C-藻蓝蛋白抑制TGF-β1诱导的宫颈癌Caski细胞上皮-间充质转化

目的：探究C-藻蓝蛋白（C-phycocyanin，C-PC）对转化生长因子β1（transforming growth factor beta 1，TGF-β1）诱导的 宫颈癌Caski细胞上皮-间充质转化（epithelial-mesenchymal transition, EMT）的影响。方法：根据处理方法不同将Caski细胞分为 3组即10 ng/ml TGF-β1处理组、 10 ng/ml TGF-β1+300 μg/ml C-PC共处理组和对照组（未加药处理），处理24 h后观察细胞的形态 变化；采用划痕实验及Transwell实验分别检测TGF-β1和C-PC对Caski细胞迁移和侵袭的影响；Western blotting方法检测C-PC 对TGF-β1诱导的Caski细胞上皮表型标志蛋白E-cadherin、间质表型标志蛋白N-cadherin表达水平的影响；qPCR 法检测间质表 型相关锌指转录因子Snail、Zeb1、Twist mRNA的表达。结果：TGF-β1处理组Caski细胞失去原有的上皮表型的特征， 而TGF-β 1+C-PC共处理组细胞保持正常的上皮表型的特征；TGF-β1处理组Caski细胞的迁移率[（60.0±1.4） % vs（33.5±2.2） %、（40.0± 2.8） %， 均P<0.05]和侵袭穿膜细胞数[（108.2±6.2）vs（25.2±3.1）、（39.8±5.4）个， 均P<0.01]较共处理组和对照组显著升高；与对照 组相比，TGF-β1处理组Caski细胞中E-cadherin表达显著降低（P<0.05），Twist、Snail、Zeb1的mRNA表达水平显著升高（P<0.01）， 而加入C-PC共处理可逆转上述改变（P<0.05或P<0.01），同时显著降低N-cadherin的蛋白表达水平（均P<0.05）。结论：C-PC处 理能够抑制TGF-β1诱导的Caski细胞侵袭转移能力进而影响EMT过程，其机制可能与C-PC处理降低Twist、Snail、Zeb1的 mRNA表达有关。     

ROR1-AS1调控Notch1信号通路对乳腺癌MCF-7细胞增殖和凋亡影响

摘 要：［目的］ 研究酪氨酸蛋白激酶跨膜受体 1 反义 RNA 1（tyrosine protein kinase transmembrane receptor 1 antisense RNA 1，ROR1-AS1）调控Notch1信号通路对乳腺癌MCF-7细胞增殖、凋亡及上皮-间质转化(epithelial-mesenchymal transition，EMT)的影响。［方法］ 实时荧光定量PCR方法检测乳腺癌MCF-7、MDA-MB-231、MDA-MB-361细胞和正常乳腺上皮MCF10A细胞中ROR1-AS1表达变化。乳腺癌MCF-7细胞分成对照、sh-NC（转染shRNA 对照）、sh-ROR1-AS1（转染ROR1-AS1 shRNA）、sh-ROR1-AS1+Jagged1组（转染ROR1-AS1 shRNA，Notch1通路激活剂Jagged1处理）。CCK-8实验检测细胞增殖，流式细胞术检测细胞凋亡，Transwell小室检测细胞迁移和侵袭，Western blot方法检测细胞中活化的半胱氨酰天冬氨酸特异性蛋白酶9（cleaved cysteinyl aspartate-specific protease-9，C-Caspase-9）、上皮型钙黏蛋白（E-cadherin）、活化的半胱氨酰天冬氨酸特异性蛋白酶3（cleaved cysteinyl aspartate-specific protease-3，C-Caspase-3）、神经型钙黏蛋白（N-cadherin）、Notch1、Hes1蛋白表达。 ［结果］ 乳腺癌MCF-7、MDA-MB-231、MDA-MB-361细胞中ROR1-AS1水平明显高于正常乳腺上皮MCF10A细胞（P<0.05）。与对照组、sh-NC组比较，sh-ROR1-AS1组乳腺癌MCF-7细胞存活率降低(100.00%±9.82%，99.74%±12.05%，58.91%±6.33%)，细胞凋亡率升高(4.81%±0.26%，4.93%±0.34%，20.58%±2.11%），细胞迁移数目( 225.36±12.84个，224.89±19.56个，140.89±13.64个）和侵袭数目(180.47±16.32个，177.54±16.92个，110.44±10.87个)减少，细胞中C-Caspase-9、E-cadherin、C-Caspase-3蛋白表达水平增加，N-cadherin、Notch1、Hes1蛋白表达水平降低（P<0.05）。与sh-ROR1-AS1组比较，sh-ROR1-AS1+Jagged1组乳腺癌MCF-7细胞存活率升高(100.00%±11.58% vs 147.36%±12.05%），细胞凋亡率降低(19.54%±1.74% vs 8.36%±0.75%)，细胞迁移数目(139.58±12.78个 vs 175.26±16.94个)和侵袭数目（107.45±9.35个 vs 162.04±13.67个）均升高，C-Caspase-9、E-cadherin、C-Caspase-3蛋白水平降低，N-cadherin、Notch1、Hes1蛋白水平升高（P<0.05）。［结论］ 下调ROR1-AS1通过抑制Notch1信号减弱乳腺癌MCF-7细胞增殖、迁移、侵袭和EMT能力，激活细胞凋亡。     

