Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and pr...Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.展开更多
Antisense oligonucleotide(ASO)for anti-apoptosis is emerging as a highly promising therapeutic agents for ischemic stroke with complex pathological environment.However,its therapeutic efficacy is seriously limited by ...Antisense oligonucleotide(ASO)for anti-apoptosis is emerging as a highly promising therapeutic agents for ischemic stroke with complex pathological environment.However,its therapeutic efficacy is seriously limited by a number of challenges including inefficient internalization,low blood-brain barrier(BBB)penetration,poor stability,and potential toxicity of the carrier.Herein,a carrier-free programmed spherical nucleic acid nanostructure is developed for effective ischemic stroke therapy via integrating multifunctional modules into one DNA structure.By co-encoding caspase-3-ASO and transferrin receptor(TfR)aptamer into circle template,the spherical nucleic acid nanostructure(TD)was obtained via self-assembly.The experimental results demonstrated that the developed TD displayed efficient BBB penetration capability(6.4 times)and satisfactory caspase-3 silence effect(2.3 times)due to the dense DNA packaging in TD.Taken together,our study demonstrated that the carrier-free programmed spherical nucleic acid nanostructure could significantly improve the therapeutic efficacy of ischemic stroke and was a promising therapeutic tool for various brain damage-related diseases.展开更多
目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),...目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib+300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.展开更多
3′ O (甲硫甲基 )缩醛的脱氧核苷 (1)与N 碘代丁二酰亚胺 (NIS)和二苯基次膦酸反应得到相应的 3′ O (二苯膦酰氧 )甲基缩醛 (2 )。在三甲基硅三氟甲磺酸酯 (TMSOTf)的条件下 ,后者与 3′ 位保护的脱氧核苷 (5 )缩合 ,得到 (3′→ 5′...3′ O (甲硫甲基 )缩醛的脱氧核苷 (1)与N 碘代丁二酰亚胺 (NIS)和二苯基次膦酸反应得到相应的 3′ O (二苯膦酰氧 )甲基缩醛 (2 )。在三甲基硅三氟甲磺酸酯 (TMSOTf)的条件下 ,后者与 3′ 位保护的脱氧核苷 (5 )缩合 ,得到 (3′→ 5′)甲缩醛连接的二聚体d[(B1 m B2 ) ](m =次甲基 ) (6)。同样的 ,2 (B =T或B =C)与 3′ 位保护的二聚体 9(T m T或C m T)缩合 ,可得到相应的三聚体 10 (T m T m T ,C m T m T和T m C m T)。展开更多
Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal mus...Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.展开更多
目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分...目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分为6组:①空白对照组;②脂质体转染对照组;③正义链转染对照组;④200nmol/L AS ODN转染组;⑤400nmol/L AS ODN转染组;⑥600nmol/L AS ODN转染组。作用20小时后收获各组细胞,并进行后续实验:①倒置显微镜下观察细胞形态变化;②western blot法检测各组细胞survivin蛋白的表达情况;③流式细胞术检测细胞增殖和凋亡指数;④比色法检测各组细胞caspase-3的活性变化。结果:①AS ODN转染组细胞survivin表达减弱,以600nmol/L转染组最为明显,各对照组survivin蛋白表达无明显改变;②AS ODN转染组细胞形态出现变圆、折光增强、漂浮、细胞碎片形成等变化,而各对照组细胞生长良好;③各AS ODN转染组细胞凋亡指数明显高于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义(P>0.05);④各AS ODN转染组细胞增殖指数明显低于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义;⑤在各AS ODN转染组中,可见到不同程度的caspase-3的活化,而对照组细胞内无明显的变化。结论:survivin AS ODN转染能下调survivin蛋白表达,诱导肝癌细胞凋亡,抑制细胞增殖,其作用途径可能涉及抑制survivin表达,诱导caspase-3活化,启动凋亡信号传导。展开更多
文摘Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.
基金This work was funded by the National Natural Science Foundation of China(Nos.82073395 and 319009919)Postdoctoral Science Foundation of China(Nos.2020TQ0288).
文摘Antisense oligonucleotide(ASO)for anti-apoptosis is emerging as a highly promising therapeutic agents for ischemic stroke with complex pathological environment.However,its therapeutic efficacy is seriously limited by a number of challenges including inefficient internalization,low blood-brain barrier(BBB)penetration,poor stability,and potential toxicity of the carrier.Herein,a carrier-free programmed spherical nucleic acid nanostructure is developed for effective ischemic stroke therapy via integrating multifunctional modules into one DNA structure.By co-encoding caspase-3-ASO and transferrin receptor(TfR)aptamer into circle template,the spherical nucleic acid nanostructure(TD)was obtained via self-assembly.The experimental results demonstrated that the developed TD displayed efficient BBB penetration capability(6.4 times)and satisfactory caspase-3 silence effect(2.3 times)due to the dense DNA packaging in TD.Taken together,our study demonstrated that the carrier-free programmed spherical nucleic acid nanostructure could significantly improve the therapeutic efficacy of ischemic stroke and was a promising therapeutic tool for various brain damage-related diseases.
文摘目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib+300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.
文摘3′ O (甲硫甲基 )缩醛的脱氧核苷 (1)与N 碘代丁二酰亚胺 (NIS)和二苯基次膦酸反应得到相应的 3′ O (二苯膦酰氧 )甲基缩醛 (2 )。在三甲基硅三氟甲磺酸酯 (TMSOTf)的条件下 ,后者与 3′ 位保护的脱氧核苷 (5 )缩合 ,得到 (3′→ 5′)甲缩醛连接的二聚体d[(B1 m B2 ) ](m =次甲基 ) (6)。同样的 ,2 (B =T或B =C)与 3′ 位保护的二聚体 9(T m T或C m T)缩合 ,可得到相应的三聚体 10 (T m T m T ,C m T m T和T m C m T)。
基金supported by the National Natural Science Foundation of China,Nos.82171172(to RZ)and 81771366(to RZ)Fundamental Research Funds for the Central Universities of Central South University,Nos.2021zzts1095(to SZ)and 2022zzts0832(to HY)。
文摘Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.
文摘目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分为6组:①空白对照组;②脂质体转染对照组;③正义链转染对照组;④200nmol/L AS ODN转染组;⑤400nmol/L AS ODN转染组;⑥600nmol/L AS ODN转染组。作用20小时后收获各组细胞,并进行后续实验:①倒置显微镜下观察细胞形态变化;②western blot法检测各组细胞survivin蛋白的表达情况;③流式细胞术检测细胞增殖和凋亡指数;④比色法检测各组细胞caspase-3的活性变化。结果:①AS ODN转染组细胞survivin表达减弱,以600nmol/L转染组最为明显,各对照组survivin蛋白表达无明显改变;②AS ODN转染组细胞形态出现变圆、折光增强、漂浮、细胞碎片形成等变化,而各对照组细胞生长良好;③各AS ODN转染组细胞凋亡指数明显高于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义(P>0.05);④各AS ODN转染组细胞增殖指数明显低于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义;⑤在各AS ODN转染组中,可见到不同程度的caspase-3的活化,而对照组细胞内无明显的变化。结论:survivin AS ODN转染能下调survivin蛋白表达,诱导肝癌细胞凋亡,抑制细胞增殖,其作用途径可能涉及抑制survivin表达,诱导caspase-3活化,启动凋亡信号传导。
