Objective To investigate the inhibitory effects of RNA interference (RNAi) expression vector on the expression of survivin in pancreatic cancer cell PANC-1. Methods The protein and mRNA expressions of survivin were examined with immunofluorescence and RT-PCR. The survivin gene was cloned into the T-vector and sequenced. The RNAi expression vectors targeting survivin, named si-svv-1 and si-svv-2 respectively according to whether they harbored a mutation or no mutation, were constructed and transfected into PANC-1 cells with liposome. The expression of survivin mRNA was detected with RT-PCR. Apoptosis of PANC-1 cells was analyzed with DNA ladder and FACS. Results There was a high degree expression of survivin in PANC-1 cells. The expression of survivin was not inhibited by RNAi expression vectors si-svv-1, but inhibited about (72.43±8.04)% by si-svv-2 and the apoptosis rate of PANC-1 cells increased to （12.36±1.44）% after 72 h. Conclusion The RNAi expression vector can effectively inhibit the expression of survivin in pancreatic cancer cell PANC-1 cells and induce the apoptosis in PANC-1 cells.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To investigate the radiation-sensitizing effects of Ku80 silencing by siRNA interference for A549 lung cancer cells. Methods The sequences of Ku80 siRNA and negative siRNA were chemically synthesized and transfected into A549 lung cancer cells by lipofectamine. RT-PCR and Western bolt analysis were used to determine Ku80 gene expression. The transfected cells in culture dishes were irradiated with X ray at doses of 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, respectively. Once all treatments were completed, the cells were processed with the colony formation assay. Results RT-PCR detection showed that Ku80 mRNA levels in A549 lung cancer cells were reduced after transfected with Ku80 siRNA at 24 h, 48 h and 72 h time points. Western blot analysis showed that Ku80 protein content decreased at 48 h and 72 h time points compared with the control group ( P lt; 0. 05 ) . Cloning formation assay indicated that radiosensitivity of A549 lung cancer cells was enhanced after transfected with Ku80 siRNA. Conclusion Ku80 siRNA can effectively inhibit Ku80 gene expression of A549 lung cancer cells, and therefore enhance its radiosensitivity.
Objective To construct the expression vector of HLA-G-shRNA and investigate the effect of HLA-GshRNA from NK cell lysis. Methods Four HLA-G shRNA plasmids were constructed and transiently transfected to Bel-7402 cell lines， the levels of mRNA and protein of HLA-G were detected by Real-Time PCR and Western blot. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay. Results The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one which we designed, and no aberrations such as mutation，deletion or insertion occurred. The expressions of HLA-G confirmed by Real Time-PCR and Western blot were significantly down-regulated. Bel-7402 cell lines transfected HLA-G shRNA showed higher lytic activity （P＜0.01）. After KIR2DL4 receptor blocked，lytic activity of NK-92 MI cell were decreased （P＜0.01）. Conclusions HLA-G shRNA plasmids are successfully constructed and HLA-G down-regulated can increase NK cytolysis against Bel-7402 cell. After HLA-G combines with KIR2DL4 receptor at the surface of NK cells， the inhibition effect is transferred.
Objective To study the inhibitory effect of RNA interference (RNAi) on bcl-2 expression of vascular smooth muscle cells （VSMCs) in rabbit. Methods The expression vector of bcl-2 gene-targeting small interference RNA (pshRNA-bcl-2) was constructed and was transfected into VSMCs by lipofectamine, and the unloaded vector was used as control. The expression of bcl-2 mRNA was identified by RT-PCR and Western blot, respectively. The growth of the transfected VSMCs was examined by MTT. Results The pshRNA-bcl-2 may inhibit the expression of bcl-2 gene at the levels of transcription and translation. There were significant differences (P＜0.01) of the expressions of bcl-2 mRNA between the VSMCs that were transfected with pshRNA-bcl-2 and the ones in plasmid transfected group and control group, respectively. There was a significant difference (P＜0.01) in the growth of VSMCs between the plasmid transfected and the control groups. Conclusion The plasmid containing the small interference RNA of bcl-2 may have an inhibitory effect on the cell growth and endogenous expression of bcl-2 gene at the levels of transcription and translation in VSMCs.
Objective To investigate the role of calcium- and integrin-binding protein-1（CIB1） in oxidized lowdensity lipoprotein（OX-LDL） inhibiting migration of mouse macrophages. Methods To silence CIB1 express of mouse macrophages by RNA interference, then incubating mouse macrophages with OX-LDL, cell migration and cell spreading of mouse macrophages were analyzed. Results At 24-72h after macrophages transfected CIB1 siRNA, the express of CIB1 protein was restrained obviously. To silence CIB1 express could increase migration and spreading of mouse macrophages significantly. Conclusions CIB1 plays the important role in intracellular modulating mechanism of OX-LDL inhibiting mouse macrophages migration.
