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Objective To discuss the effect of G-protein-coupled receptor 49 (GPR49)gene on proliferation and invasive ability of hepatoma cell line Huh7 and its molecular biological mechanism.Methods According to the different transfected small interfering RNA(si-RNA),Huh7 cells were divided into the GPR49-siRNA(si-GPR49)group and the NC-siRNA (si-NC)group.Untransfected Huh7 cells were set as the control group. Messenger RNA (mRNA )and protein expression of GPR49, cyclin D1 and matrix metalloproteinase 9 (MMP9)in the cells of the three groups were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot method.The proliferation and invasive ability of the cells of each group were respectively detected by MTT method and Transwell method.Results The relative expression of GPR49 mRNA of the si-GPR49 group was (23.8 ±3.1)% of the control group (P <0.05). Compared with the control group,the protein expression of GPR49,cyclin D1 and MMP9 of the si-GPR49 group decreased significantly (all in P <0.05).The proliferation experiment results by MTT indicated that the optical density(OD)of the cells of the si-GPR49 group at 72 h was (0.53 ±0.12),which was significantly lower than that of the control group (1.35 ±0.28).The difference had statistical significance (P <0.05). The average invaded cell counts of the si-GPR49 group were (13.6 ±2.5),which was significantly lower than (65.3 ±6.1 )of the control group.The difference had statistical significance (P <0.05 ).Conclusions GPR49-siRNA may inhibit the gene expression of GPR49 in Huh7 cells.Its mechanism may be that the proliferation of Huh7 cells is inhibited by reducing the level of cyclin D1;the migration and invasive ability of Huh7 cells is inhibited by affecting the expression level of MMP9.
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Objective To inhibit the gene expression of signal transducer and activator of transcription factor 1 (STAT1) in human esophageal squamous cell carcinoma cell line Eca109 by RNA interference and investigate its effect on the radiosensitivity and cell cycle of Eca109 cells.Methods Interference vector pSTAT1-shRNA for STAT1 gene was designed and constructed.After being mixed with lentiviral packaging plasmids,the interference vectors were used to transfected 293T cells.Virus solution was collected to infect ECA109 cells.Real-time PCR and Western blot were used to measure the mRNA and protein expression levels of STAT1 in Eca109 cells.Colony formation assay and flow cytometry were used to evaluate the radiosensitivity and cell cycle distribution of Eca109 cells.Results All Eca109 cells were divided into blank control group,transfection-positive group,and transfection-negative group.The transfection-positive group showed significantly lower mRNA and protein expression levels of STAT1 than the other two groups.The values of D0,SF2,and Dq of transfection-positive group were 2.03 Gy,0.83,and 1.20 Gy,respectively,lower than those of blank control group (2.98 Gy,0.88,and 1.39 Gy) and those of transfection-negative cells (3.02 Gy,0.88,and 1.57 Gy).At 12 h,24 h,and 48 h after 4 Gy-irradiation,the transfection-positive group showed significantly higher percentage of G0 + G1 than the blank control group and transfection-negative group (34.13％ vs 22.03％ vs 22.27％,F =7.56,P =0.023 ; 43.80％ vs 28.40％ vs28.63％,F=10.01,P=0.012;53.20％ vs42.2％ vs41.83％,F＝10.73,P=0.010) and significantly lower percentage of G2 + M than the blank control group and transfection-negative group (14.33％ vs 32.23％ vs 32.23％,F=16.86,P=0.003;27.73％ vs 43.53％ vs 44.00％,F=26.62,P=0.001;14.23％ vs27.97％ vs27.93％,F=40.34,P=0.000).Conclusions RNAinterference of STAT1 in Eca109 cells does not affect the proliferation ability of Eca109 cells,and it can increase the radiosensitivity of Eca109 cells probably by regulating cell cycle after irradiation.
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ObjectiveTo explore the effects of small interfering RNA (siRNA) specific to Bcl-2gene on radiosensitivity of esophageal cancer cells. Methods Bcl-2 gene siRNA ( Bcl-2 siRNA ) was induced into esophageal cancer EC9706 cells by lipofectamine.Bcl-2 protein expression and apoptosis of EC9706 cells were detected by flowcytometer. Clone forming assay was used to determine the inhibitory effects of X-ray radiation combined with Bcl-2 siRNA interference. ResultsWhen Bcl-2 siRNA had been induced into EC9706 cells, Bcl-2 protein expression in EC9706 cells was inhibited, and cell apoptosis was increased. Bcl-2 protein expression rates of EC9706 cells induced with Bcl-2 siRNA1, A2, A3 (25.13％ ±2. 04％ ,38.87％ ± 3.34％ , 30.55％ ± 2. 73％ ) were lower than the control group ( 84.28％ ± 1. 47％ )(t =4. 01,3.04,3.64, P ＜ 0. 05 ). After interference, the apoptosis rate of EC9706 cells ( 33.86％ ±1.04％ ) was higher than the control group and siRNA negative group (5.51％ ±0. 14％ and 5.59％ ±0. 46％ ) (t =6. 55,6. 54,P ＜0. 01 ). Bcl-2 gene siRNA interference enhanced X-ray inducing apoptosis of EC9706 cells (56.76％ ± 1.24％ ), which was higher than the radiation alone group ( 24.51％ ± 0. 48％ )(t =3.59,P ＜ 0. 05 ). The D0, Dq, and SF2 of combined treatment group were much lower than those of irradiation alone group . The sensitization enhancing ratio was 1.32 ( ratio of D0 values ) . Conclusions Bcl-2 gene siRNA could enhance the radiosensitivity of esophageal cancer EC9706 cells and may has a good future in clinical practice.
