Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21...Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.展开更多
Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion...Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.展开更多
Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expr...Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.展开更多
目的探讨运用RNA干扰技术沉默FAK基因表达对人舌鳞癌细胞株CAL-27增殖与迁移能力的影响。方法使用si RNA干扰技术瞬时转染构建FAK si RNA。将实验细胞分为空白对照组、阴性对照组和实验组。运用q PCR和Western blotting法检测FAK m RNA...目的探讨运用RNA干扰技术沉默FAK基因表达对人舌鳞癌细胞株CAL-27增殖与迁移能力的影响。方法使用si RNA干扰技术瞬时转染构建FAK si RNA。将实验细胞分为空白对照组、阴性对照组和实验组。运用q PCR和Western blotting法检测FAK m RNA和蛋白的表达情况,MTT法检测细胞的增殖情况,Transwell小室法研究细胞的迁移能力。结果 q PCR和Western blotting实验结果显示,实验组细胞FAK m RNA和蛋白表达量明显低于空白对照组和阴性对照组(Pm RNA<0.01,P蛋白<0.05);MTT实验结果显示在48和72 h时,空白对照组和阴性对照组的吸光度值均明显高于实验组(P<0.01);Transwell实验结果显示实验组穿膜迁移细胞数明显低于空白对照组和阴性对照组(P<0.05)。结论沉默FAK基因表达可有效抑制人舌鳞癌CAL-27细胞的增殖和迁移能力。展开更多
基金Supported by grants from National Science Foundation of China （No. 30873038） and Young Research Foundation of Health Department of Hubei Province, China （No. QJX2008-3）. We are grateful to Dr. Zhaokang Hu （North Carolina State University） for helpful discussion.
文摘Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.
文摘Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.
基金supported by the National Natural Science Foundation of China Fund Project(82272956).
文摘Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.
文摘目的探讨运用RNA干扰技术沉默FAK基因表达对人舌鳞癌细胞株CAL-27增殖与迁移能力的影响。方法使用si RNA干扰技术瞬时转染构建FAK si RNA。将实验细胞分为空白对照组、阴性对照组和实验组。运用q PCR和Western blotting法检测FAK m RNA和蛋白的表达情况,MTT法检测细胞的增殖情况,Transwell小室法研究细胞的迁移能力。结果 q PCR和Western blotting实验结果显示,实验组细胞FAK m RNA和蛋白表达量明显低于空白对照组和阴性对照组(Pm RNA<0.01,P蛋白<0.05);MTT实验结果显示在48和72 h时,空白对照组和阴性对照组的吸光度值均明显高于实验组(P<0.01);Transwell实验结果显示实验组穿膜迁移细胞数明显低于空白对照组和阴性对照组(P<0.05)。结论沉默FAK基因表达可有效抑制人舌鳞癌CAL-27细胞的增殖和迁移能力。
