Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C
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Abstract:
Objective
To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC).
Methods
Recombinant adeno-associated virus( rAAV) encoding cdc2-siRNA was packaged, and then was injected into the cerebellum of 2 weeks old npc-/- mice. Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks. Purkinje cells visualized by HE staining in cerebellum were counted, and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology.
Results
(1) Footprint test showed that the stride length in cdc2-siRNA npc-/- group((4.92±0.31) cm) was markedly longer than that in empty vector npc-/- group((4.05±0.19) cm)(P<0.05). (2)Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/- group((26.01±1.82) s, (50.93±1.98) s) was significantly shorter than that in the empty vector npc-/- group((31.96±3.47)s, (56.89±2.97)s), respectively(P<0.05 for all comparisons). (3)The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/- group(11.0±2.5) compared with the empty vector npc-/- group(5.1±2.2)(P<0.05). (4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/- group(1.42±0.22, 0.95±0.31)were significantly lower than those in the empty vector npc-/- group(2.11±0.29, 2.61±0.62), respectively(P<0.05 for all comparisons).
Conclusion
Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/- mice by ameliorating Purkinje cell’s loss and reducing the hyperphosphorylation of Tau in cerebellum.
Key words:
Niemann-Pick disease type C; Coordination; Cell division cycle 2; Purkinje cellObjective To evaluate the effect of Rab23 on the proliferation of a squamous cell carcinoma cell line Sa3,and to investigate its mechanisms.Methods Cultured Sa3 cells were classified into four groups:normal control group transfected with green fluorescent protein (GFP),Rab23-overexpressing group transfected with a GFP-labelled Rab23-overexpressing plasmid,Rab23-silencing group transfected with a plasmid carrying a Rab23-targeting shRNA,empty vector group transfected with an empty vector.After additional culture for different durations,plate colony formation assay and flow cytometry were performed to evaluate the proliferative activity of Sa3 cells,and Western blot was conducted to detect the expression of Erl/phosphorylated-Erk in Sa3 cells.Statistical analysis was carried out by t test,one-way analysis of variance and Bonferroni's multiple comparison test.Results Stable Sa3 cell lines with overexpression or silencing of Rab23 were established by plasmid construction and lentivirus-mediated transfection.The plate colony formation assay showed that the colony formation rate was significantly lower in the Rab23-overexpressing group than in the normal control group (2.3％ ± 0.2％ vs.3.6％ ± 0.3％,P ＜ 0.05),but higher in the Rab23-silencing group than in the empty vector group (4.1％ ± 0.2％ vs.1.8％ ± 0.03％,P ＜ 0.01).Rab23 overexpression induced G1 phase arrest in Sa3 cells.The proliferation index was significantly decreased in the Rab23-overexpressing group compared with the normal control group (0.581 ± 0.035 vs.0.698 ± 0.018,P ＜ 0.05),but increased in the Rab23-silencing group compared with the empty vector group (0.567 ± 0.015 vs.0.444 ± 0.014,P ＜ 0.01).As Western blot showed,there were no significant changes in the expression of Erk in the Rab23-silencing or-overexpressing group compared with the normal control group,whereas the expression of p-Erk was attenuated in the Rab23-overexpressing group compared with the normal control group,but enhanced in the Rab23-silencing group compared with the empty vector group.Conclusions Rab23 could inhibit the proliferation of Sa3 cells,which may be associated with the Erk pathway.
Key words:
Rab23; Lentivirus infections; Carcinoma, squamous cell; Cell line, tumor; Cell proliferation
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To assess whether the Pax6 gene is involved in the development of retinoblastoma.Three human retinoblastoma cell cultures were transfected with human Pax6 specific double-stranded, small interfering siRNA molecules RH-1 and RH-2. In addition, untreated control groups and negative control groups (CT groups) transfected with siRNA without homology to the human genome were formed for all three cell culture lines.After Pax6 gene was silenced by siRNA, the percentage of tumor cell survival decreased significantly (P < 0.05). Correspondingly, the percentage of apoptotic cells to total cells was significantly (P < 0.05) higher in the three retinoblastoma cell lines transfected with siRNA than in the CT control groups and the untreated control groups. In a parallel manner, the cell cycle was significantly (P < 0.01) altered in the transfected study groups, with reduced percentages of retinoblastoma cells in the S-phase. The cell-cycle-associated protein P21 was upregulated, and the protein P27 was slightly upregulated in the transfected retinoblastoma cell lines, in comparison to the control groups.Silencing the Pax6 gene with short interfering RNA resulted in an inhibited growth and an increased apoptosis of cultured human retinoblastoma cells. It was paralleled by upregulation of the P21 and P27 proteins.
