KAP-1 knock down by shRNA imparts sphere-forming activity of Panc-1 cells
10.3760/cma.j.issn.1007-8118.2013.07.010
- VernacularTitle:shRNA干扰KAP-1基因表达对胰腺癌肿瘤干细胞自我更新的影响
- Author:
Jianxin JIANG
;
Lei ZHAN
;
Yaozhen PAN
;
Chengyi SUN
- Publication Type:Journal Article
- Keywords:
Pancreatic neoplasm;
Stem cells;
Gene expression
- From:
Chinese Journal of Hepatobiliary Surgery
2013;19(7):520-525
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate whether KAP-1 could induce CSC-self renewal in pancreatic cancer cell lines.Methods KAP-1 expression was examined in 14 cases of pancreatic cancer with immunohistochemistry.KAP-1 shRNA amplified by PCR was inserted into pGC-LV in vector,and then identified by restriction endonuclease digestion and nucleotide sequencing.The lentiviral vector pGC-shRNA-KAP-1 was co-transfected with pHelper 1.0 and pHelper 2.0 packaging plasmids into HEK 293T cells,and the lentivirus was collected and virus titer was measured.The expressions of KAP-1 and vimentin were detected by Western blot and RT-qPCR when human pancreatic cancer cell line Panc-1 was infected by the lentivirus.Sphere forming assay was conducted to assess the capacity of CSC or CSC-like cell self-renewal in this study.Results The KAP-1 expression level in cancerous tissues on immunohistochemistry was significantly higher than in the corresponding normal tissues (P=0.002).After infected by lentivirus,the expressions of KAP-1 and vimentin were knocked down,which could be detected by Wesren blot and RT-qPCR.Compared to Panc-1-GFP (NC) cells,the outcomes suggested that knocking down the expression of KAP-1 could decrease the formation of pancreatospheres,which further suggested the capacity of CSC-self-renewal in primary and secondary pancreatospheres of Panc-1-shRNA-KAP-1 and NC cells.Conclusions KAP-1 expression in pancreatic cancer tissues has been identified.The lentiviral vector for shRNAs targeting KAP-1 was constructed successfully.The formation of pancreatospheres decreased by knocking down the KAP 1 gene.KAP-1 is involved in the regulation of CSC phenotype.The mechanism is probably related to the upregulated expression of the EMT marker vimentin.
