第10号染色体上磷酸酶和张力蛋白同源缺失的基因对人卵巢癌A2780细胞侵袭和迁移能力的影响及相关机制

目的 探讨第10号染色体上磷酸酶和张力蛋白同源缺失的基因(PTEN)对人卵巢癌A2780细胞侵袭和迁移能力的影响及相关机制.方法 将携带外源性人PTEN基因的野生型质粒(WT-PTEN)和磷酸酶域突变型质粒(C124A-PTEN)转入人卵巢癌细胞株A2780中,利用Transwell小室法和划痕愈合实验检测转染前后细胞侵袭和迁移能力的变化,应用Western blot检测各组细胞PTEN及其下游相关蛋白表达的变化.以空载体质粒和未转染细胞作为对照.结果 细胞侵袭实验显示,WT-PTEN/A2780、C124A-PTEN/A2780、GFP/A2780和A2780细胞穿透Matrigel胶的细胞数,分别为(24.3±2.5)个、(43.7±3.8)个、(44.7±2.1)个和(45.0±3.0)个,WT-PTEN/A2780细胞的穿膜数明显少于其他各组(P＜0.05).划痕愈合实验显示,WT-PTEN/A2780、C124A-PTEN/A2780、GFP/A2780和A2780细胞的迁移距离分别为(54.1±3.7)μm、(78.7±3.4)μm、(78.1±3.1)μm和(76.8±3.5)μm,WT-PTEN/A2780细胞的迁移速度明显慢于C124A-PTEN/A2780、GFP/A2780和A2780细胞(P＜0.05).WT-PTEN/A2780细胞中基质金属蛋白酶9(MMP-9)蛋白的表达明显低于C124A-PTEN/A2780、GFP/A2780和A2780细胞(P＜0.05).结论 野生型PTEN可有效抑制A2780细胞的侵袭和迁移,该抑制作用与A2780细胞中MMP-9表达下调有关.

Abstract：

Objectiye To evaluate the effects of PTEN on invasive and migration ability of human ovarian cancer cell line A2780 and related mechanisms. Methods The plasmid including WT-PTEN and mutant PTEN were transferred into A2780 cells. The invasive and migration ability were measured before and after transfection by transwell chamber and wound-healing assays. The expression of PTEN protein and related proteins in the cells were detected by Western blot analysis. Empty plasmid-transfected A2780 and normal A2780 cells were used as control (the different four groups were named as WT-PTEN/A2780,C124A-PTEN/A2780, GFP/A2780 and A2780 ). Results The number of penetrating cells was significantly less in WT-PTEN/A2780 cells(24.3 ± 2.5 ) than those in C124A-PTEN/A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8, 44.7 ± 2. 1 and 45.0 ± 3.0 ) ( P ＜ 0.05 ). The migration distance was markedly shorter in WT-PTEN/A2780 cells (54.1 ± 3.7) than those in C124A-PTEN/A2780, GFP/A2780 and A2780 cells (78.7 ± 3.4, 78.1 ± 3.1 and 76.8 ± 3.5 ) ( P ＜ 0. 05 ). Conclusions Transfection with PTEN may suppress the invasive and migration ability of ovarian cancer cell line A2780 depending on its phosphatase activity, and the suppressive effect may be due to the down-regulation of MMP-9 in the cancer cells.     

PTEN基因增强卵巢癌A2780细胞对紫杉醇敏感性的研究

背景与目的:近来的研究表明PTEN基因(phosphatase and tensin homology deleted on chromosome ten,第10号染色体上磷酸酶和张力蛋白同源缺失的基因)失活在肿瘤化疗耐药发挥了重要作用。本文研究外源性PTEN基因稳定转染人卵巢癌A2780细胞后对紫杉醇敏感性的影响。方法:将野生型PTEN基因真核表达质粒在脂质体介导下转染人卵巢癌A2780细胞,同时以转染空载体和未转染细胞作为对照(3组细胞分别命名为WT-PTEN/A2780,GFP/A2780和A2780),分别应用RT-PCR和蛋白印迹技术检测各组细胞PTEN mRNA转录和蛋白表达的变化;四甲基偶氮唑蓝实验观察转染PTEN基因对肿瘤细胞生长的抑制作用和A2780细胞对紫杉醇药物敏感性的影响,流式细胞仪(FACS)分析细胞凋亡情况。结果:与对照组相比,转染野生型PTEN基因能明显增加A2780细胞PTENmRNA转录和蛋白的表达,差异有显著性(P<0.05);MTT法显示,WT-PTEN/A2780细胞生长明显慢于GFP/A2780和A2780细胞;紫杉醇对WT-PTEN/A2780,GFP/A2780和A2780的IC50值分别为(7.6±0.40)nmol/l、(22.5±0.9)nmol/l和(22.7±0.8)nmol/l,WT-PTEN/A2780细胞对紫杉醇药物敏感性显著增加(P<0.05);FACS分析显示,紫杉醇作用24h后,WT-PTEN/A2780,GFP/A2780和A2780的凋亡率分别为(34.6±1.6)%、(13.5±0.9)%和(12.7±1.0)%,差异有显著性(P<0.05)。结论:转染野生型PTEN重组质粒能有效提高A2780细胞内PTEN的表达,诱导A2780细胞凋亡,显著增加A2780细胞对紫杉醇杀伤的敏感性。     