Retinoblastoma
Retinoblastoma protein
PAX6
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Objective
To explore the effect of Rab26 on the Proliferation and migration of human in small cell lung cancer cells(H446).
Methods
The H446 cells were transfected with over-expression vector of Rab26, or Rab26 siRNA. Blank group was used as a negative control, respectively. After transfection 48 h, the protein level and mRNA of Rab26 were determined by RT-PCR and Western blot. Proliferation of the H446 cells was assayed by flow cytometry and the migration rate was detected by the scratch test.
Results
The transfection efficiency of Rab26 overexpression plasmid group was (76.8±4.3) % and Rab26 siRNA group was (79.5±3.57)%. After transfection 48 hours, the expression of Rab26 in overexpression group was significantly higher than control group in mRNA and protein level (P<0.05), however, the Rab siRNA group showed that the expression of Rab26 was significantly lower than control group (P<0.05). MTT assay showed that the cell viability of blank control group was (56.4±3.23)%, (100±4.31)%, Rab26 overexpression group was (42.5±3.59)%, (62.3±2.97)% and Rab26 siRNA group was (75±3.86)%, (123.31)% in 24 and 48 hours, respectively. The cell viability of Rab26 overexpression group was significantly lower than that of blank group (P<0.05), while Rab26 siRNA group was significantly higher than that of blank group (P<0.05). Scratch test showed that the cell migration rate of blank control group was (0.53±0.03)μm/min and (0.32±0.04)μm/min, Rab26 plasmid overexpression group was (0.21±0.04)μm/min and (0.22±0.04)μm/min, the Rab26 siRNA group was (0.61±0.02) μm/min, (0.42±0.03) um/min after 24 and 48 hours. The cell migration rates of Rab26 plasmid overexpression group was significantly lower than the blank group, but Rab26 siRNA group was significantly higher than the blank group (P<0.05).
Conclusions
Rab26 is associated with the cellular proliferation and migration in H446 cell. Rab26 may be a novel therapy target for the treatments of small cell lung cancer.
Key words:
Rab26; Small cell lung cancer; H446 cells; Proliferation; Migration
Viability assay
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Objective To construct lentiviruses targeting mice connexin 37 (CX37) small interfeting RNA (siRNA) and modelling the gastric cancer with murine gastric cancer cell line.The lentiviruses were transfected into gastric cancer.Methods Lentiviruses carrying siRNA of CX37 were constructed,which knocked down mRNA and protein expression of CX37 significantly in vitro.Sixty mice with gastric cancer were randomly divided into CX37 siRNA group,mock-siRNA group and the control group,receiving injection of CX37 siRNA,mock-siRNA and normal saline respectively.After 6 weeks,the expression of CX37 mRNA and protein was detected by using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively.Apoptosis was examined by using TdT-mediated dUTP nick end labeling (TUNEL).Results Six weeks after lentivirus transfection,the results showed that the CX37 mRNA levels in CX37 siRNA group (42％) were significantly lower than in the mock group (63％)and control group (58 ％) (P ＜ 0.05).Western blotting revealed the expression of CX37 protein in CX37siRNA group was lower than in other three groups (0.21 ± 0.07 vs.0.65 ± 0.06 vs.0.54 ± 0.07).At the same time,the apoptotic index in CX37 siRNA group was higher than in mock-siRNA group and control group [(19.7±5.1)％ vs.(9.8±6.4)％ vs.(10.5±7.2)％,P＜0.05].Conclusion CX37 siRNA can significantly inhibit the growth of the gastric cancer cells.
