The Effects Of PIAS3 Overexpression On Proliferation,Migration And Invasion Abilities Of Lung Adenocarcinoma Cell Line A549

Lung cancer is one of the important diseases that threaten the health of human being. The pathological types of lung cancer include small cell lung cancer(SCLC)and nonsmall cell lung cancer(NSCLC). NSCLC includes squamous cell carcinoma, adenocarcinoma, gland squamous cell carcinoma and so on. As a new way of treatment, molecular targeted drug has a higher selectivity and a relatively small side effect,comparing with the traditional anti-tumor chemotherapy drugs. The molecular targeted therapy drug used on NSCLC is mostly targeting on EGFR. Comparing with western countries, the rate of EGFR gene mutation is as high as 50% in our country. However, there are still generous patients of mutate negative. In addition, many patients show primary drug resistance and secondary drug resistance, which makes the existing molecular targeted drugs to a certain limit. Therefore, it is of great significance to explore new target molecules in tumor signaling pathway for treating lung cancer. STAT3(signal transducers and activators of transcription 3) is a member of STATs family. STAT3 exists in most tissues and it plays an important role in the occurrence and development of tumor. The quantity and activity of STAT3 was positively associated with the growth, proliferation and invasion abilities of tumor. STAT3 mediates signal transduction of many cytokines and growth factor and STAT3 is the transcription factor of many genes. PIAS3(protein inhibitor of activated STAT3) is an endogenous inhibitor of STAT3. Some study found that the expression of PIAS3 is higher in many human normal tissues and cells while very low or absent in the tumor tissue and cells. When silences the expression of PIAS3 in tumors, the activity of STAT3 increase persistently and the abilities of growth, proliferation and invasion increase significantly. In this paper, we transfected p CMV-Sport6-PIAS3 to A549 cells to explore the change of proliferation, migration and invasion abilities of A549. We cotransfect p CMV-Sport6-PIAS3 and STAT3 expression vector(PRC-STAT3) or STAT3 phosphorylation sites continued activation vector(PRC-STAT3-CT) to observe whether STAT3’s activity is inhibited. We are willing to explore the special inhibition of PIAS3 and the feasibility of PIAS3 as a new target for lung adenocarcinoma.Objective: To study the effect of PIAS3 overexpression on proliferation, migration and invasion of A549 cells and the special inhibition of STAT3; to explore the feasibility of using PIAS3 as a new target for lung adenocarcinoma.Methods: 1.Compared the transfection efficiency of Lipofectamine TM 2000 and Vigo Fect in A549 cells, and then choose the higher one for the next experiment. 2. Use Western Blot to detect PIAS3 overexpression of A549 cells after being transfected p CMV-Sport6-PIAS3. 3. Use MTT experiment to detect the change of survival and growth abilities in A549 cells of PIAS3 overexpression. 4. Use colony formation assay to detect the change of proliferation ability in A549 cells of PIAS3 overexpression. 5. Use scratch wound assay to study the change of migration ability in A549 cells of PIAS3 overexpression. 6. Use Transwell assay to study the change of invasion ability in A549 cells of PIAS3 overexpression. 7. Use indirect immunofluorescence staining experiments to reveal the effect of PIAS3 overexpression to p-STAT3 in the nucleus.Results: 1.The transfection efficiency of LipofectamineTM 2000 was obviously higher than Vigo Fect in lung adenocarcinoma A549 cells. 2. Western Blot showed that the expression of PIAS3 in A549 was significantly increased after being transfected with p CMV-Sport6-PIAS3. 3. The MTT assay result showed that the survival rate of A549 cells after being transfected with p CMV-Sport6-PIAS3 was decreased significantly comparing with Mock group and p CMV-Sport6 group. 4. The CFE results showed that the formation ability of lung adenocarcinoma A549 cells was decreased after being transfected with p CMV-Sport6-PIAS3, the survival fraction was only 9.78%. 5. The scratch wound assay showed that the migration ability was decreased by PIAS3 overexpression on A549. 6. The Transwell assay showed that the invasion ability was decreased by PIAS3 overexpression on A549. 7. The result of immunofluorescence staining indicated that the nucleus translocation of p-STAT3 was significantly decresed compared with the p CMV-Sport6 group after transfected with p CMV-Sport6-PIAS3; the nucleus translocation of p-STAT3 was still decresed comparing with the p CMVSport6 group, even after being co-transfected with PRC-STAT3 or PRC-STAT3-CT.Conclusions: 1.The transfection efficiency of LipofectamineTM 2000 is higher than Vigo Fect in lung adenocarcinoma A549 cells. 2.PIAS3 expression is significantly increased in A549 cells after being transfected p CMV-Sport6-PIAS3. 3.PIAS3 overexpression can significantly inhibit the survival, proliferation, migration and invasion abilities of lung adenocarcinoma A549 cells. 4. PIAS3 overexpression can specifically inhibite STAT3 activity in A549 cells, even over expression or persistent activation of STAT3. 5. PIAS3 is an important potential target for the treatment of lung adenocarcinoma...