Background:Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking.Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTP...Background:Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking.Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins(GAPs)and guanine nucleotide exchange factors(GEFs).Arhgap39/Vilse/Porf-2 is a newly identified GAP.The role of Arhgap39 in migration and invasion has not been addressed thoroughly.Methods:The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion.Results:Loss of Arhgap39 noticeably increased the migration and invasive potential.Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro.RNA-seq analysis revealed that matrix metalloproteinase 13(MMP13)and Laminin subunit beta 1(LAMB1)were increased in Arhgap39^(^(-/-))cells.We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas.Intriguingly,the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl(KPA)mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl(KP)mice.The cell migration and invasion of KPA mice were also similar to those of control KP mice.Conclusion:Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells,but not in those isolated from KPA mice.展开更多
目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293...目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293T细胞,收集细胞上清获得的病毒过滤后感染Siha细胞,RT-qPCR和Western blot检测敲减效率,以及转染后Bax及Bcl-2的表达变化;CCK-8法检测敲减后细胞的增殖水平。结果成功构建敲减ARHGAP30基因的慢病毒质粒,并建立Siha稳转细胞,ARHGAP30在Siha细胞中的转录和翻译减少(P<0.01),Bax/Bcl-2明显降低(P<0.01),凋亡减少,细胞增殖水平升高(P<0.01)。结论ARHGAP30参与Siha细胞的增殖与凋亡,调控ARHGAP30基因或将干扰宫颈癌的发生和发展。展开更多
ARHGAP36是Rho GAPs家族的一员,在成神经管细胞瘤中超表达,可能参与调节细胞增殖。为了研究ARHGAP36在羊精子发生过程中的作用,采用RACE(rapid amplification of c DNA ends)技术从绵羊睾丸组织中克隆了SARHGAP36的c DNA全长序列。内部...ARHGAP36是Rho GAPs家族的一员,在成神经管细胞瘤中超表达,可能参与调节细胞增殖。为了研究ARHGAP36在羊精子发生过程中的作用,采用RACE(rapid amplification of c DNA ends)技术从绵羊睾丸组织中克隆了SARHGAP36的c DNA全长序列。内部区域c DNA序列与5'RACE和3'RACE扩增的两端序列测序后拼接,得到了2698 bp的绵羊ARHGAP36 c DNA全长,其中包含一个长1593 bp的开放阅读框(ORF),编码530 aa,与Uni Prot中预测的牛ARHGAP36氨基酸序列(A7MB27)同源性为98%,说明我们克隆的c DNA是Rho GAP家族的成员ARHGAP36。对所克隆的绵羊ARHGAP36的氨基酸序列和Uni Prot数据库中所收集的所有其它ARHGAP成员的氨基酸序列比对分析显示,ARHGAP36没有其它ARHGAP成员都有的标志性"精氨酸指"基序,而它的对应基序中的保守精氨酸残基被苏氨酸替代了,暗示ARHGAP36很可能并不依靠酶催化活性发挥其功能。当把所克隆的c DNA与Gen Bank中发布的绵羊arhgap36序列比对时发现绵羊arhgap36基因组测序结果的第一个外显子中缺少一小段DNA,导致如果用该基因组序列预测绵羊ARHGAP36的c DNA,无法得到能编码正确氨基酸的c DNA,因此,实验为进一步研究ARHGAP36这个特殊ARHGAP成员的功能提供了正确的c DNA序列。绵羊睾丸总蛋白Western blot检测发现绵羊睾丸内的ARHGAP36有3种长度,免疫组织化学检测发现ARHGAP36蛋白在绵羊睾丸精细管中的精子发生过程中各类细胞中都有分布。展开更多
ARHGAP17(Rho GTPase activating protein 17)作为小G蛋白家族成员调控蛋白参与众多生理过程,如细胞骨架重组、细胞极性的维持、细胞的黏附与迁徙、基因的转录、肿瘤血管生成、上皮-间质转化、细胞的增殖分化和凋亡等。近年来,ARHGAP17...ARHGAP17(Rho GTPase activating protein 17)作为小G蛋白家族成员调控蛋白参与众多生理过程,如细胞骨架重组、细胞极性的维持、细胞的黏附与迁徙、基因的转录、肿瘤血管生成、上皮-间质转化、细胞的增殖分化和凋亡等。近年来,ARHGAP17在各种恶性肿瘤组织中表达及其传导通路的研究已取得了初步进展,包括结肠癌、宫颈癌、乳腺癌等相关恶性肿瘤。本文分别从ARHGAP17对各种相关肿瘤的发生、发展的调控作用及预后影响进行综述。展开更多
文摘Background:Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking.Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins(GAPs)and guanine nucleotide exchange factors(GEFs).Arhgap39/Vilse/Porf-2 is a newly identified GAP.The role of Arhgap39 in migration and invasion has not been addressed thoroughly.Methods:The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion.Results:Loss of Arhgap39 noticeably increased the migration and invasive potential.Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro.RNA-seq analysis revealed that matrix metalloproteinase 13(MMP13)and Laminin subunit beta 1(LAMB1)were increased in Arhgap39^(^(-/-))cells.We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas.Intriguingly,the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl(KPA)mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl(KP)mice.The cell migration and invasion of KPA mice were also similar to those of control KP mice.Conclusion:Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells,but not in those isolated from KPA mice.
文摘目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293T细胞,收集细胞上清获得的病毒过滤后感染Siha细胞,RT-qPCR和Western blot检测敲减效率,以及转染后Bax及Bcl-2的表达变化;CCK-8法检测敲减后细胞的增殖水平。结果成功构建敲减ARHGAP30基因的慢病毒质粒,并建立Siha稳转细胞,ARHGAP30在Siha细胞中的转录和翻译减少(P<0.01),Bax/Bcl-2明显降低(P<0.01),凋亡减少,细胞增殖水平升高(P<0.01)。结论ARHGAP30参与Siha细胞的增殖与凋亡,调控ARHGAP30基因或将干扰宫颈癌的发生和发展。
文摘ARHGAP17(Rho GTPase activating protein 17)作为小G蛋白家族成员调控蛋白参与众多生理过程,如细胞骨架重组、细胞极性的维持、细胞的黏附与迁徙、基因的转录、肿瘤血管生成、上皮-间质转化、细胞的增殖分化和凋亡等。近年来,ARHGAP17在各种恶性肿瘤组织中表达及其传导通路的研究已取得了初步进展,包括结肠癌、宫颈癌、乳腺癌等相关恶性肿瘤。本文分别从ARHGAP17对各种相关肿瘤的发生、发展的调控作用及预后影响进行综述。