siRNA 干扰胰岛素样生长因子1 受体表达对缺氧环境下肝癌HepG2 细胞周期及凋亡的影响

目的：探讨siRNA技术干扰胰岛素样生长因子-1受体（insulin-like growth factors-1 receptors, IGF-1R）表达对缺氧环境下肝癌HepG2 细胞周期和凋亡的影响。方法：采用氯化钴处理制备实验所需的肝癌缺氧细胞模型。设计并合成3 对siRNA序列和1 对阴性对照序列，转染缺氧的肝癌HepG2 细胞24 h 后以荧光显微镜观察转染效果，采用WB法检测IGF-1R蛋白表达筛选出干扰效率最高的siRNA序列。选用该序列再次转染缺氧肝癌细胞，以流式细胞术、MTT法检测细胞的周期、凋亡和增殖变化，以WB法检测HepG2 细胞中CDK1、CDK2 和Caspase-3 蛋白的表达。结果：成功建立缺氧HepG2 细胞模型，siRNA转染缺氧HepG2 细胞后以IGF-1R-siRNA-2 转染效率最高且敲减IGF-1R表达最明显（均P<0.01）。IGF-1R-siRNA-2 转染的HepG2 细胞的增殖被明显抑制（P<0.05 或P<0.01）、细胞周期被阻滞在G0/G1（P<0.05）、细胞凋亡率明显增加至（25.3±1.3）%（P<0.01），同时发现敲减IGF-1R后缺氧HepG2 细胞中CDK1、CDK2 蛋白表达明显降低而Caspase-3 表达则明显增加（P<0.05或P<0.01）。结论：siRNA干扰IGF-1R表达通过调控细胞周期和凋亡相关蛋白而抑制缺氧环境中HepG2细胞的恶性生物学行为，IGF-1R可能是HCC潜在的治疗靶点。     

过表达miR-497靶向细胞周期蛋白E1抑制肺癌A549细胞的上皮间质转化

目的：探讨过表达 miR-497 靶向细胞周期蛋白 E1（cyclin E1，CCNE1）对肺癌 A549 细胞上皮间质转化（epithelialmesenchymal transition，EMT）的影响。方法：常规培养人肺癌A549细胞，细胞实验分为正常组（不加干预）、对照组（转染miR497 mimics-NC）、实验组（转染miR-497 mimics）。采用Transwell小室实验、免疫荧光染色、qPCR、Western blotting法分别检测各组细胞迁移和侵袭能力、蛋白间质标志物α-SMA和上皮标志物E-cadherin的表达、miR-497和CCNE1的表达水平，荧光素酶基因基因报告实验验证miR-497和CCNE1的靶向关系。结果：与对照组和正常组相比，实验组A549细胞迁移和侵袭的数量明显减少（均P<0.05），细胞的间质标志物α-SMA的绿色荧光强度明显减弱（[ 36.95±5.81）vs（98.69±2.36）、（97.94±2.63），均P<0.05]，上皮标志物E-cadherin的绿色荧光强度明显增强（[ 388.41±10.93）vs（100.95±6.37）、（102.55±3.18），均P<0.05]，miR-497 的表达明显升高（均P<0.05），CCNE1的表达均明显下降（均P<0.05）。miR-497能够靶向调控CCNE1的表达。结论：在肺癌A549细胞中miR-497能够靶向调控CCNE1的表达，上调miR-497的表达后能明显抑制A549细胞迁移和侵袭能力，影响EMT相关蛋白的表达。     