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Objective To discuss the role of DNA-dependent protein kinase catalytic subunit (DNA-DPKCS) in human lung adenocarcinoma cell line (A549) by using small interfering RNA (siRNA) to specifically knockdown DNA-DPKCS expression and its effects on cell proliferation, cell cycle and radio-sensitivity. Methods The DNA-DPKCS-siRNA expression vector was constructed and transfected into A549 cell line. The transformed clones were randomly selected and isolated. The cell cycle distribution and apop-tesis were analyzed by flowcytometry analysis. Cell survival was detected by using clonogenic formation as-say. Results With specific inhibition of DNA-DPKCS expression, stable transfected cell line 549pRNA-DNA-DPKCS was constructed by RNA interference technique. The 549pRNA-C and 549pSUPER cell lines were the control cell lines tansfected with control and blank plasmids, respectively. Compared with A549 cells, the expression levels of DNA-DPKCS mRNA (0.110: 1. 000), protein (0. 870: 2.967) and activity of DNA-DPKCS (0.004: 0.266) in 549pRNA-DNA-DPKCS cells were significantly lower (F = 80.55 ,P < 0.01;F=63.96, P<0.01;F=51.62,P<0.01, respectively). The analysis of SF_2(0.25:0.76), D_0 (1.42:1.62) and D_q (0.06: 1. 00) showed significant difference between 549pRNA-DNA-DPKCS and A549 cells (F = 996.86, P < 0.01 ; F = 17.41, P < 0.05 ; F = 68.92, P < 0.01). The number of 549pRNA-DNA-DPKCS cells in S (24.5%: 35.5%) and G_2 (10.7%: 11.0%) phases was significantly decreased (F = 4.83, P<0.05 and F=32.04, P <0.01, respectively). Conclusions In A549 cells, inhibit of DNA-DPKCS gene expression can enhance the radiosensitivity and affect cell cycle distribution.
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Vascular endothelial growth factor(VEGF) is one of the best characterized angiogenic regulators which has many effects in promoting endotheliocyte proliferation and differentiation,increasing the microvascular permeability,inducing the angiogenesis,triggering the growth,survival and migration of tumor cells by combining its specificity receptor (VEGFR).Dysregulation of VEGF expression and signaling pathways therefore plays a pivotal role in the pathogenesis and clinical features of hematologic malignancies,direct and indirect targeting of VEGF and its receptors therefore may provide a potent novel therapeutic approach to overcome bone marrow angiogenesis and multidrug resis-tance thereby improve patient outcome.Recent years,a novel VEGF blockade system using RNA interference attracts more and more people's attention.The small interfering RNA (siRNA) targeting VEGF/VEGFR can completely inhibits the expression of VEGF and induce the silence of corresponding genes.
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Objective To study the construction of the lentiviral-mediated RNA interference(RNAi) vector targering rat suppressors of cytokine signaling 3(SOCS3) gene.Methods Three target sequences were selected by on-line designer software on Ambion according to rat SOCS3 mRNA sequence(NM053565),the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized.After annealing,these double strands DNA were cloned to pRNA-Lenti-green fluorescent protein(GFP),which contained U6 promoter and GFP.The resulting Lentiviral vector containing SOCS3 shRNA was named pRNA-Lenti-SOCS3-GFP.After the rat glioma cells(C6)were transduced with the constructed 1entiviral vectors,real-time polymerase chain reaction was used to evaluate the level of SOCS3 expression(including siRNA1 group,siRNA2 group,siRNA3 group,vacuity group and siRNA-Negative group).The pRNA-Lenti-SOCS3-GFP and Lentivector Pakaging plasmid mix were cotransfected into 293T to package Lentivirus particles.Culture supematant was harvested,then the virus titer was determined by serial dilution assay.Results The SOCS3 mRNA sequence was successfully cloned to pRNA-Lenti-GFP,which was proved by PCR and DNA sequence.Compared with control group,the SOCS3 mRNA expressions were obviously suppressed in all 3 experimental groups,especially the expression rate in siRNA1 group was reduced by 80%.The Lentiviral particle titer was determined by serial dilution assay with 1.0?1010 TU?L-1.Conclusion The lentiviral-mediated RNAi vector of rat SOCS3 gene has been constructed successfully,this may provide a potential tool for studying and treating SOCS3-related diseases.