Knockdown of survivin using siRNA promotes prostatic apoptosis in cancer cell DU145

AIM: To investigate the promoting effects of survivin-siRNA on apoptosis of DU145 cells. METHODS: The DNA template coding siRNA against survivin was synthesized and recombinant plasmid pSi-sur was constructed. The recombinant and the two controls, liposome and pSi-scrambled plasmid, were transfected into DU145 cells. The expression of survivin in mRNA and protein was detected by RT-PCR and Western blotting, respectively. Apoptosis was measured by acridine orange staining, Annexin V-FITC labeled flow cytometry and TUNEL assay.RESULTS: Compared to the liposome control, the levels of survivin mRNA and protein in siRNA group were 0.28±0.07 and 0.34±0.05 (n=3, P<0.05) respectively 72 h after transfection. The apoptotic rate of the cells in pSi-sur group was significantly higher than that in two control groups as showed in acridine orange staining, Annexin V-FITC labeled flow cytometry and TUNEL assay.CONCLUSION: Survivin siRNA significantly inhibits the expression of survivin both in mRNA and protein levels, and induces apoptosis of DU145 cells in vitro.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Effect of siRNA for survivin gene on growth and apoptosis in A549 cells

AIM:To observe the influence of lentiviral vectors expressing siRNA for survivin gene knockdown in A549 cells, sequentially as tools to explore the molecule pathogenesis and new gene therapy of lung adenocarcinoma. METHODS:The lentiviral vectors, which express survivin siRNA, were constructed and transfected into A549 cell strain. The titers of the lentiviruses were determined by 293T cells. The expressions of survivin and caspase-3 were detected by Western blotting and RT-PCR. The cell cycle and cell growth of A549 cells were examined by MTT and FCM．RESULTS:The expression of survivin was suppressed effectively by siRNA targeting survivin. The expression of survivin mRNA decreased by 97%. The expression of survivin protein decreased by 94%. The rate of cell growth was decreased. The G1 phase cells were increased, whereas S phase cells were decreased. CONCLUSION:The lentivirus vectors expressing siRNA for survivin can significantly inhibit gene expression and the cell growth, and markedly induce the apoptosis. It is hopeful to be a new gene therapy of lung adenocarcinoma.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels. METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z). Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels. The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope. The chloride current was recorded by the patch-clamp technique. RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK1 potassium. The mRNA expression of ClC-3 was increased by the IK1 siRNA. IK1 siRNA inhibited the expression of ClC-3 protein. A chloride current was activated by hypotonic challenges, and the hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.     

Curcumin inhibits migration and invasion of lung cancer PC-9 cells via down-regulation of nectin-4 expression

AIM: To investigate the effects of curcumin on the abilities of migration and invasion in the lung cancer PC-9 cells, and to observe the relationship between curcumin and nectin-4 expression.METHODS: The viability, migration and invasion of lung cancer PC-9 cells treated with curcumin or transfected with siNectin-4 were measured by MTT assay, wound healing test and Transwell assay, respectively. The protein levels of nectin-4, p-AKT and AKT in the PC-9 cells treated with curcumin or transfected with siNectin-4 were detected by Western blot.RESULTS: Curcumin inhibited the viability of PC-9 cells. The wound healing rates and the numbers of the transmembrane cells in curcumin 10 μmol/L and 20 μmol/L groups were decreased compared with control group without curcumin treatment. The expression level of nectin-4 was reduced after curcumin treatment for 24 h. The viability of the PC-9 cells was significantly inhibited after transfected with siNectin-4 for 48 h or 72 h (P<0.01), and the wound healing rates was decreased in siNectin-4 group compared with NC group (P<0.01). The numbers of the transmembrane cells in siNectin-4 group was significantly reduced (P<0.01). Curcumin and knockdown of nectin-4 suppressed the activation of AKT pathway in PC-9 cells. In siNectin-4+curcumin group, the cell viability reduced compared with curcumin group, and wound healing rates, cell invasive ability and AKT phosphorylation levels were decreased.CONCLUSION: Curcumin inhibits migration and invasion of the lung cancer PC-9 cells via down-regulation of nectin-4 expression and inhibition of AKT pathway.     