Key words:
Gastric cancer; Connexin 37; Small interfering RNA
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Objective To evaluate the inhibitory effect of CDC20 siRNA on the expression of CDC20 mRNA and protein in mouse Hepal-6 hepatocarcinoma cell lines.Methods The siRNAs which aimed at CDC20 mRNA of Hepal-6,were designed and synthesized.Using cation lipofectamine 2000 embedding techniques,the siRNAs were transfected into Hepal-6 cell lines for 48 h,and then the expression of CDC20 mRNA and protein was detected by RT-PCR and Western blot.Results Compared with normal and negative control groups,CDC20 mRNA expression in the siRNA group was obviously decreased with the increased concentrations of siRNAs.Under the concentrations of 100,50 and 25 nmol/L siRNA,mRNA expression levels in mixed siRNA group and normal control group were 0.370±0.058 vs 0.840±0.044,0.480±0.081 vs 0.900±0.068,and 0.830±0.062 vs 1.010±0.054 respectively(P＜0.01).CDC20 protein expression levels in the siRNA groups were obviously decreased also(siRNA-1,siRNA-2,siRNA-3 and mixed siRNA groups vs normal or negative control group:0.482±0.035,0.402±0.003.0.479±0.026 and 0.446±0.017 vs 0.826±0.031 or 0.835±0.024,P＜0.01).Conclusion CDC20 siRNA has remarkable inhibitory effect on the expression of CDC20 mRNA and protein in mouse Hepal-6 cell lines.
Key words:
CDC20; RNA interference; Carcinoma,hepatoeellular; Mitosis
Lipofectamine
CDC20
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To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.本研究旨在阐明猪miR-331-3p 对细胞增殖的影响，探讨其对细胞增殖的作用机制首先构建了miR-331-3p 的过表达载体pcDNA 3.1 (+)-miR-331-3p，并将将PK15 细胞分为4 组，分别为实验组、实验组对照组、抑制剂组和抑制剂对照组。实验组和对照组分别转染pcDNA 3.1(+)-miR-331-3p 和pcDNA 3.1(+)。抑制剂组和抑制剂对照组分别转染miR-331-3p Inhibitor 和miR-331-3p 阴性对照(miR-331-3p NC)。通过在各组添加CCK-8试剂绘制细胞增殖曲线，并使用PI 染色检测细胞所处周期比例。同时，利用实时荧光定量PCR (Quantitative real-time PCR，qPCR) 检测生长抑制蛋白家族成员5 (Inhibitor of growth family member 5，ING5)、细胞周期蛋白依赖性激酶2 (Cyclin dependent kinase 2，CDK2)、细胞周期蛋白依赖性激酶3 (Cyclin dependent kinase 3，CDK3)、细胞周期蛋白依赖性激酶4 (Cyclin dependent kinase 4，CDK4)、细胞周期蛋白B (Cyclin B) 和细胞周期蛋白依赖性激酶抑制剂1A (Cyclin dependent kinase inhibitor 1A，CDKN1A) 的表达变化。结果表明，实验组miR-331-3p表达量显著升高，细胞增殖曲线表明48 h 和72 h 时细胞数目均呈现出实验组>实验对照组和抑制剂对照组>抑制剂组的趋势 (P<0.05)。与实验对照组相比，实验组处于G0/G1 期的细胞比例下调，S 期和G2/M 细胞的比例上调，抑制剂对照组趋势与之相反；同时，实验组中与促进增殖的基因CDK2、CDK3、CDK4 和Cyclin B 的mRNA 表达水平均显著升高，而抑制增殖的基因ING5 和CDKN1A 均表现出显著下降的趋势。本研究成功构建了miR-331-3p过表达载体，且发现miR-331-3p 具有促进猪肾上皮细胞增殖的能力，研究结果为深入研究miR-331-3p 在猪生长发育中的作用机制奠定了基础。.
