Effects of KAP -2

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2. Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed. Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LV-plenti-GFP-KAP-1), negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10% fetal calf serum). Capan2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor), empty vector control group (cells transfected with microRNAs inhibitor), blank control group 2 (cells cultured in 1640 medium plus 10% fetal calf serum). The lentivirus was identified by double endonuclease restriction and sequencing ,and the virus titer was detected. The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells. The expressions of KAP-1, genes of EMT proteins and mRNA of miR-100-5p were detected by realtime quantitative polymerase chain reaction. The protein expressions of KAP-1, EMT proteins in all the groups were detected by Western blot. The measurement data were presented by mean±standard deviation, and were analyzed using the analysis of variance. Results The lentiviral vector of LV-plenti-GFP-KAP-1 was successfully constructed, and the virus titer was 2×108 TU/ml. Compared with the control group, the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours. The relative mRNA expressions of KAP-1, N-cadherin, vimentin, E-cadherin, miR-100-5p were 1.77±0.83, 2.62±0.71, 2.50±0.21, 7.20±1.17 and 1.81±0.40 in theexperimentalgroup, 5.03±0.29, 5.07±1.53, 3.83±0.57, 7.83±0.78, 7.01±0.96 in the negative control group, 5.13±1.14, 5.81±1.49, 4.92±0.90, 3.07±0.36, 6.87±0.35 in the blank control group 1, with significant difference among the 3 groups (F=5.99, 7.62, 7.88, 6.62, 4.64, P<0.05). The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group, empty vector controlgroup, blank control group 2 were 1.56±0.42, 4.89±0.61, 5.20±0.38, with significant difference among the 3 groups (F=5.14, P<0.05). The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group, empty vector control group, blank control group 2 were 3.10±1.37, 3.44±0.94, 3.08±1.16,with no significant difference among the 3 groups (F=0.49, P>0.05). The results of western blot showed that the relative protein expressions of KAP-1, N-cadherin, vimentin, E-cadherin were 2.77±1.99, 1.31±0.38, 4.25±0.63, 0.62±0.06 in the experimental group, 0.83±0.46, 0.41±0.37, 1.03±0.33, 1.17±0.45 in the negative control group, 0.71±0.26, 0.08±0.04, 1.37±0.92, 3.04±0.65 in the blank control group 1, with significant difference among the 3 groups (F=5.54, 4.68, 3.19, 8.18, P<0.05). The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group, empty vector control group, blank control group 2 were 2.27±0.71, 0.56±0.43, 0.61±0.39, with significant difference among the 3 groups (F=4.81, P<0.05). The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group, empty vector control group, blank control group 2 were 3.19±0.55, 3.93±0.06, 3.61±0.73, with no significant difference among the 3 groups (F=0.04, P>0.05). Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression.