OncoTargets and Therapy (Jul 2019)
Research on the establishment of a TPM3 monoclonal stable transfected PANC-1 cell line and the experiment of the EMT occurrence in human pancreatic cancer
Abstract
Shuo Zhou,1 Xiang Ma,2 Zhen-Jie Wang,1 Wei-Yue Zhang,3 Hai Jiang,1 San-Dang Li,1 Tai-Zhe Zhang,1 Jie Du,1 Zheng Lu21Department of Emergency Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, People’s Republic of China; 2Department of Hepatobiliary Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, People’s Republic of China; 3Department of Emergency Medicine, The Second People’s Hospital of Bengbu City, Bengbu 233000, Anhui, People’s Republic of ChinaBackground: Pancreatic cancer is one of the most aggressive human malignancies that is associated with early metastasis and chemoresistance. Tropomyosin (TPM) is an indispensable regulatory protein for muscle contraction, Abnormal expressions of TPM gene are closely related to the carcinogenesis and metastasis of malignant tumors.Purpose: In this experiment, a monoclonal stable transfected cell line was established by the knock-down of TMP3 expression in PANC-1 cells with the lentivirus method, and the impacts of the downregulated TPM3 gene expression on the EMT-related molecules and biological behaviors of PANC-1 cells were explored.Methods: Based on the TPM3 gene sequence, we designed the RNA interference sequence, constructed and screened out the recombinant plasmid segment TPM3-shRNA with the optimal silencing effect, and carried out lentivirus titer determination and packaging. The recombinant lentiviral interference vector LV-TPM3-shRNA was transfected into PANC-1 cells; the transfection efficiency was then evaluated to screen out the monoclonal stable transfected PANC-1 cell line with downregulated TPM3 expression. The qRT-PCR and Western blot were used to detect the changes in the gene- and protein-levels expressions of EMT-related transcription factors in the target cell line and to respectively test the variations of the invasion and proliferation capacities.Results: It is shown that the monoclonal stable transfected PANC-1 cell line with downregulated TPM3 expression was successfully established with the recombinant lentiviral vector. After knocking down the expression of TPM3 gene in PANC-1 cells, EMT occurred in the cells; the cell phenotype showed malignant transformation, and the in vitro biological behaviors of the cells (such as proliferation and invasion) were enhanced to different degrees.Conclusion: It is indicated that the TPM3 gene can be a potential target spot for the treatment of pancreatic cancer.Keywords: pancreatic cancer, TPM3, lentivirus, EMT, proliferation, invasion
