目的探讨C3G基因对H9C2心肌细胞凋亡、增殖的影响及其机制。方法构建RNA干扰慢病毒,购买p CXN2-Flag空质粒和p CXN2-Flag-h C3G过表达人C3G m RNA质粒。分别用空白试剂、阴性慢病毒、C3G si RNA慢病毒、C3G si RNA慢病毒+空质粒、C3G si...目的探讨C3G基因对H9C2心肌细胞凋亡、增殖的影响及其机制。方法构建RNA干扰慢病毒,购买p CXN2-Flag空质粒和p CXN2-Flag-h C3G过表达人C3G m RNA质粒。分别用空白试剂、阴性慢病毒、C3G si RNA慢病毒、C3G si RNA慢病毒+空质粒、C3G si RNA慢病毒+h C3G质粒随机感染和转染H9C2心肌细胞,实验被随机分为5个组,即空白对照组、阴性对照组、沉默C3G组、沉默C3G+空质粒组和沉默C3G+过表达人C3G组。转染质粒72h后,采用RTPCR法检测目的基因C3G m RNA的表达,流式细胞术检测H9C2心肌细胞的凋亡情况,MTT比色法测定细胞增殖率,Western blotting检测细胞C3G、p-ERK1/2、Bax及Flag的蛋白表达水平。结果阴性对照组和沉默C3G组经嘌呤霉素筛选,85%以上细胞被绿色荧光蛋白标记,提示细胞被慢病毒感染。与空白对照组和阴性对照组比较,沉默C3G组和沉默C3G+空质粒组的细胞中Bax蛋白表达水平和细胞凋亡率均明显增加(P<0.01,P<0.05),且这两组细胞C3G m RNA、C3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率均明显降低(P<0.01,P<0.05);与沉默C3G组和沉默C3G+空质粒组比较,沉默C3G+过表达人C3G组的细胞中Bax蛋白表达水平和细胞凋亡率明显降低(P<0.01,P<0.05),且细胞中C3G m RNA、C3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率则明显增加(P<0.01,P<0.05)。结论沉默C3G基因能有效抑制细胞的增殖,促进H9C2心肌细胞凋亡;过表达C3G基因能逆转沉默C3G基因对H9C2心肌细胞的影响,表现为细胞凋亡减少,增殖明显,其机制可能与C3G基因对p-ERK1/2蛋白和促凋亡分子Bax的调控有关。展开更多
C3G is a GEF(guanine nucleotide exchange factor)for Rap GTPases,among which the isoform Rap1b is an essential protein in platelet biology.Using transgenic mouse models with platelet-specific overexpression of C3G or m...C3G is a GEF(guanine nucleotide exchange factor)for Rap GTPases,among which the isoform Rap1b is an essential protein in platelet biology.Using transgenic mouse models with platelet-specific overexpression of C3G or mutant C3GΔCat,we have unveiled a new function of C3G in regulating the hemostatic function of platelets through its participation in the thrombin-PKCRap1b pathway.C3G also plays important roles in angiogenesis,tumor growth,and metastasis through its regulation of the platelet secretome.In addition,C3G contributes to megakaryopoiesis and thrombopoiesis.Here,we used a platelet-specific C3G-KO mouse model to further support the role of C3G in hemostasis.C3G-KO platelets showed a significant delay in platelet activation and aggregation as a consequence of the defective activation of Rap1,which resulted in decreased thrombus formation in vivo.Additionally,we explored the contribution of C3G-Rap1b to platelet signaling pathways triggered by thrombin,PMA or ADP,in the referenced transgenic mouse model,through the use of a battery of specific inhibitors.We found that platelet C3G is phosphorylated at Tyr504 by a mechanism involving PKC-Src.This phosphorylation was shown to be positively regulated by ERKs through their inhibition of the tyrosine phosphatase Shp2.Moreover,C3G participates in the ADP-P2Y12-PI3K-Rap1b pathway and is a mediator of thrombin-TXA2 activities.However,it inhibits the synthesis of TXA2 through cPLA2 regulation.Taken together,our data reveal the critical role of C3G in the main pathways leading to platelet activation and aggregation through the regulation of Rap1b.展开更多
