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 According to heparanase’s gene sequence of GenBank, to construct heparanase gene-targeted small interfering RNA (siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant breast cancer MDA-MB-231 cell. Methods Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector, which was identified by sequence identify. Human malignant breast cancer MDA-MB-231 cell was transfected with the constructed vector with lipofectamine method. Fluorescence photograph was taken. Real-time PCR (RT-PCR) was performed to evaluate the level of heparanase mRNA expression. Results Four kinds of heparanase gene-targeted hairpin siRNA were designed, then were inserted into pGPU6/GFP/Neo vector after annealing. Sequencing indicated the construction was successful. Fluorescence photographs showed MDA-MB-231 cells were transfected successfully. RT-PCR showed that heparanase mRNA expression levels were inhibited significantly （Plt;0.05）. Conclusion The heparanase gene-targeted siRNA and its vector are successfully constructed and MDA-MB-231 cells are transfected successfully. Heparanase mRNA expression levels are significantly inhibited by siRNA vector, which provide a new method for the treatment of cancer.
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 the expression short hairpin RNA （shRNA） targeting gene kir2. 1 in rat myocardial cells, named pEGFP6 kir2. 1, and to observe the effects on the expression of messenger RNA（mRNA） and protein of gene kir2. 1 as well as the changes of myocardial beating rates. Methods Five RNA interference （RNAi） sites targeting the rat kir2. 1 gene was selected, designed and synthesized five pairs of oligonucleotides fragments ,annealed them to double-strand, then cloned them into the vectors containing U6 promoter,obtained the vector expressing five aim genes. Rat myocardial cells were divided into three groups： Experimental group, negative plasmid control group and normal control group. Reverse transcription-polymerase chain reaction（RT PCR） and Western-blot were carried out to detect the expression of the mRNA and protein of gene kir2.1 and the beating rates of myocardial cells were observed after 72 h. Results The expression of mRNA and protein of gene kir2. 1 of experimental group were markedly lower than that of other two control groups after 72 h（P〈0.01）. There was no statistically significant between two control groups. The beating rate in experimental group was much faster than other two control groups （P〈0.01）, remained unchanged in both negative plasmid control group and normal control group. Conclusion Plasmid pEGFP6-kir2.1 could suppress the expression of the mRNA and protein of kir2.1 and increase the rat cardiac muscle cell beats.
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 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.
ObjectiveTo evaluate the inhibitory effect of small interfering RNA (siRNA) targeting peroxisome-proliferator-activated receptor-γcoactivator-1α(PGC-1α) on retinal neovascularization in the mouse. MethodsEighty seven-day-old C57BL/6J mice were divided into normal group, model blank group, model control group and PGC-1αsiRNA group, twenty mice in each group. Mice in the normal group were kept in normal room air. Mice in the model blank group, model control group and PGC-1αsiRNA group were induced for retinal neovascularization by hypoxia. Liposome with PGC-1αsiRNA (1 μl) and liposome with negative control siRNA (1 μl) were injected into the vitreous in the PGC-1αsiRNA group and model control group respectively when mice were moved out to room air from the cabin (Postnatal 12). No injection were performed in the model blank group. At postnatal 17, fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. PGC-1αand vascular endothelial growth factor (VEGF) level in retina were measured by real-time polymerase chain reaction (real-time PCR) and Western blot. Inhibition efficiency of PGC-1αsiRNA on PGC-1αand VEGF was calculated. ResultsMice in the normal group showed reticular distribution of retinal blood vessels. Central nonperfused retina, neovascular tufts and fluorescein leakage were seen in the model blank group and model control group. Neovascular tuft and fluorescein leakage were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group. The neovascular nuclei were increased in the model blank group and model control group compared to the normal group (P < 0.05). The neovascular nuclei were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group (P < 0.05). The expression of PGC-1αmRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased 54% and 53% respectively in the PGC-1αsiRNA group as compared with model blank group and model control group (P < 0.05). The expression of VEGF mRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased significantly in the PGC-1αsiRNA group (decreased 48% and 40% respectively) as compared with model blank group and model control group (P < 0.05). ConclusionsIntravitreal injection of PGC-1αsiRNA mediated by liposome can inhibit retinal neovascularization in the mouse effectively.
ObjectiveTo observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells. MethodsCultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively. ResultsThe mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05). ConclusionKnockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
Objective To construct gene silence adenovirus vector targeting both transglutaminase 2 （TG2） and Mer receptor tyrosine kinase （Mertk） synchronously and detect the gene silence function of it. Methods The interfering plasmids targeting TG2 protein and Mertk protein were constructed firstly， then the H1 promoter and RNA interfering （RNAi） sequence were cut and ligated to pAdTrack for constructing pAdTrack/TG2/Mertk. The pAdTrack/TG2/Mertk was transfected into BJ5183 bacterial cells which contained pAdEasy-1， then the plasmid was detected by enzyme digestion after recovery. Adenovirus were harvested after that pAdTrack/TG2/Mertk was infected into HEK293 cells. The virus titer was measured after repeated amplification. The RAW264.7 cells were infected by pAdTrack/TG2/Mertk， pAdTrack/TG2， pAdTrack/Mertk， and pAdTrack/green fluorescent protein （GFP）， respectively. Then the expression levels of TG2 protein and Mertk protein of mouse macrophages were detected by Western blot after infection. Results The virus titer of pAdTrack/TG2/Mertk plasmid was 6.13×1010GFU/mL. The pAdTrack/TG2/Mertk plasmid which contained 2 promoters and 2 RNAi sequences was identified successfully by enzyme digestion. Compared with pAdTrack/GFP group and pAdTrack/Mertk group （there was no significant differece between the 2 groups）， the expression levels of TG2 protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/TG2 decreased obviously （P＜0.01）， but there was no significant difference between the later 2 groups. Compared with pAdTrack/GFP group and pAdTrack/TG2 group （there was no significant difference between the 2 groups）， the expression levels of Mertk protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/Mertk decreased obviously too （P＜0.01）， but there was no significant difference between the later 2 groups. Conclusion Gene silence adenovirus vector plasmid targeting both TG2 and Mertk synchronously is constructed successfully， and the pAdTrack/TG2/Mertk can reduce the expressions of TG2 protein and Mertk protein of mouse macrophages obviously.
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.