Cyclin E
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Objective To selectively down-regulate the expression of paired box gene 8(Pax-8) in rat myocytes by RNA interference,in order to investigate the effect on cellular apoptosis and proliferation.Methods The primary cultured H9C2(2-1) myocytes were divided into 3 groups: short interference RNA targeting Pax-8(Pax-8 siRNA) group,non-specific siRNA group as the negative control(NC siRNA),and the blank control group.The former two groups were treated with siRNA-liposome compounds consisting of siRNA(5μl) and Lipofectamine 2000(5μl),and the blank control group was treated with equal volume of culture medium.RT-PCR and Western blotting were performed to analyze the expressions of Pax-8 mRNA and protein.Fluorescence spectrophotometry was used to detect the activity of Caspase-3 which was wed to reflect cell apoptosis.The inhibitory rate of cell proliferation was detected by CCK-8 assay.Results In comparison with NC siRNA group and blank control group,the expression of Pax-8 mRNA in Pax-8 siRNA group was inhibited by 60.30% and 63.09% respectively(both P0.05),and of Pax-8 protein by 50.38% and 54.17%(both P0.05),while no significant difference was found between NC siRNA group and blank control group.The activity of Caspase-3 was elevated and significantly higher in Pax-8 siRNA group than in NC siRNA group and blank control group(P0.01),while no significant difference was found between the latter 2 groups.CCK-8 assay revealed that the proliferation inhibitory rate was higher in Pax-8 siRNA group than in NC siRNA group(P0.01).Conclusion Pax-8 siRNA may selectively down-regulate the expression of Pax-8 in myocardial cells,and conspicuously promote apoptosis and inhibit proliferation.
Lipofectamine
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Objective
To investigate the molecular mechanism of P75NTR gene-induced apoptosis in tongue squamous cell carcinoma Tca8113 cell lineage.
Methods
P75NTR specific siRNA was transferred into P75NTR positive tongue squamous cell carcinoma Tca8113 cells. P75NTR positive Tca8113 cells were divided into 4 groups: blank group (without transfection), negative control group (transfected with negative control siRNA), experiment group-776 (transfected with siRNA-P75NTR-776) and experiment group-1234 (transfected with siRNA-P75NTR-1234). Transfection efficiency and cell apoptosis were detected by flow cytometry. The interference effect of P75NTR mRNA expression was detected by fluorescence quantitative PCR. 3-(4, 5-dimethyl-2-thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide assay was applied in measuring cell prolife-ration. The protein changes of P75NTR were detected by Western blotting. The distributions of nuclear factor-κB(NF-κB) of cells were observed by cell immunofluorescence labeling method.
Results
The transfection efficiency was 30%. The apoptosis rate of experiment group-776, experiment group-1234 and negative control group was (20.35±0.18) %, (12.32±1.51)% and (2.63±0.10)% respectively. Compared with the negative control group, the differences of the former two group had statistical significance (t=177.20, P<0.005; t=37.12, P<0.005). The P75NTR gene interference was successful. The inhibition rate of P75NTR protein reached 31% in experiment group-776. The cell viability of Tca8113 cells after P75NTR-siRNA interference was 70.02%, 78.01% and 95.81% in experiment group-776, experiment group-1234 and negative control group. And there were significant differences between experiment group-776 and negative control group(χ2= 235.3, P<0.010), and between experiment group-1234 and negative control group (χ2= 117.5, P<0.005). NF-κB distribution was increased in cell cytoplasm in the interference group than that in control group.
Conclusion
P75NTR may promote the proliferation or inhibit the apoptosis of tongue squamous cell carcinoma, and the molecular mechanism may be correlated with hindering the transportion of NF-κB into cell nuclear.
Key words:
Apoptosis; Nerver growth factor; NF-Kappa B
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[Effect of siRNA-mediated beta-catenin gene on Wnt signal pathway in lung adenocarcinoma A549 cell].