短发卡状RNA抑制人Bubl基因表达及其对人卵巢癌A2780细胞药物敏感性和周期的影响

背景与目的:既往研究提示,紫杉醇类药物作用的发挥有赖于功能完整的纺锤体检查点,而Bubl是纺锤体检查点的重要组成部分.本研究旨在探讨Bubl短发卡状RNA真核表达载体稳定转染对人卵巢癌A2780细胞紫杉醇敏感性及细胞周期的影响.方法:构建Bubl基因短发卡状RNA真核表达载体pEGFP-Bubl-shRNA,以脂质体Lipofectamine~(TM)2000包裹空质粒转染体外培养的人卵巢癌细胞株A2780后进行稳定筛选.应用RT-PCR、Western印迹法分析目的基因mRNA及其蛋白水平的表达情况:MTT法、流式细胞术等方法检测转染前后,A2780细胞对紫杉醇敏感性及其细胞周期的变化;Hoechst33342染色法检测细胞分裂指数.结果:与对照组pEGFP-Cl/A2780及A2780组相比,pEGFP-Bubl-ShRNA/A2780组细胞中mRNA和蛋白水平的表达均明显下降,其差异具有统计学意义(P＜0.05);MTT检测结果提示,转染pEGFP-Bubl-shRNA质粒后细胞对紫杉醇的敏感性明显降低,与对照组相比,差异具有统计学意义(P＜0.05);流式细胞术检测结果显示,紫杉醇1 μ mol/L处理细胞24及48 h后,与pEGFP-Cl/A2780及A2780组相比,该组细胞凋亡率明显降低(P＜0.05),G_2/M期细胞比例下降,差异具有统计学意义(P＜0.05);Hoechst33342染色提示,与对照组相比,pEGFP-Bubl-shRNA/A2780组的分裂指数(MI)明显降低,差异具有统计学意义(P＜0.05).结论:Bubl基因的下调可导致人卵巢癌A2780细胞对紫杉醇敏感性及G_2/M期细胞比例下降.pEGFP-Cl-shBubl质粒的成功构建,为研究Bubl基因的功能及定位的研究提供了进一步的实验条件.     

抑癌基因PTEN对肝癌细胞增殖的抑制及作用机制

目的探讨抑癌基因PTEN对肝癌细胞增殖和细胞周期的调控作用。方法构建野生型PTEN基因和突变型PTEN基因的真核表达载体pEGFP—WT—PTEN和pEGFP—PTEN;G129R。采用脂质体介导的基因转染法,分别将上述载体转染不表达PTEN蛋白的人肝细胞肝癌细胞系HHCC,经G418筛选,获得稳定表达PTEN蛋白的细胞克隆。以流式细胞仪测定细胞周期,以Western blot法分析稳定表达PTEN蛋白的肝癌细胞内源性的磷酸化AKT表达水平,同时与未进行基因转染和转染空载体pEGFP-C1的HHCC细胞进行对照。结果稳定表达野生型PTEN蛋白的肝癌细胞,细胞生长受到明显抑制,与转染空载体的HHCC细胞比较,G1期细胞比例显著增高,G2期和S期细胞比例显著降低,且差异有统计学意义（P〈0．05）;而转染突变型PTEN基因的HHCC细胞,与转染空载体的HHCC细胞比较,差异无统计学意义（P〉0.05）。与转染空载体的HHCC细胞比较,稳定表达野生型PTEN蛋白的HHCC细胞,其内源性磷酸化AKT水平明显减低;而转染突变型PTEN基因的HHCC细胞,其AKT水平无明显变化。结论野生型PTEN基因对肝癌细胞周期具有调控作用,而突变型PTEN基因丧失对肝癌细胞周期的调控作用;野生型PTEN基因可能通过降低AKT的活化而实现对肝癌细胞周期的调控。     

