Lentiviral Vector For PHLPP2 Small-hairpin RNA And Its Influence On Tca8113 Cell Proliferation

At present, tongue cancer is the highest rates in the oral cavity cancer,it has a high malignant degree, early lymph node metastasis, postoperative recurrence and survival quality is poorer, it poses a great threat to patient’s life. In recent years, because of human play more and more attention on tongue cancer, they have made certain achievements on the various research aspects, but the 5-year survival rate of tongue cancer patients has been stalled middle level,this mainly due to the lack of specific biological markers in the early and the recurrence of tongue cancer is easy to transfer and other causes. Because of the tongue is a important function of organ in human body,therefore, how to prolong the life of the survival of patients with tongue cancer,improve the quality of the survival of patients with postoperative, this exploration is tongue cancer research emphasis.PHLPP is a kind of protein kinase, it has the function of inhibition of tumor formation,and it is a typical tumor-suppressor genes. When PHLPP is activated, it can be make the protein phosphatase Akt phosphorylation, result in conduction affected certain signaling pathways in cells. PHLPP2 is an isomer of PHLPP, its loss is associated with many diseases, especially in some malignant tumors, if happened it lack of different level, will cause the increase of the malignant tumor cell proliferation,apoptosis of decreased, but PHLPP2 in malignant tumor cells is not very clear is how to play a role, especially in the specific mechanism in the tongue cancer research. Cell contains an important pathway is phosphatidyl inositol- 3- threonine kinase/silk protein kinase(phosphatidyl inositol- 3- kinase/serine- threonine kinase, PI3K/AKT)signal transduction pathway, it mainly through activation of AKT affected by a variety of downstream effects of molecular activation status. Akt pathway is the central link inthe pathway, it is play an very important role in the signaling pathway and it can be affect cell function, closely related to the occurrence of a variety of malignant tumor development. The carcinogenic potential of Akt phosphorylation on major Ser with 473 points, studies have shown that Akt activity in malignant tumor cell proliferation,metastasis and the radiation and chemotherapy antagonism in the role is critical.Our experiment studies the function of PHLPP2 and Akt Ser 473 in the role of tongue cancer, and some meaningful results are obtained from this experiment, made It’s a beneficial exploration to the potential therapy of tongue cancer. To construct the lentiviral vector for PHLPP2 small-hairpin RNA(shRNA),and to detect its effect on tongue cancer Tca8113 cell growth and the phosphorylation level of Akt Ser473.PHLPP2 shRNA was designed and constructed to PSIH-H1 lentiviral vector.After sequencing, PSIH-H1-PHLPP2 was packaged to lentivirus in 293 T cells. After infected with lentivirus and selected by puromycin,mixed Tca8113 colonies stably expressing PHLPP2 shRNA were obtained and the fusion PHLPP2 expression was detected byreal-time PCR(qRT-PCR) and Western blot. Finally,the effect of PHLPP2 shRNA on Tca8113 growth was determined by Cell growth assay.The effect of knock down of PHLPP2 on the phosphorylation of Akt Ser473 was determined by Western blot.qRT-PCR and Western blot showed that PSIH-H1-PHLPP2 could suppress the PHLPP2 gene expression. Suppression of PHLPP2 could promote the growth of Tca8113 cells.Knock down of PHLPP2 could markedly promote the phosphorylation of Akt Ser473. The lentivirus-mediated PHLPP2 shRNA were obtained, which laysthe foundation for further research on the function and mechanism of PHLPP2 in tongue cancer.