer is a major disease threatening women's health. According to statistics, breast cancer accounts for one-third in all new-onset tumors in female (
PTEN (phosphatase and tensin homolog deleted on chromosome ten) has been reported to inhibit progression and development of various cancers, and it was established as a tumor suppressor (
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
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) (
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 (
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 (
lncRNA is a transcription RNA with the length of more than 200 nucleotides. lncRNA can not be translated into protein because of lack of a promoter. More and more studies have confirmed that lncRNA regulates the proliferation, apoptosis, differentiation and migration of tumor cells through its participation in regulating chromatin modification, transcriptional activation, intranuclear interference and silencing of X chromosome (
Via directly inhibiting phosphoinositide 3-kinase (PI3K) signaling pathway and negatively regulating miR-21, PTEN could affect a series of cellular processes (
MAPK is a serine/threonine protein kinase in cells, which plays an important role in breast cancer. Abnormal changes of MAPK signaling pathway play an important role in the growth, differentiation and other physiological activities of breast cancer. To investigate the role of PTENP1 in MAPK signaling pathway in breast cancer cells, the protein expression levels of p-p44/42 MAPK and p-p38 MAPK in MCF7 expressing PTENP1 were detected, and it was found that PTENP1 could downregulate the phosphorylation of Erk1/2 (P44/42 MAPK) and p38 MAPK protein, important factors in MAPK signaling pathway in breast cancer cells, indicating that PTENP1 can also regulate the proliferation and migration of breast cancer cells via regulating MAPK signaling pathway, in addition to inhibiting the activity of AKT signaling pathway in breast cancer cells.
In recent years, some scholars have proposed the competitive endogenous RNA (ceRNA) hypothesis; in other words, there are common microRNA binding sites in lncRNA and mRNA in corresponding functional network, which weakens the inhibiting effect of microRNA on homologous mRNA via microRNA adsorption (
In conclusion, lncRNA PTENP1 can inhibit the proliferation and migration of breast cancer cells, which may be realized through downregulating cyclin A2 and CDK2, the important proteins in cell cycle, and AKT and MAPK signaling pathways. lncRNA PTENP1 plays an important role in the occurrence and development of breast cancer, and the in-depth study on it can reveal not only the related mechanism of PTEN, its cancer suppressor gene, but also the role and mechanism of lncRNA in breast cancer. In addition, the in-depth study on lncRNA can provide new ideas for the prevention and treatment of breast cancer.
The growth curve of breast cancer MCF7 cells.
Colony-forming assay of breast cancer MCF7 cells. (A) Control group; (B) experimental group; (C) compared with control group, colony number was significantly reduced in the experimental group (**P<0.01).
Influence of lncRNA PTENP1 on migration capacity of breast cancer MCF7 cells between control group and experimental group (**P<0.01).
Influence of lncRNA PTENP1 on cyclin of breast cancer MCF7 cells between control group and experimental group.
Influence of lncRNA PTENP1 on AKT and MAPK signaling pathways of breast cancer MCF7 cells between control group and experimental group.