短发卡状RNA抑制人Bub1基因表达及其对人卵巢癌A2780细胞药物敏感性和周期的影响

背景与目的:既往研究提示,紫杉醇类药物作用的发挥有赖于功能完整的纺锤体检查点,而Bubl是纺锤体检查点的重要组成部分.本研究旨在探讨Bubl短发卡状RNA真核表达载体稳定转染对人卵巢癌A2780细胞紫杉醇敏感性及细胞周期的影响.方法:构建Bubl基因短发卡状RNA真核表达载体pEGFP-Bubl-shRNA,以脂质体Lipofectamine~(TM)2000包裹空质粒转染体外培养的人卵巢癌细胞株A2780后进行稳定筛选.应用RT-PCR、Western印迹法分析目的基因mRNA及其蛋白水平的表达情况:MTT法、流式细胞术等方法检测转染前后,A2780细胞对紫杉醇敏感性及其细胞周期的变化;Hoechst33342染色法检测细胞分裂指数.结果:与对照组pEGFP-Cl/A2780及A2780组相比,pEGFP-Bubl-ShRNA/A2780组细胞中mRNA和蛋白水平的表达均明显下降,其差异具有统计学意义(P＜0.05);MTT检测结果提示,转染pEGFP-Bubl-shRNA质粒后细胞对紫杉醇的敏感性明显降低,与对照组相比,差异具有统计学意义(P＜0.05);流式细胞术检测结果显示,紫杉醇1 μ mol/L处理细胞24及48 h后,与pEGFP-Cl/A2780及A2780组相比,该组细胞凋亡率明显降低(P＜0.05),G_2/M期细胞比例下降,差异具有统计学意义(P＜0.05);Hoechst33342染色提示,与对照组相比,pEGFP-Bubl-shRNA/A2780组的分裂指数(MI)明显降低,差异具有统计学意义(P＜0.05).结论:Bubl基因的下调可导致人卵巢癌A2780细胞对紫杉醇敏感性及G_2/M期细胞比例下降.pEGFP-Cl-shBubl质粒的成功构建,为研究Bubl基因的功能及定位的研究提供了进一步的实验条件.     

增强c-Jun氨基末端激酶活性可逆转卵巢癌的顺铂耐药性

目的: 探讨卵巢癌的顺铂(cisplatin,CDDP)耐药以及如何促使其逆转的机制.方法:将携带c-Jun氨基末端激酶1(c-Jun NH2-terminal kinase 1,JNK1)显性突变体的质粒pcDNA3(FLAG)-JNK1-dn及表达野生型JNK1的质粒pcDNA3(FLAG)-JNK1-wt分别转染卵巢癌A278细胞和A2780/DDP耐药细胞株.20 μmol/L CDDP作用后,检测细胞中p-JNK及下游p-c-Jun的表达,观察2株细胞的凋亡率及CDDP作用后2株细胞中Fas/FasL的表达.结果:CDDP作用后A2780细胞中p-JNK及下游p-c-Jun持续表达,而A2780/DDP细胞中仅有短暂表达.pcDNA3(FLAG)-JNK1-dn转染A2780细胞可抑制CDDP诱导的JNK1活性,并使细胞的凋亡率明显低于未转染组[(18.3±0.4)% vs (47.3±4.7)%, P <0.01].pcDNA3(FLAG)-JNK1-wt转染可使JNK1持续活化,FasL表达上调,A2780/DDP细胞凋亡率明显高于未转染组[(36.9±1.6)% vs (10.9±1.7)%, P <0.01]. 结论:JNK信号转导通路在CDDP诱导的卵巢癌细胞凋亡中起重要作用.野生型JNK1基因转染使A2780/DDP细胞对CDDP的耐药性得到逆转.     