Primary liver cancers constitute the fourth leading cause of cancer mortality worldwide,due to their high morbidity,late diagnosis and lack of effective treatments.Hepatocellular carcinoma(HCC)represents 80%and cholan...Primary liver cancers constitute the fourth leading cause of cancer mortality worldwide,due to their high morbidity,late diagnosis and lack of effective treatments.Hepatocellular carcinoma(HCC)represents 80%and cholangiocarcinoma(CCA)15%of liver cancers.Several genetic and epigenetic gene alterations(e.g.,TERT,TP53 or CTNNB1)are HCC drivers,although many additional gene alterations contribute to HCC initiation and/or progression.Rho and Ras GTPases have been widely implicated in tumorigenesis and their activators(GEFs)have recently emerged as putative key players in liver cancer.The Ras GEF,C3G(RAPGEF1),a GEF mainly for Rap proteins,has recently been uncovered as a relevant gene in HCC.Its upregulation promotes tumor growth,although a decrease in C3G levels favors migration/invasion and lung metastasis.Rap1A/1B/2A/2B are overexpressed in HCC tumors,but their effects are controversial and not equivalent to those of C3G.The C3G partner,CRKL,is also overexpressed in HCC,promoting proliferation,migration and invasion.Various Rho GEFs are also deregulated in liver cancer.Tiam1 and Tiam2 expression is upregulated in HCC,promoting proliferation,migration and metastasis.In addition,ARHGEF-10L/9/19/39 are overexpressed in HCC tumors,facilitating migration,invasion,metastasis and proliferation.Another Rho GEF,Vav2,is also involved in metastasis.Little is known about the participation of these GEFs and GTPases in CCA.However,analysis of cancer databases uncovered deregulations or genetic alterations in several of these genes,in both CCA and HCC.Hence,GEFs function appear essential for liver homeostasis,although future studies are needed to define their precise function in liver cancer.展开更多
文摘目的探讨C3G基因对H9C2心肌细胞凋亡、增殖的影响及其机制。方法构建RNA干扰慢病毒,购买p CXN2-Flag空质粒和p CXN2-Flag-h C3G过表达人C3G m RNA质粒。分别用空白试剂、阴性慢病毒、C3G si RNA慢病毒、C3G si RNA慢病毒+空质粒、C3G si RNA慢病毒+h C3G质粒随机感染和转染H9C2心肌细胞,实验被随机分为5个组,即空白对照组、阴性对照组、沉默C3G组、沉默C3G+空质粒组和沉默C3G+过表达人C3G组。转染质粒72h后,采用RTPCR法检测目的基因C3G m RNA的表达,流式细胞术检测H9C2心肌细胞的凋亡情况,MTT比色法测定细胞增殖率,Western blotting检测细胞C3G、p-ERK1/2、Bax及Flag的蛋白表达水平。结果阴性对照组和沉默C3G组经嘌呤霉素筛选,85%以上细胞被绿色荧光蛋白标记,提示细胞被慢病毒感染。与空白对照组和阴性对照组比较,沉默C3G组和沉默C3G+空质粒组的细胞中Bax蛋白表达水平和细胞凋亡率均明显增加(P<0.01,P<0.05),且这两组细胞C3G m RNA、C3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率均明显降低(P<0.01,P<0.05);与沉默C3G组和沉默C3G+空质粒组比较,沉默C3G+过表达人C3G组的细胞中Bax蛋白表达水平和细胞凋亡率明显降低(P<0.01,P<0.05),且细胞中C3G m RNA、C3G蛋白、p-ERK1/2蛋白的表达水平和细胞增殖率则明显增加(P<0.01,P<0.05)。结论沉默C3G基因能有效抑制细胞的增殖,促进H9C2心肌细胞凋亡;过表达C3G基因能逆转沉默C3G基因对H9C2心肌细胞的影响,表现为细胞凋亡减少,增殖明显,其机制可能与C3G基因对p-ERK1/2蛋白和促凋亡分子Bax的调控有关。
基金supported by grants from the Spanish Ministry of Economy and Competitiveness[SAF2013-48210-C2-1-R,SAF2016-76588-C2-2-R to C.Guerrero,SAF2013-48210-C2-2-R,SAF2016-76588-C2-1-R to A.P.]by two grants from the Council of Education of Junta de Castilla y León,Spain[SA157A12-1,SA017U16 to C.Guerrero].All funding was cosponsored by the European FEDER Program.
