Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
目的 构建沉默环氧化酶-2（COX-2）基因重组慢病毒，观察其体外侵袭的抑制作用，从而探讨干扰COX-2抑制喉癌细胞增殖的作用机理，为喉癌的治疗提供新的思路。 方法 逆转录聚合酶链反应（RT-PCR）检测COX-2基因在人表皮样喉癌细胞（Hep-2）中的表达情况。利用上海吉凯公司RNA干扰（RNAi）慢病毒表达载体系统，构建针对COX-2基因慢病毒RNAi表达载体。转染Hep-2细胞，干扰COX-2基因的表达，实时定量PCR检测干扰前后基因表达变化。利用生长曲线测定干扰载体转染前后细胞生长速度变化。流式细胞仪检测细胞的生长周期。Boyden侵袭小室法测定体外侵袭力。 结果 成功构建了COX-2慢病毒RNAi表达载体，并建立了干扰COX-2基因的Hep-2细胞系。实时定量PCR检测COX-2基因在Hep-2细胞系中过表达被显著抑制。生长曲线测定，COX-2基因干扰后细胞增殖明显变慢。流式细胞仪检测细胞的生长周期可见干扰组诱导Hep-2细胞凋亡，转染G0～G1期细胞数量明显上升，S期细胞减少，表明siRNA干扰Hep-2细胞后，细胞由G0～G1期进入到S期受到阻滞，细胞增殖速度下降。体外侵袭实验中，Hep-2-AS侵袭细胞数（31.0 ± 1.8）显著低于Hep-2细胞（104.0 ± 2.6）及Hep-2-P细胞（99.0 ± 2.7），差异有统计学意义（P＜0.05）。 结论 喉癌中过表达的COX-2基因被干扰后表达明显降低并显著抑制细胞的生长速度和侵袭能力。同时验证了COX-2基因RNA干扰在进行抗肿瘤的治疗中潜在的应用前景。
目的 采用RNA干扰技术沉默CCS（copper chaperone for SOD1）基因，构建相关小干扰RNA（siRNA），探索出针对CCS的高效siRNA序列。 方法 合成用于人脐静脉内皮细胞（HUVEC）细胞中沉默CCS基因的siRNA。应用脂质体转染的方法在HUVEC细胞中对CCS基因进行RNA沉默。蛋白免疫印迹Western blotting检测沉默前后CCS蛋白表达变化的情况，甲基四唑蓝法MTT检测转染前后细胞活力。最后用单因素方差分析对数据进行统计学分析，以确定有效的siRNA序列。 结果 转染前后细胞形态无肉眼可见变化，转染后细胞活力分别为98.5%和98.8%。CCS蛋白沉默率分别为63.7%和61.4%。 结论 采用siCCS-2和siCCS-3序列转染条件对HUVEC细胞活力损伤小，CCS沉默效率高，实验条件稳定，重复性好。为我们继续研究沉默CCS后抑制血管内皮细胞的生长增殖、血管形成提供了稳定的实验基础。
ObjectiveTo construct the human small interfering RNA (siRNA) lentiviral vector who targeting inhibitor of differentiation-1 (Id1) gene, and to detect its efficiency of gene silence for the HepG2 cells. MethodsThe most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Id1 gene (included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4), who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector (Id1-RNAi-LV) and then infected human HepG2 cells. Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively. ResultsExpression level of Id1 protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group (P < 0.05), who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector (Id1-RNAi-LV) was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0×109 TU/mL.results of real time-PCR and Western blot showed that, the expression levels of Id1 mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group (P < 0.05). ConclusionsThe specific lentiviral can constantly down-regulate the expression of Id1 gene.
ObjectiveTo investigate the inhibitory effects and related mechanism of S100A4 gene silencing on oxygen-induced retinal neovascularization. Methods7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, normal-S100A4 group, oxygen induced retinopathy (OIR) group, OIR-S100A4 group, OIR-green fluorescent protein (GFP) group. To establish the OIR model, mice from all groups except normal one were exposed to (75±2)% oxygen for 5 days and then to room air. In the OIR-S100A4 group and OIR-GFP group, the OIR mice were given an intravitreal injection of 1μl of 1.0×109 PFU/ml adenovirus of Ad-S100A4-RNAi or Ad-GFP at P12, and then returned to normoxia for the next 5 days. In the OIR group, OIR was induced in C57bl/6J mice from P7 to P17. In the normal-S100A4 group, the normal P12 mice were give an intravitreal injection of 1 μl of Ad-S100A4-RNAi adenovirus, and maintained in room air from P12 to P17. In normal group, newborn mouse litters were maintained in room air from P0 to P17 without any treatment. Mice in all five groups were euthanized at P17, and retinas were collected for biochemical assays and morphological study. Retinal neovascularization (RNV) was evaluated by counting the number of pre-retinal neovascular cells and the whole mount immunofluorescent staining of the mouse retina. Protein and mRNA expression levels of S100A4, cAMP responsive element binding protein (CREB), B cell lymphoma-2 (bcl-2), Caspase-3 were determined with western blot and real-time PCR. ResultsThe number of pre-retinal neovascular cell nuclei in retinas from OIR-S100A4 group were obviously lower than those in the retinas from OIR group and OIR-GFP group (t=13.61, 14.64; P < 0.05). In OIR-S100A4 group, the retinal neovascular tufts area and the vaso-oblitertion area were both significantly smaller than those in OIR group and OIR-GFP group (P < 0.05). Protein level of CREB and bcl-2 were significantly down-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05).On the contrary, protein levels of Caspase-3 were up-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05). ConclusionAd-S100A4-RNAi transfer ameliorates RNV in mouse model of OIR maybe through down-regulating the expression of bcl-2 and CREB, and up-regulating the Caspase-3.
To investigate the effects of Snail1 gene silence on the expression of tight junction proteins and the migration ability of Hep-2 cells, Hep-2 cells were transfected with plasmids which is containing the shRNA of Snail1 gene, and cultured till the cells could be passaged stably (named Sh-snail1 cells). The expression of tight junction proteins (ZO-1, Occludin, Claudin-5) were detected by Western blot. The migration ability of Sh-snail1 cells was investigated by wound healing assay, and the protein expression of members of RhoGTPase family (RhoA, Cdc42) was detected by Western blot, which is closely related to the migration ability. Our results showed that the expression of tight junction proteins (ZO-1, Occludin, Claudin-5) was significantly increased; the migration ability of Sh-snail1 cell was inhibited; the expression of RhoA and Cdc42 was downregulated. All of these indicated that silencing the gene of Snail1 in Hep-2 cells can up-regulate the expression of tight junction proteins and down regulate the expression of Cdc42 and RhoA, and further inhibit the migration of Hep-2 cells. Furthermore, opening of the tight junctions between cells and the stronger migration ability of cancer cells are important processes in cancer metastasis. It is confirmed that the Snail1 gene is closely related to the two processes, providing an experimental basis for targeted therapy of laryngeal squamous cell carcinoma.
ObjectiveTo investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.MethodsThree lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining.ResultsAfter lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced (P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups (P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant (P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased.ConclusionSilencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.