表观遗传修饰介导抑制SOSTDC1表达促进宫颈癌细胞的恶性生物学行为

目的：探究含硬化蛋白域蛋白1（SOSTDC1）对宫颈癌细胞恶性生物学行为的调控及其分子机制。方法：收集2020年8 月至2022 年5 月间在福建省肿瘤医院活检或手术切除的53 例宫颈癌组织和相应的癌旁组织标本,免疫组化法检测SOSTDC1 蛋白在宫颈癌组织及相应癌旁组织中的表达,qPCR 法检测正常宫颈细胞、宫颈癌细胞中SOSTDC1 mRNA 表达;将SOSTDC1 过表达慢病毒（OE-sostdc1）和对照空病毒（NC）感染宫颈癌细胞SiHa 及CaSki,将其分为SiHa-OE-sostdc1、SiHa-NC、CaSki-OE-sostdc1、CaSki-NC 组,采用WST-1法、细胞集落形成实验、Transwell 实验和WB法检测转染各组SiHa 及CaSki 细胞的增殖、集落形成、迁移和侵袭能力和BMP、Wnt/β-catenin 信号途径相关蛋白及上皮-间充质转化（EMT）相关蛋白的表达。用DNA甲基化酶抑制剂5-氮杂2''-脱氧胞苷（5''-Aza-CdR）处理宫颈癌细胞后采用qPCR和WB法检测SOSTDC1 mRNA及蛋白的表达变化,用甲基化特异性PCR（MSP）检测5 例配对宫颈癌组织与癌旁组织中SOSTDC1 基因启动子区甲基化水平,同时qPCR 检测其SOSTDC1 mRNA水平。结果：与癌旁组织比较,SOSTDC1蛋白在宫颈癌组织中呈低表达（P<0.01）,且与淋巴结转移与FIGO分期有关联（均P<0.05）;与正常宫颈HUCEC细胞比较,SOSTDC1 mRNA 在宫颈癌C33A、HeLa、SiHa、CaSki 细胞中均呈低表达（均P<0.01）。过表达SOSTDC1显著抑制SiHa 及CaSki 细胞的增殖、迁移和侵袭能力（均P<0.05）。WB法结果检测显示,过表达SOSTDC1 显著抑制 SiHa 及 CaSki 细胞中磷酸化 Smad、Dvl2/3、β -catenin、VIM、N-cadherin、Snail 蛋白的表达（均P<0.05）,5''-Aza-CdR 处理后的SiHa 及CaSki 细胞中SOSTDC1 mRNA和蛋白水平均显著增加（均P<0.05）,MSP检测结果显示,相较于癌旁组织,宫颈癌组织中SOSTDC1基因启动子区呈高度甲基化,且SOSTDC1 mRNA水平降低（P<0.01）。结论：SOSTDC1在宫颈癌组织中呈低表达且与肿瘤的恶性进展关联,其表达下调与其基因启动子区高度甲基化有关,过表达SOSTDC1 可能通过阻断BMP及Wnt/β-catenin信号通路从而抑制SiHa、CaSki细胞的增殖、侵袭和迁移能力。     

miRNA通过Mcl-1基因调控HBV阳性肝癌细胞对索拉菲尼的敏感性

目的： 探讨干预 HBV 阳性肝癌细胞相关 miRNAs 对肝癌细胞索拉菲尼敏感性的影响。 方法： qPCR 检测 HepG2.2.15 （HBV阳性）和HepG2.vc （HBV阴性）肝癌细胞中miR-29、miR-101和miR-193b的表达,以HepG2.2.15细胞中低表达 的miRNA合成相应的miRNA mimics,并将目标miRNA mimics分别转染至HepG2.2.15和HepG2.vc细胞;qPCR检测两种细胞中 目标miRNA表达,Western blotting检测目标miRNA mimics转染前后Mcl-1蛋白表达。同时分别向转染和非转染的HepG2.2.15 和HepG2.vc细胞中分别加入（1×10 -9 ）~（1×10 -3 ）mol/L的索拉菲尼,72 h后测定索加菲尼对细胞作用的IC 50 值和细胞凋亡率。 结 果： 与HepG2.vc细胞比较,HepG2.2.15细胞中miR-193b的表达显著降低（P<0.05）;与miR-193b mimics转染前比较,HepG2.2.15 和HepG2.vc细胞中miR-193b的表达均有显著升高（P<0.05）。与HepG2.vc细胞比较,HepG2.2.15细胞中Mcl-1蛋白表达显著增 高（P<0.05）;miR-193b mimics转染后,HepG2.2.15和HepG2.vc细胞中Mcl-1蛋白表达较两者转染前均有显著降低（P<0.05）; miR-193b mimics 转染后,索拉菲尼可显著增加两组细胞的凋亡率（P<0.01）,同时其对两组细胞作用的 IC 50 值显著降低 [HepG2.2.15细胞： （0.215±0.028）vs (0.391±0.025) mol/L,HepG2.vc细胞： （0.315±0.027）vs (0.654±0.019) mol/L;均P<0.01]。 结 论： HBV相关肝癌细胞中miR-193b的低表达可能是癌细胞对索拉菲尼敏感性降低的原因,Mcl-1可能为miR-193b的靶点,miR- 193b mimics与索拉菲尼具有显著协同作用。     