抑癌基因OVCA1体外对卵巢癌细胞A2780迁移和侵袭的抑制作用

目的:探讨卵巢癌基因1[ovarian cancer gene 1,OVCA1;也称为DPH2L( diphthamide synthesis protein 2-like)基因]体外对卵巢癌细胞系A2780迁移和侵袭能力的抑制作用.方法:采用脂质体法将GFP标记的携带OVCA1的重组质粒pEGFP-OVCA1或GFP空白质粒分别转染A2780细胞后,G418筛选稳定转染细胞,有限稀释法筛选稳定转染细胞的单克隆细胞株,荧光显微镜检查绿色荧光蛋白的表达及RT-PCR方法检测A2780细胞OVCA1基因mRNA的表达.A2780-OVCA1细胞为实验组, A2780-GFP细胞和A2780细胞为对照组,采用划痕实验、Transwell体外迁移实验和Matrigel体外侵袭实验检测OVCA1对A2780细胞迁移和侵袭能力的影响.结果:重组质粒pEGFP-OVCA1转染后获得了稳定表达GFP标记OVCA1蛋白的A2780细胞株.A2780-OVCA1组划痕后的细胞迁移速度明显小于两对照组(P<0.05);A2780-OVCA1组穿过Transwell滤膜的迁移细胞数明显少于两对照组(P<0.05);A2780-OVCA1组穿过Matrigel滤膜的侵袭细胞数明显少于两对照组(P<0.05).结论:OVCA1在体外具有显著抑制卵巢癌A2780细胞迁移和侵袭的作用.     

Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.     

Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.     

Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.     

Radiation sensitivity of merkel cell carcinoma cell lines

: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

Inhibition of S-adenosylhomocysteine hydrolase decreases cell mobility and cell proliferation through cell cycle arrest

S-adenosylhomocysteine hydrolase (AHCY) hydrolyzes S-adenosylhomocysteine to adenosine and l-homocysteine, and it is already known that inhibition of AHCY decreased cell proliferation by G2/M arrest in MCF7 cells. However, the previous study has not indicated what mechanism the cell cycle arrest is induced by. In this study, we aimed to investigate the different cell cycle mechanisms in both p53 wild-typed MCF7 and p53 mutant-typed MCF7-ADR by suppressing AHCY. We extensively proved that AHCY knockdown has an anti-proliferative effect by using the WST-1 assay, BrdU assay, and cell cytometry analysis and an anti-invasive, migration effect by wound-healing assay and trans-well analysis. Our study showed that down-regulation of AHCY effectively suppressed cell proliferation by regulating the MEK/ERK signaling pathway and through cell cycle arrests. The cell cycle arrest occurred at the G2/M checkpoint by inhibiting degradation of cyclinB1 and phosphorylation of CDC2 in MCF7 cells and at the G1 phase by inhibiting cyclinD1 and CDK6 in MCF7-ADR cells. Finally, we determined that AHCY regulates the expression of ATM kinase that phosphorylates p53 and affects to arrest of G2/M phase in MCF7 cells. The findings of this study significantly suggest that AHCY is an important regulator of cell proliferation through different mechanism in between MCF7 and MCF7-ADR cells as p53 status.     

Mechanisms of kaposis-sarcoma cell supernatant-induced vascular cell invasion

Kaposi's sarcoma (KS) is a highly angiogenic lesion frequently associated with acquired immune deficiency syndrome. Histologically the lesions appear to contain proliferative 'spindle shaped' cells with a mixed smooth muscle-endothelial-fibroblastic histotype and a conspicuous neovascularization, derived from host cell recruitment. Media conditioned by cultured KS cells (KS-CM) have angiogenic properties. KS-CM is able to promote endothelial and smooth muscle cell migration and invasion. The mechanisms of this KS-CM activity are still unknown. We hypothesize that KS-CM contains numerous factors with different roles in inducing the neo angiogenic process. We show that AIDS-IST-KS cell supernatants induce gelatinase A production and plasminogen activator (PA) up-regulation in vascular cells. KS-CM activity in vivo is heparin dependent. Also bFGF alone, a heparin dependent factor, alone can induce endothelial and smooth muscle cell invasion, MMP-2 production and PA activity. However, antibodies to bFGF do not block KS-CM activity and do not reduce the effect on PA up-regulation. This evidence suggests that heparin-binding factors other than bFGF may be present. Chromatography of KS-CM on heparin-sepharose demonstrates the presence of two heparin-binding fractions with chemotactic and gelatinase A inducing activity. The flow through was also active. KS-CM absorption on heparin-sepharose beads did not modify its induction of PA activity, further evidence for the presence of non heparin-binding factors as well.     