Cholesterol depletion up-regulates expression of prohibitin in human prostate carcinoma PC-3 cells

AIM: To study how cholesterol delpetion affects prohibitin expression and the development of prostate cancer. METHODS: Human prostate carcinoma PC-3 cells were cultured in normal medium (NM) and cholesterol delpetion medium (CDM) for 48 h.The mRNA expression of prohibitin was detected by real-time PCR. Prohibitin promoter was cloned and inserted into PGL3-Basic to reconstruct plasmid. The plasmid was transiently transfected into PC-3 cells. The cells were cultured in the medium with different concentrations of cholesterol for 48 h and luciferase expression was detected.RESULTS: The results of real-time PCR showed that the mRNA level of prohibitin increased about 19 times in PC-3 cells in the presence of CDM. After transfected with prohibitin promoter plasmid for 48 h, PC-3 cells cultured in CDM had higher luciferase expression than the cells cultured in NM or in CDM with cholesterol. CONCLUSION: Cholesterol depletion up-regulates prohibitin expression in PC-3 cells, which may be one of the self-prophylactic and regulatory mechanisms to protect PC-3 cells from apoptosis caused by cholesterol insufficiency.     

NKX3.1 down-regulates bcl-2 gene expression in prostate cancer PC-3 cells

AIM: To investigate the effect of NKX3.1 on the gene expression of bcl-2 and apoptosis in prostate cancer PC-3 cells. METHODS: The eukaryotic expression plasmid pcDNA3.1- NKX3.1 was transiently transfected into PC-3 cells. RT-PCR and Western blotting were used to detect the effects of NKX3.1 on the expression of bcl-2 gene. Down-regulatory effect of NKX3.1 on bcl-2 gene was detected by EMSA. Flow cytometry and apoptotic body staining were carried out to study the effects of NKX3.1 on apoptosis of PC-3 cells. RESULTS: The mRNA and protein expression of bcl-2 in PC-3 cells was down-regulated by over-expression of NKX3.1. The EMSA result showed that NKX3.1 interacted with the NKX3.1 binding elements in upstream regulatory region of bcl-2 gene. The results of flow cytometry showed that the number of apoptotic PC-3 cells increased by 1.41-fold after NKX3.1 transfection to PC-3 cells. NKX3.1 increased the apoptotic bodies stained by Hoechst 33258 significantly. CONCLUSION: NKX3.1 down-regulates the expression of anti-apoptotic gene bcl-2 and induces the apoptosis of prostate cancer PC-3 cells.     

Effect of ginsenoside Rh2 on cultured PC-3M cells in vitro

AIM:To explore the influence of ginsenoside Rh₂ on cultured PC-3M cell cycle in vitro. METHODS:DNA content was measured using [³H]-TdR incorporation assay. To analysis the cycle changes of PC-3M cells, flow cytometry was used in the experiment.RESULTS:The results indicated that the rate of [³H]-TdR incorporation was lower in Rh₂-treated cells than the control in a dose-dependent manner. Flow cytometry demonstrated that the pecent of G₁ phase treated with Rh₂ was higher than that of control. CONCLUSION:These results suggested that PC-3M cells cultured with Rh₂ for 24 h were blocked at G₁ phase, and Rh₂ inhibited the growth of PC-3M cells in vitro.     

Knockdown of astrocyte elevated gene-1 by siRNA inhibits cell proliferation and induces apoptosis in human neuroblastoma cell line

AIM: To investigate the effect of siRNA-induced astrocyte elevated gene-1 ( AEG-1 ) down-regulation on the proliferation, apoptosis and cell cycle of neuroblastoma cells. METHODS: An siRNA targeting to AEG-1 mRNA (AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000. A non-specific siRNA (control siRNA) and non-treatment were used as negative control and blank control,respectively . The cell proliferation was detected by MTT method and colony formation assay. The apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Compared with control groups, the expression of AEG-1 mRNA was evidently declined in the cells transfected with AEG-1 siRNAs (P<0.05). AEG-1 siRNA significantly decreased the cell proliferation. After treated with AEG-1 siRNA for 48 h, the percentage of apoptotic cells was significant increased and the cell cycle was arrested in G₀/G₁ phase compared with the control cells (P<0.05). CONCLUSION: The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in neuroblastoma cells. Knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits cell proliferation and apoptosis, and induces cell arrest in G₀/G₁ phase of the cell cycle.     