小核核糖核蛋白多肽A对肝细胞癌细胞恶性生物学行为的影响及其机制

目的：探究小核核糖核蛋白多肽A（SNRPA）在肝细胞癌（HCC）组织和细胞中的表达及其调控HCC 细胞HepG2 和Hep3B恶性生物学行为的作用及其机制。方法: 数据库分析SNRPA在泛癌组织中的表达及其与病理分期、HCC 患者预后的相关性。常规培养HepG2 和Hep3B 细胞，将si-NC ，si-SNRPA#1、si-SNRPA#2转染HepG2 和Hep3B 细胞，实验分为si-NC 组、 si-SNRPA#1 组和si-SNRPA#2 组；将SNRPA-vector 和SNRPA-oe 载体转染LO2 细胞，分为SNRPA-vector 组和SNRPA-oe 组。 qPCR法检测正常肝细胞和肝癌细胞以及转染各组HepG2和Hep3B细胞中SNRPA mRNA的表达，MTT法、Transwell 法和WB法分别检测转染后各组HepG2 和Hep3B细胞的增殖、迁移和侵袭能力以及EMT相关蛋白表达的变化。结果: 数据库分析显示，SNRPA mRNA在多数肿瘤组织中均呈高表达（均P<0.001）且与病理分期有关联（P<0.05或P<0.01）。SNRPA在HCC组织和细胞中均呈高表达（P<0.05 或P<0.01），且与HCC患者的预后有关联（P<0.01）。敲减SNRPA表达明显抑制HepG2 和Hep3B细胞增殖（P<0.05或P<0.01）而过表达SNRPA则能促进LO2细胞增殖（P<0.01），敲减SNRPA表达明显抑制HepG2和Hep3B细胞的迁移和侵袭能力（均P<0.01），明显促进E-cadherin 的表达上调（P<0.01），而抑制N-cadherin、vimentin 的表达（P<0.01）。结论: SNRPA在HCC组织及细胞中呈明显高表达，其可能通过调控上皮间质转化（EMT）进程进而促进HepG2和Hep3B细胞的增殖、迁移和侵袭。     

CAAP1抑制肝癌HepG2细胞凋亡而促进其增殖、迁移和侵袭

目的：探讨CAAP1对肝癌HepG2细胞凋亡、增殖、迁移和侵袭的影响及其作用机制。方法：构建CAAP1过表达载体 pcDNA3/CAAP1 和敲降载体 pSilencer 2.1-U6 neo/shR-CAAP1,转染肝癌 HepG2 细胞后,qPCR 和 WB 实验分别检测 CAAP1 mRNA和蛋白的表达水平。实验分为过表达对照组（pcDNA3）、过表达组（pcDNA3/CAAP1）、敲降对照组（pSilencer 2.1-U6 neo,pSilencer）和敲降组（pSilencer 2.1-U6 neo/shR-CAAP1,shR-CAAP1）,流式细胞术检测各组细胞凋亡水平,WB 检测 cleaved caspase 3蛋白表达水平,CCK-8实验检测各组细胞的增殖情况,克隆形成实验检测各组细胞的集落形成能力,划痕和Transwell小室实验检测各组细胞迁移和侵袭能力。检索TCGA数据库,分析CAAP1对肝癌患者OS的影响。结果：成功构建CAAP1的过表达载体pcDNA3/CAAP1和敲降载体shR-CAAP1,转染后pcDNA3/CAAP1组和shR-CAAP1组细胞中CAAP1mRNA及蛋白表达水平均明显增高或降低（均P<0.05）。与pcDNA3组比较,pcDNA3/CAAP1组细胞凋亡率下降32%、cleaved caspase 3蛋白表达水平显著降低（均P<0.05）;与pSilencer组比较,shR-CAAP1 组细胞凋亡率上升 73%,cleaved caspase 3 蛋白表达水平显著升高（均P<0.05）。pcDNA3/CAAP1组细胞增殖显著增强（P<0.05）,shR-CAAP1组细胞增殖显著降低（P<0.05）。pcDNA3/CAAP1组细胞迁移数增加48%、细胞迁移距离增加59%、细胞侵袭数增加52%（均P<0.05）,shR-CAAP1组细胞迁移数减少53%、细胞迁移距离减少29%、细胞侵袭数减少45%（均P<0.05）。TCGA数据库数据分析显示,肝癌组织中CAAP1的高表达与肝癌患者OS呈负相关（P<0.05）。结论：CAAP1能够抑制肝癌HepG2细胞凋亡从而促进其增殖、迁移和侵袭,其可能与肝癌的发生发展密切相关。     

