The Effect Of Of Hccr Gene On Biological Characters In Pancreatic Cancer Panc1 Cells And Its Up-stream Signal Pathway

Objective To obtain the PANC1 pancreatic cell lines which were stably transfected with HCCR siRNA plasmid. To investigate the effect of proliferation and invasion on PANC1 cell by knockdown of HCCR expression through RNA inference. To reasearch the mechanism of PI3K/Akt signal pathway on regulation of HCCR.Methods1. siRNA targeting HCCR was designed and plasmid of pGCsi-HCCR was synthesized. Plasmid of pGCsi-HCCR was transfected into PANC1 cells. The vector pGCsi was used as negtive control. G418-resistant clones were selected and obtained the stably transfected cell lines. Protein of the stably transfected cell lines(HCCRsiRNA cell lines and vector cell lines)were detected and identified by western blot using HCCR antibody for many times.2. Protein of p53 HCCRsiRNA cell lines and vector cell lines were also detected.3.Flow cytometry(FCM) ,MTT, and Transwell assay were respectively examined to observe the apoptotic rate, proliferation and invasion of HCCRsiRNA transfectant.4. PANC1 cells stably transfected with constitutively active Akt and dominant negative Akt were cultured and transfected with three HCCR promotors using Lipofectamine 2000.The luciferase activity was measured and normalized after 24h of transfection with the luciferase assay kit.Results1. Obtain the PANC1 pancreatic cell lines which were stably transfected with HCCR siRNA plasmid and vector plasmid. Conpared with the control,HCCR protein in PANC1 cells transfected with siRNA decreased by western blot as expected. 2. Plasmid of pGCsi-HCCR can inhibit the expression of HCCR.The protein level of p53 was also decreased in HCCRsiRNA cells compared with the vector cells.3. Plasmid of pGCsi-HCCR can induce a more significant G0/G1 stage arrest and higher apoptotic rate compared with pGCsi vector. The proliferation of HCCRsiRNA cells were decreased by 0.65 times and 0.68 times in HCCRsiRNA cells compared with pGCsi vector.The number of invasion cells were respectively 24.4±9.9 and 49.1±15.4, which were significant difference.4 .we generated three reporter constructs containing different proximal promoter regions of HCCR-1 (pGL3/HCCR-1-P1196, pGL3/HCCR-1-P504, and pGL3/HCCR-1-P423). The stable PANC-1 cell lines carrying either CA-Akt , DN-Akt or vector were respectively transfected with reporter constructs and they were assayed for luciferase activity.The luciferase activity of the three constructs (pGL3/HCCR-1-P1196, pGL3/HCCR-1-P504, and pGL3/HCCR-1-P423) in CA-Akt PANC1 cells were respectively increased by 1.53,1.80,1.50 folds compared with the vector PANC1 cells. The luciferase activity of the three constructs in DN-Akt PANC1 cells were respectively decreased by 0.76,0.75,1.0 folds compared with the vector PANC1 cells.Conclusions:1. Down-regulating the expression of HCCR gene by siRNA can inhibit proliferation and invasion and induce apoptosis in pancreatic cancer PANC1 cells.2. The HCCR promoters can be activated in PANC1 stably transfected with CA-Akt, but not in PANC1 stably transfected with DN-Akt.