ObjectiveTo investigate the influence of EZH2 gene down-regulation by RNA interference on the proliferation and invasion of human glioma cell line U251.
MethodsThe recombinant plasmid of small hairpin RNA targeting EZH2 gene was constructed, and transfected into gioma U251 cells by electroporation. The expression of EZH2 mRNA and protein in the cells was detected by using reverse transcriptase-polymerase chain reaction and Western blot respectively; the viability of cells was determined by using methyl thiazol tetrazo1ium assay; and the invasiveness of U251 cells was tested by Transwell cabin.
ResultsThe expression levels of EZH2 mRNA in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.19±0.02) than that in the untransfected group (1.13±0.05) and the control-shRNA-GFP group (1.15±0.05). The expression levels of EZH2 protein in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.20±0.02) than that in the untransfected group (1.03±0.03) and the control-shRNA-GFP group (0.97±0.06). The proliferation rates in EZH2-shRNA group were significantly decreased as compared with those in the untransfected group and control-shRNA-GFP group (24 hours after transfection:60.13%±3.15%, 100.00%±9.31%, 100.03%±9.35%; 48 hours after transfection:53.01%±3.14%, 100.00%±9.13%, 99.58%±9.27%; P<0.05) and Transwell cabin suggested that the invasiveness of U251 cells was significantly decreased (46.00±2.82, 60.67±5.71, 61.00±2.48; P<0.01).
ConclusionEZH2-targeted RNA interference can reduce the proliferation and invasion of human glioma cells, which suggests that EZH2 shRNA may be a potential gene therapeutic target of human glioma.
Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.
Objective
To investigate the effect of TNF-related weak inducer of apoptosis/fibroblast growth Factor-inducible 14 (TWEAK/Fn14) on the cell proliferation by transfecting Fn14 shRNA to PANC-1 cells.
Methods
The shRNA gene targeting Fn14 gene was constructed and transfected into pancreatic cancer cell line PANC-1 to specifically silence the expression of Fn14 gene. The effect of shRNA interference sequence on the expression of Fn14 was detected by flow cytometry and immunofluorescence. CCK-8 was used to detect the cell proliferation of PANC-1 after blocking TWEAK-induced signal pathway. Western blotting method was used to detect the expressions of downstream factors such as nuclear factor-kappa B (NF-κB), TWEAK and caspase-3 to explore the pathway mechanism of TWEAK/Fn14.
Results
The absorbance value (A value) in the Fn14 shRNA group was significantly lower than the control groups in 24 hours after transfected (P<0.000 1). After the specific shRNA sequences transfected PANC-1 cells, NF-κB, TWEAK and caspase-3 protein expressions were also significantly lower than the control group (P<0.05), and the apoptosis of PANC-1 cells increased after inhibition of TWEAK/Fn14 signaling pathway.
Conclusions
TWEAK/Fn14 involved in the progression of pancreatic cancer. The Fn14 expression could influence the process of cell apoptosis.
Objective To establish a cell culture model in vitro of acute lung injury and investigate the effects of NF-κB p65 on the inflammation and oxidative stress in TNF-α-activated type Ⅱ alveolar epithelial cells. Methods A549 cells were treated with TNF-α ( 10 ng/mL, 24 h) in the absence or presence of NF-κB p65 siRNA ( 50 nmol /L) . RT-PCR and Western blot were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The contents of IL-1β, IL-4, and IL-6 in the culture supernatant were measured by ELISA. The concentration of MDA and SOD were detected by colorimetric method. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium ( MTT) assay. Results P65 RNAi significantly decreased the transcription and translation of NF-κB p65 induced by TNF-α( P lt; 0. 05) . The levels of IL-1β, IL-4, and IL-6 were significantly lower in the supernatants of A549 cells pretransfected with NF-κB p65 siRNA ( P lt;0. 05) , while the concentration of MDA markedly decreased ( P lt; 0. 05) , and the activation of SOD increased dramatically ( P lt; 0. 05) . Consequently, the survival rate of A549 in the p65 siRNA group improved( P lt; 0. 05) . Conclusions NF-κB p65 plays a key role in the oxidative stress induced by TNF-α. NF-κB p65 silencing can down-regulate the inflammation and oxidative stress induced by TNF-αand enhance the proliferation of alveolar epithelial cells.