GSTP1基因对人肝癌细胞系HepG2增殖和侵袭能力的影响

目的 探讨过表达/沉默GSTP1基因对人肝癌细胞系HepG2增殖及侵袭能力的影响。 方法 采用腺病毒载体转染人肝癌细胞系HepG2,获得过表达/沉默GSTP1基因的HepG2细胞。实时荧光定量PCR（qPCR）法检测细胞中GSTP1 mRNA表达水平;CCK-8法和Transwell小室法分别检测细胞的细胞活力和侵袭能力;免疫印迹法（Western blot）检测细胞中Akt、mTOR、p-Akt和p-mTOR的蛋白表达。 结果 经腺病毒载体转染后,成功获得过表达/沉默GSTP1基因的HepG2细胞;过表达GSTP1 基因后,HepG2细胞的细胞活力和侵袭能力显著降低（P<0.05）,而沉默GSTP1基因后,HepG2细胞的细胞活力和侵袭能力则显著增高（P<0.05）;过表达GSTP1基因后,p-Akt和p-mTOR的蛋白表达水平显著降低（P<0.05）,而沉默GSTP1基因的结果则相反。 结论 过表达GSTP1基因可抑制HepG2细胞的增殖和侵袭能力,沉默GSTP1基因则促进HepG2细胞的增殖和侵袭能力,其作用机制可能与Akt/mTOR信号通路有关。     

HDAC6对人肝癌HepG2细胞迁移和侵袭的影响及其机制

目的:明确HDAC6对人肝癌HepG2细胞迁移和侵袭的影响及其机制。方法:应用Western blot技术检测正常肝细胞系LO2和肝癌细胞系HepG2细胞中 HDAC6的表达。应用HDAC6抑制剂Tubastatin A抑制HepG2细胞中HDAC6的表达,运用Western blot及荧光定量PCR技术检测HepG2细胞中转化生长因子β1（transforming growth factor-β1,TGF-β1）和上皮间充质转化（epithelial mesenchymal transition,EMT）相关分子标志物（N-cadherin,β-catenin,Vimentin）的表达;用TGF-β1抑制剂SB431542刺激HepG2细胞后,检测HepG2细胞中 EMT标志蛋白的表达,应用TGF-β1刺激HepG2细胞后观察HepG2细胞的形态变化。应用过表达HDAC6的质粒P3-HDAC6、P3-HDAC6+TGF-β1分别作用于HepG2细胞,通过Western blot检测HepG2细胞中EMT相关分子标志物的表达,应用划痕及Transwell技术检测HepG2细胞处理前后的迁移和侵袭能力变化。结果:肝癌细胞系HepG2细胞内HDAC6的蛋白表达量明显低于正常肝细胞系LO2（P＜0.05）。 HepG2细胞Tubastatin A组中N-cadherin、β-catenin、Vimentin、TGF-β1的mRNA和蛋白表达水平相较于空白对照组明显增高（P均<0.05）。SB431542组中EMT标志蛋白表达水平相较于空白对照组明显降低（P均<0.01）。 TGF-β1刺激HepG2细胞后细胞变得分散且狭长。同时发现P3-HDAC6+TGF-β1组的细胞迁移和侵袭能力在48 h后明显强于P3-HDAC6组且弱于空白对照组（P均<0.05）。结论:HDAC6可通过下调TGF-β1的表达从而抑制HepG2细胞的EMT进而抑制HepG2细胞的迁移和侵袭。     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.     

hSav1蛋白高表达对Mst1介导的细胞凋亡的影响

Objective To elucidate the effect of hSavl expression on Mstl-mediated apoptosis in HeLa cells. Methods Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 μmol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. Results Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSavl could be detect from the anti-Mstl immunoprecipitation complex. The immunofluorescent labeling showed that hSavl and Mstl had the same localization in cells. Overexpressed protein hSavl did not induce a significant cell apoptosis. However, co-expression of hSavl with Mstl resulted in a significant increase of apoptosis above the level seen with Mstl alone (24. 5% ± 2.4% vs. 39.3% ± 4.0%, P < 0. 05). Conclusion Our findings indicate that hSavl is a newly identified protein that interacts with Mst1 and a, augments Mst1-mediated apoptosis.