To study the effects of siRNA-mediated beta-catenin down-regulation on Wnt/beta-catenin signal transduction pathway in lung adenocarcinoma A549 cells.A549 cell was divided into three groups: PGCsil-CTNNB1-siRNA group (transfection with beta-catenin interference plasmids), negative control group (transfection with negative control plasmids) and non-transfection group (blank control). For each group, real-time PCR was employed to detect the expression of beta-catenin and cyclin D1 (a target gene of Wnt/beta-catenin pathway). A MTT cell proliferation assay was performed to study the proliferating capacity. Flow cytometry was used for cell cycle analysis. Clone formation and Millicell chamber experiment were performed to evaluate the clone formation and migration capacities of cells.The expression of beta-catenin and cyclin D1 in PGCsil-CTNNB1-siRNA cell decreased at the mRNA level. It was lower than negative control (0.002+/-0.001 vs 0.023+/-0.002, P<0.01; 0.005+/-0.002 vs 0.040+/-0.020, P<0.05) and blank control groups (beta-catenin mRNA: 0.021+/-0.003, P<0.01; cyclin D1 mRNA: 0.042+/-0.004, P<0.05). Also the proliferating capacity at Days 5-7 was inhibited in comparison with negative control and blank control groups (P<0.05, P<0.01). The cell-doubling time (58.1 h) was markedly longer than those of negative control group (37.9 h, P<0.05) and blank control group (34.2 h, P<0.05). The number of cells in G0-G1 phrase increased in PGCsil-CTNNB1-siRNA group (86.4%+/-2.6%) in comparison with negative control (73.8%+/-0.9%, P<0.01) and blank control groups (75.8%+/-1.5%, P<0.01). In PGCsil-CTNNB1-siRNA group, the clone formation ratio (31.6%+/-7.7%) was lower than negative control (46.9%+/-7.3%, P<0.05) and blank control groups (44.2%+/-2.5%, P<0.05). And the number of cells (16.0+/-3.8) passing through the membrane of Millicell chamber was smaller than negative control (32.7+/-3.1, P<0.01) and blank control groups (33.0+/-2.7, P<0.01).Knocking down beta-catenin not only inhibits the Wnt/beta-catenin signal transduction pathway in lung adenocarcinoma A549 cells but also decreases cell proliferation, clone formation and migration capacity. Thus it may become a novel target for lung cancer therapy.
clone (Java method)
Beta-catenin
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The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)-derived exosomes (SNPC-Exo) and the roles of the P53/P21 pathway on the senescence of NPC.The senescent phenotypes of NPC were induced by interleukin-1β treatment. SNPC-Exo was extracted from the culture medium of senescent NPC and purified by differential centrifugation. The structure of SNPC-Exo was identified by transmission electron microscopy and western blot analysis was used to determine the exosomal marker proteins CD63 and Tsg101. Western blot analysis was performed to determine the relative expression levels of P16, P21, and P53 in NPC. Senescence-associated β-galactosidase (SA-β-gal) staining was used to stain the senescent NPC and a phase contrast microscope was used to observe and count the SA-β-gal staining of NPC. The proliferation of SNPC-Exo-treated NPC was assessed using growth curve analysis and the colony formation assay. The cell cycle of SNPC-Exo-treated NPC was determined by flow cytometry. NPC were transfected with siRNA to knock down P53 and P21 expression.Interleukin-1β-treated NPC had a higher percentage of SA-β-gal positive cells (45%) than the control group (20%) and showed an increase in the relative expression of P16, P21, and P53 (P < 0.05). SNPC-Exo were positive for exosomal marker protein CD63 and Tsg 101 and negative for calnexin, and successfully internalized as previously described. SNPC-Exo-treated NPC showed an increase in the relative expression of P21 and P53 (P < 0.05). Compared with the control group, the SNPC-Exo-treated NPC showed a lower growth rate (3 times lower on the 5th day and 2 times lower on the 7th day), fewer colony-forming units (12.0%), and a higher percentage of SA-β-gal-positive NPC (50.0%). The SNPC-Exo-treated NPC contained more G1 phase cells (68.0%) and fewer S phase (15.5%) cells than the control group (53.0% in G1 phase, 33.5% in S phase). The expression of P21 and P53 significantly decreased in SNPC-exo-treated NPC after siRNA transfection (P < 0.05), followed by a higher growth rate (2 times higher on the 5th day and 1.5 times higher on the 7th day) and lower percentage of SA-β-gal-positive NPC (22.5%). Moreover, the inhibition of the P53/P21 pathway promoted the SNPC-Exo-treated NPC to enter the S phase (from 15.5% to 25.3%).The inhibition of the P53/P21 pathway attenuated the senescence of NPC induced by SNPC-Exo.
CD63
Senescence
Exosome
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