Ginsenoside Rh2 enhances the apoptotic effect ofcisplatin on PC-3M prostatic cancer cells

AIM:To study the inducing apoptotic effect of Cisplatin (DDP) combined with Ginsenoside Rh₂ on PC-3M prostatic cancer cells in vitro.METHODS:Cell viability was examined by MTT test and the apoptosis of PC-3M by electrophoresis of NP-40 extracting fragmental DNA and flowcytometry.RESULTS:Pc-3M cells were exposed to 15.0 mg/L Rh₂ combined with 0.4 mg/L DDP: ①Cell viabililty inhibitory rate was increased by 1.44 times from 25.0% (with 0.4 mg/L DDP) to 61.03%. ②Characteristic apoptotic DNA laddering was found more obviously than using either agent alone. ③Apoptotic rate of PC-3M was increased from 0.32% (with 0.4 mg/L DDP) to 52.2%, which showed similar effect with 4.0 mg/L DDP, it was much higher than that of 15.0 mg/L Rh₂ alone (13.6%) (P＜0.01).CONCLUSION:Rh₂ greatly enhances the apoptotic effect of DDP on PC-3M prostatic cancer cells.     

Effects of RUNX3 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of RUNX3 gene on the growth and drug sensitivity of SH-SY5Y cells.METHODS: The siRNA plasmid of RUNX3 was constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting. The growth curve, cell cycle distribution, drug sensitivity assay and accumulation of adriamycin in cells were detected by MTT assay and flow cytometry. The expressions of cyclin D1, CDK4, CDK6, p21, p27, Bcl-2, Bax, P-gp and MRP were analyzed by Western blotting. RESULTS: mU6pro-RUNX3 siRNA was successfully constructed and transfected into SH-SY5Y cells. Down-regulation of RUNX3 significantly promoted the cellular proliferation, inhibit the drug sensitivity and intracellular adriamycin accumulation of cells, compared with that in the controls (P<0.05). The expressions of P-gp, Bcl-2 and cyclin D1 in transfected cells were increased, while p21 decreased.CONCLUSION: RUNX3 might play important roles in the development of neuroblastoma.     

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones

AIM: To inhibit specifically the u-PAR expression in highly invasive cell subclones and block its function in those cells invasion. METHODS: A cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in the reverse direction. Then an antisense RNA expression plasmid for u-PAR was constructed and transfected into highly invasive cell subclones. The u-PAR expression in resistant cells was examined by RT-PCR and immunohistochemical assay. RESULTS: Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively. CONCLUSION: The results indicated that an antisense vector for u-PAR might played a specific inhibitory role in the cells. This model is useful for observing the inhibitory effects of the antisense vector for u-PAR on invasion by highly invasive cell subclones of human prostate carcinoma.     

3-MA combined with TSA inhibits growth of triple-negative breast cancer cells

AIM: To investigate the effect of 3-methyladenine (3-MA) combined with trichostatin A (TSA) on triple-negative breast cancer cells and the mechanism involved. METHODS: The viability of MDA-MB-231 cells was detected by MTT assay, the migration ability was determined by scratch assay, and the expression of autophagy-related proteins was detected by Western blot. RESULTS: TSA significantly inhibited the viability of MDA-MB-231 cells in a time-and dose-dependent manner. The results of scratch assay showed that TSA inhibited cell migration ability. Western blot data indicated that TSA resulted in a moderate increase in LC3-Ⅱ expression. Moreover, 3-MA inhibited cell autophagy induced by TSA, and combination of 3-MA and TSA further inhibited the viability of MDA-MB-231 cells. CONCLUSION: Combination of 3-MA and TSA may effectively inhibit the growth of triple-negative breast cancer cells.     

