【Abstract】 Objective The seed cells source is a research focus in tissue engineered cartilage. To observe whether the post-RNA interference (RNAi) chondrocytes could be used as the seed cells of tissue engineered cartilage. Methods Chondrocytes were separated from Sprague Dawley rats. The first passage chondrocytes were used and divided into 2 groups: normal chondrocytes (control group) and post-RNAi (experimental group). Normal and post-RNAi chondrocytes were seeded into chitosan/gelatin material and cultured in vitro to prepare tissue engineered cartilage. The contents of Aggrecan and Aggrecanase-1, 2 were measured by HE and Masson staining, scanning electron microscope (SEM), and RT-PCR. Results The histological results: no obvious difference was observed in cell number and extracellular matrix (ECM) between 2 groups at 2 weeks; when compared with control group, the secretion of ECM and the cell number increased in experimental group with time. The RT-PCR results: the expression of Aggrecan mRNA in experimental group was significantly higher than that in control group (P lt; 0.05); but the expressions of Aggrecanase-1, 2 mRNA in experimental group were significantly lower than those in control group (P lt; 0.05). The SEM results: the cell number in experimental group was obviously more than that in control group, and the cells in experimental group were conjugated closely. Conclusion The post-RNAi chondrocytes can be used as the seed cells for tissue engineered cartilage, which can secrete more Aggrecan than normal chondrocytes. But their biological activities need studying further.
Objective To observe the effect of RNA interference (RNAi) on HepG2 hepatic cancer cell by small interfering RNA (siRNA). Methods siRNA targeting vascular endothelial growth factor (VEGF) gene was transfected into HepG2 cells by LipofectimineTM 2000. The VEGF mRNA and protein were respectively detected by real-time quantitive PCR and Western blot, and the concentration of VEGF protein in the cell culture supertant was determined by ELISA at 48 h after culture. Results The average efficiency of siRNA transfection was (90.4±2.9)% after 6 h cell culture. The expressions of VEGF mRNA and protein in HepG2 cells could be effectively suppressed by siRNA, and the concentration of VEGF protein in the cell culture supertant was also decreased. Conclusion siRNA can knock down the expression of VEGF gene and decrease the concentration of VEGF protein in HepG2 cells.
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 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.
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 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 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.
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 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 observe whether Nogo-66 can inhibit the neurite outgrowth during the neuronal differentiation of the neural stem cells (NSCs) and remove such an inhibitory effect by the small interfering RNA (siRNA) mediated knockdown of the Nogo66 receptor (NgR). Methods NSCs derived from the rat spinal cord were collected, and were cultured by the suspension culture in vitro. NSCs were transfected by siRNA to knock downtheexpression of NgR. Immunofluorescence and Western blot were used to assess the knockdown efficiency. NSCs were divided into four groups and differentiated in the medium containing 10% FBS. In the control group, no intervention was applied to NSCs; in the Nogo-P4 group, NSCs were differentiated in the presence of Nogo-P4 (active segment of Nogo-66); in the siRNA group, NSCs were transfected by siRNA to knock down NgR before they were differentiated; in the siRNA and Nogo-P4 group, NSCs were transfected by siRNA to knock down NgR before they were differentiated in presence of Nogo-P4. The differentiated neurons were labeled by immunofluorescence, and the neurite length was measured by the ImagePro Plus 5.0 software. The differentiation of the neurite length was compared in each group. Results The suspension-cultured cells became the nerve bulb, which could positively expresses Nestin by immunofluorescence. At 1 week of the differentiation in the medium containing 10% FBS, the positively-labeled neuron specific enolase, the glial fibrillary acidic protein, and the myelin basic protein were observed. Both immunofluorescence and Western blot approved that the expression of NgR was knocked down by transfection of siRNA at 24 hours after the transfection. The knockdown efficiency was 90.35%±3.10%. The neurite length was 97.80±6.97 μm, 80.54±6.75 μm,92.14±7.27 μm, and 94.01±8.37 μm in the control group, the Nogo-P4 group, the siRNA group, and the siRNA and Nogo-P4 group, respectively. The Nogo-P4 group had a significant difference when compared with the otherthree groups (Plt;0.01), and the other three groups had no significant difference when compared with each other（Pgt;0.05）. ConclusionNogo-66 can inhibit the neuronal neurite outgrowth during the differentiation ofNSCs. Such an inhibitory effect can be removed by the siRNA mediated knockdown of NgR.
