Zhou Shuo, Ma Xiang, Wang Zhen-Jie, Zhang Wei-Yue, Jiang Hai, Li San-Dang, Zhang Tai-Zhe, Du Jie, Lu Zheng
Department of Emergency Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, People's Republic of China.
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233000, Anhui, People's Republic of China.
Onco Targets Ther. 2019 Jul 11;12:5577-5587. doi: 10.2147/OTT.S212689. eCollection 2019.
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. 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. 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. 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. It is indicated that the TPM3 gene can be a potential target spot for the treatment of pancreatic cancer.
胰腺癌是最具侵袭性的人类恶性肿瘤之一，与早期转移和化疗耐药相关。原肌球蛋白（TPM）是肌肉收缩所必需的调节蛋白，TPM基因的异常表达与恶性肿瘤的发生和转移密切相关。在本实验中，采用慢病毒法敲低PANC-1细胞中TMP3的表达，建立了单克隆稳定转染细胞系，并探讨了TPM3基因表达下调对PANC-1细胞上皮-间质转化（EMT）相关分子及生物学行为的影响。根据TPM3基因序列设计RNA干扰序列，构建并筛选出具有最佳沉默效果的重组质粒片段TPM3-shRNA，进行慢病毒滴度测定和包装。将重组慢病毒干扰载体LV-TPM3-shRNA转染至PANC-1细胞；随后评估转染效率，筛选出TPM3表达下调的单克隆稳定转染PANC-1细胞系。采用qRT-PCR和蛋白质免疫印迹法检测靶细胞系中EMT相关转录因子的基因和蛋白水平表达变化，并分别检测侵袭和增殖能力的变化。结果显示，利用重组慢病毒载体成功建立了TPM3表达下调的单克隆稳定转染PANC-1细胞系。敲低PANC-1细胞中TPM3基因的表达后，细胞发生EMT；细胞表型呈现恶性转化，细胞的体外生物学行为（如增殖和侵袭）均有不同程度增强。表明TPM3基因可能成为治疗胰腺癌的潜在靶点。