文摘C3G is a GEF(guanine nucleotide exchange factor)for Rap GTPases,among which the isoform Rap1b is an essential protein in platelet biology.Using transgenic mouse models with platelet-specific overexpression of C3G or mutant C3GΔCat,we have unveiled a new function of C3G in regulating the hemostatic function of platelets through its participation in the thrombin-PKCRap1b pathway.C3G also plays important roles in angiogenesis,tumor growth,and metastasis through its regulation of the platelet secretome.In addition,C3G contributes to megakaryopoiesis and thrombopoiesis.Here,we used a platelet-specific C3G-KO mouse model to further support the role of C3G in hemostasis.C3G-KO platelets showed a significant delay in platelet activation and aggregation as a consequence of the defective activation of Rap1,which resulted in decreased thrombus formation in vivo.Additionally,we explored the contribution of C3G-Rap1b to platelet signaling pathways triggered by thrombin,PMA or ADP,in the referenced transgenic mouse model,through the use of a battery of specific inhibitors.We found that platelet C3G is phosphorylated at Tyr504 by a mechanism involving PKC-Src.This phosphorylation was shown to be positively regulated by ERKs through their inhibition of the tyrosine phosphatase Shp2.Moreover,C3G participates in the ADP-P2Y12-PI3K-Rap1b pathway and is a mediator of thrombin-TXA2 activities.However,it inhibits the synthesis of TXA2 through cPLA2 regulation.Taken together,our data reveal the critical role of C3G in the main pathways leading to platelet activation and aggregation through the regulation of Rap1b.
基金supported by grants from the Spanish Ministry of Economy and Competitiveness(SAF2016-76588-C2-1-R and PID2019-104143RB-C22 to Porras A,SAF2016-76588-C2-2-R and PID2019-104143RB-C21 to Guerrero G and PID2019-104991RB-I00 to Bragado P)by two grants from the Council of Education of Junta de Castilla y León,Spain(SA017U16 and SA078P20 to Guerrero C)+2 种基金cosponsored by the European FEDER ProgramSequera C was supported by a fellowship from Complutense University from Madrid.Gutierrez-Uzquiza A is supported by Madrid Community Program for Talent Attraction(MRF 2017-T1/BMD-5468)Bragado P received support from BBVA(Becas Leonardo 2018,BBM-TRA-0041).
文摘Primary liver cancers constitute the fourth leading cause of cancer mortality worldwide,due to their high morbidity,late diagnosis and lack of effective treatments.Hepatocellular carcinoma(HCC)represents 80%and cholangiocarcinoma(CCA)15%of liver cancers.Several genetic and epigenetic gene alterations(e.g.,TERT,TP53 or CTNNB1)are HCC drivers,although many additional gene alterations contribute to HCC initiation and/or progression.Rho and Ras GTPases have been widely implicated in tumorigenesis and their activators(GEFs)have recently emerged as putative key players in liver cancer.The Ras GEF,C3G(RAPGEF1),a GEF mainly for Rap proteins,has recently been uncovered as a relevant gene in HCC.Its upregulation promotes tumor growth,although a decrease in C3G levels favors migration/invasion and lung metastasis.Rap1A/1B/2A/2B are overexpressed in HCC tumors,but their effects are controversial and not equivalent to those of C3G.The C3G partner,CRKL,is also overexpressed in HCC,promoting proliferation,migration and invasion.Various Rho GEFs are also deregulated in liver cancer.Tiam1 and Tiam2 expression is upregulated in HCC,promoting proliferation,migration and metastasis.In addition,ARHGEF-10L/9/19/39 are overexpressed in HCC tumors,facilitating migration,invasion,metastasis and proliferation.Another Rho GEF,Vav2,is also involved in metastasis.Little is known about the participation of these GEFs and GTPases in CCA.However,analysis of cancer databases uncovered deregulations or genetic alterations in several of these genes,in both CCA and HCC.Hence,GEFs function appear essential for liver homeostasis,although future studies are needed to define their precise function in liver cancer.