CD147 affects autophagy of prostate cancer PC-3 cells

AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.     

矮化中间砧‘宫藤富士’苹果栽植密度对树体生长、冠层光照和果实产量的影响

以2011年春季定植的矮化中间砧苹果成品苗(3年根1年干的‘宫藤富士’/SH6/平邑甜茶)为试材,设置7种不同的栽植密度(株行距分别为1 m×3 m、1.5 m×3 m、2 m×3 m、0.75 m×4 m、1 m×4 m、1.25 m×4 m和1.5 m×4 m),细纺锤形整枝修剪,自栽植第2年,连续7年调查7种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长,不同栽植密度下树干粗度和总枝量逐年增加,不同处理间树干粗度无显著差异,第7年1 m×3 m和0.75 m×4 m两个栽植密度下树体总枝量超过140万条·hm-2,第8年均超过140万条·hm^-2。栽植前期(第2～4年)各栽植密度树体短枝比例不断增加,长枝比例不断减少,第5年各栽植密度枝类组成趋于稳定;综合稳产3年(第6～8年)树体的枝类组成数据,4 m行距的短枝比例明显高于3 m行距,长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为1.5 m×4 m(63.87%)、1.25 m×4 m(61.44%)、2 m×3 m(61.27%)、1 m×4 m(59.19%)、...     

Inhibition of survivin expression in BGC-823 cells by RNA interference

AIM: To investigate the feasibility and specificity of gastric carcinoma gene therapy by utilizing RNA interference (RNAi) to inhibit survivin expression in vitro and in vivo. METHODS: Small interference RNA (siRNA) homologous to survivin was designed. pTZU6+1-siRNA-survivin vector was constructed and transfected into BGC-823 cells. The transplanted BGC-823 tumor in nude mice was established to induce RNAi. The changes of survivin gene expression, tumor cell cycle and cell apoptosis were detected by flow cytometry, RT-PCR, Western blotting, immunochemistry and TUNEL. RESULTS: The expression of survivin was obviously inhibited by RNAi in vitro. The phase of cell cycle indicated the reduction of S phase, while G1/G0 phase increased. Cell apoptosis was obvious. Both the mRNA level and the protein expression of survivin decreased obviously. The tumor size reduced after treated with pTZU6+1-siRNA-survivin vector in vivo. The expression of survivin decreased in siRNA treatment group. In contrast, little change in control group in vitro and in vivo was observed. CONCLUSION: RNA interference down-regulates survivin gene expression, inhibits BGC-823 cell proliferation and induces cell apoptosis with good specificity, which may be a possible new approach for neoplasm gene therapy.     

Knockdown of growth hormone receptor prevents growth hormone induced inflammatory cytokine production in 3T3-L1 adipocytes

AIM: To investigate the effect of growth hormone receptor (GHR) knockdown on nuclear factor-κB (NF-κB) activity and inflammatory cytokine production stimulated by growth hormone (GH) in 3T3-L1 adipocytes. METHODS: The specific siRNA for GHR was transfected into 3T3-L1 adipocytes to silence GHR expressions. The effects of GH on NF-κB activation and inflammatory cytokine production in 3T3-L1 adipocytes transfected with siRNA-GHR or siRNA-control were measured by dual-luciferase system analysis, real-time RT-PCR and ELISA. RESULTS: The protein expression of GHR was diminished after transfection with GHR specific siRNA. Dual-luciferase reporter system analysis revealed that GHR knockdown resulted in attenuation of GH-stimulated NF-κB activation in the 3T3-L1 adipocytes. GHR knockdown ameliorated the GH-induced production of inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in the 3T3-L1 adipocytes. CONCLUSION: Knockdown of GHR might be efficacious to prevent GH-induced inflammatory responses in the 3T3-L1 adipocytes.