Landscape of immune cell infiltration in clear cell renal cell carcinoma to aid immunotherapy

The tumor microenvironment, comprised of tumor cells and tumor-infiltrating immune cells, is closely associated with the clinical outcome of clear cell renal cell carcinoma (ccRCC) patients. However, the landscape of immune infiltration in ccRCC has not been fully elucidated. Herein, we applied multiple computational methods and various datasets to reveal the immune infiltrative landscape of ccRCC patients. The tumor immune infiltration (TII) levels of 525 ccRCC patients using a single-sample gene were examined and further categorized into immune infiltration subgroups. The TII score was characterized by distinct clinical traits and showed a significant divergence based on gender, grade, and stage. A high TII score was associated with the ERBB signaling pathway, the TGF-β signaling pathway, and the MTOR signaling pathway, as well as a better prognosis. Furthermore, patients with high TII scores exhibited greater sensitivity to pazopanib. The low TII score was characterized by a high immune infiltration level of CD8⁺ T cells, T follicular helper cells, and regulatory T cells (Tregs). Moreover, the immune check point genes, including CTLA-4, LAG3, PD-1, and IDO1, presented a high expression level in the low TII score group. Patients in the high TII score group demonstrated significant therapeutic advantages and clinical benefits. The findings in this study have the potential to assist in the strategic design of immunotherapeutic treatments for ccRCC.     

NDRG2 suppresses the proliferation of clear cell renal cell carcinoma cell A-498

Background

Recently, the anti-tumor activity of N-myc downstream-regulated gene 2 (NDRG2) was shown decreased expression in clear cell renal cell carcinoma (CCRCC), but the role of the down-expression of NDRG2 has not been described.

Methods

The NDRG2 recombinant adenovirus plasmid was constructed. The proliferation rate and NDRG2 expression of cell infected with recombinant plasmid were mesured by MTT, Flow cytometry analysis and western blot.

Results

The CCRCC cell A-498 re-expressed NDRG2 when infected by NDRG2 recombinant adenovirus and significantly decreased the proliferation rate. Fluorescence activated cell sorter analysis showed that 25.00% of cells expressed NDRG2 were in S-phase compared to 40.67% of control cells, whereas 62.08% of cells expressed NDRG2 were in G1-phase compared to 54.39% of control cells (P < 0.05). In addition, there were much more apoptotic cells in NDRG2-expressing cells than in the controls (P < 0.05). Moreover, upregulation of NDRG2 protein was associated with a reduction in cyclin D1, cyclin E, whereas cyclinD2, cyclinD3 and cdk2 were not affected examined by western blot. Furthermore, we found that p53 could upregulate NDRG2 expression in A-498 cell.

Conclusions

We found that NDRG2 can inhibit the proliferation of the renal carcinoma cells and induce arrest at G1 phase. p53 can up-regulate the expression of NDRG2. Our results showed that NDRG2 may function as a tumor suppressor in CCRCC.     

肿瘤细胞自噬的诱导及其细胞周期分析

背景与目的:自噬作为Ⅱ型程序性死亡——自噬性死亡过程中的主要现象,与细胞的自噬性死亡有着密切的关系。研究者们对凋亡与细胞周期的关系进行了深入而细致的研究,但对自噬性细胞死亡与细胞周期的关系却知之甚少。本研究的目的是探讨不同方法诱导的细胞自噬与细胞周期之间的关系。方法:用Hanks’液替代培养基的饥饿诱导和用长春新碱诱导两种方法分别处理对数生长期的HeLa细胞、SW480细胞以及经过和未经过植物血凝素(phytohemagglutinin,PHA)刺激的健康人外周血淋巴细胞;应用激光共聚焦显微镜和透射电镜检测细胞自噬的发生,兔抗人微管相关蛋白1轻链3Ⅱ(microtubule-associatedprotein1lightchain3,MAP1-LC3-Ⅱ)/DNA双参数流式细胞术分析自噬细胞的细胞周期。结果:HeLa细胞和SW480细胞用饥饿和长春新碱两种方法诱导的细胞自噬在G1、S、G2/M期均可以发生,且自噬发生率随诱导时间的延长逐渐增加。处于静止期(未经PHA刺激)的外周血淋巴细胞没有自噬的发生,48h时LC3-Ⅱ表达率<2.62%(HanksL液饥饿诱导)或<6.16%(长春新碱诱导);经PHA刺激48h进入细胞周期的外周血淋巴细胞,2h时已有明显的自噬发生。结论:MAP1-LC3-Ⅱ/DNA双参数流式细胞术是对细胞自噬与细胞周期进行同步分析的一种新的简便可靠的方法;细胞自噬只发生在细胞进入周期后,而静止期细胞对自噬诱导因素不敏感。