PTEN Overexpression Can Inhibit Canine Mammary Tumor Cells Growth By Down-regulating PI3K/Akt Pathway

PTEN, the first discovered tumor suppressor gene, has dual phosphatase activity and plays a key role in a variety of signaling pathways regulating cell cycle, apoptosis, cell invasion and metastasis. Various tumor tissues, especially the malignant tumors h ave PTEN gene mutation or deletion. PTEN is closely related to tumorgenesis, development, invasio n and metastasis. The PI3K/Akt pathway, negatively regulated by PTEN, was abnormally activated in a variety of tumors to promote cell proliferation, differentiation and accelerate cell cycle progression. Thus, PTEN gene and PI3K/Akt pathway are important targets for cancer therapy. Canine mammary tumor is the most common tumor disease in canine, which is also an ideal model for human breast tumor. In the view of above factors, to study the value of PTEN and PI3K/Akt pathway in the treatment of breast cancer, to explore gene treatment for breast cancer through PTEN gene, following experiments were conducted.In this study, we amplified full CDS of canine PTEN gene by RT-PCR, and constructed PTEN eukaryotic expression vector(pc DNA3.1-PTEN), used liposome to transfecte pc DNA3.1-PTEN into canine mammary tumor cells(CHMp and CHMm) and G418 to screen these cells 2-3 weeks. Cells proliferation was assayed by CCK-8, cells apoptosis was measured by flow cytometry analysis, Western blotting and RT-PCR assays were used to test protein expression and mRNA of PI3K/Akt pathway and the PI3K/Akt pathway related factors. This study aims to explore the effect of overexpressed PTEN on proliferation of the canine mammary tumor cells and on the PI3K/Akt pathway and its related factors.Results: 1, RT-PCR and Western blot demonstrated that m RNA and protein expression of PTEN in transfected PTEN gene canine mammary tumor cells was significantly higher than control group and empty vector group(p<0.05); 2, Cell proliferation assaye d that transfected PTEN gene can apparently inhibit canine mammary tumor cells proliferation(p<0.05), and cell apoptosis assay detected that transfected PTEN gene can promote canine mammary tumor cells apoptosis(p<0.05); 3, Total Akt mRNA and protein expression level of transfected PTEN gene group compared with control group and empty vector group do not have significant difference(p>0.05), but p-Akt(Ser473) protein, mTOR mRNA and protein expression level of transfected PTEN gene group is significantly lower than control group and empty vector group(p<0.05). It showed that PTEN overexpression can down-regulate PI3K/Akt pathway; 4, m RNA and protein expression of anti-apoptotic gene Bax, caspase-3 and caspase-9 of transfected PTEN gene group significantly higher than control group and empty vector group(p<0.05), pro-apoptotic gene Bcl-2 mRNA and protein expression of transfected PTEN gene group significantly lower than control group and empty vector group(p<0.05); 5, Cell cycle gene CyclinD 1 mRNA and protein expression of transfected PTEN gene group significantly lower than control group and empty vector group(p<0.05), P27 kip mRNA and protein expression of transfected PTEN gene group significantly higher than control group and empty vector group(p<0.05).In summary, this experiment successfully constructed PTEN eukaryotic expression vector, and successfully expressed PTEN in canine mammary tumor cell line(CHMp and CHMm). PTEN overexpression inhibits canine mammary tumor cell proliferation and induce s apoptosis. PTEN overexpression can down-regulate PI3K/Akt pathway, then regulate expression level of downstream factors of apoptosis and cell cycle, and inhibit canine mammary tumor cells growth, providing a theoretical basis for gene therapy in breast tumor.