BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,t...BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.展开更多
Background:Circular RNA(circRNA)plays key regulatory roles in the development of many diseases.However the biological functions and potential molecular mechanisms of circRNA in the injury and repair of intestinal muco...Background:Circular RNA(circRNA)plays key regulatory roles in the development of many diseases.However the biological functions and potential molecular mechanisms of circRNA in the injury and repair of intestinal mucosa in mice after severe burns are yet to be elucidated.Methods:Cell counting kit-8(CCK-8),5-ethynyl-2-deoxyuridine(EdU),wound healing and transwell assays were used to detect cell proliferation and migration ability.Real-time quantitative PCR was used to identify the expression of circRNA,microRNA and messenger RNA.Nuclear and cytoplas-mic separation experiments were employed to perceive the location of circRNA_Maml2.Finally,in vitro and in vivo experiments were conducted to study the repairing effect of circRNA_Maml2 on the intestinal mucosa of mice after severe burns.Results:When compared with the control group,the expression of circRNA_Maml2 was sig-nificantly reduced in the severe burn group.Furthermore,overexpression of circRNA_Maml2 promoted the proliferation and migration of CT26.wt cells in vivo and the repair of damaged intestinal mucosa in vitro.CircRNA_Maml2 acted as a sponge adsorption molecule for miR-93-3p to enhance the expression of frizzled class receptor 7 and activate the downstream Wnt/β-catenin pathway,thereby promoting the repair of the intestinal mucosa.Conclusions:Our findings demonstrate that circRNA_Maml2 regulates the miR-93-3p/FZD7/Wnt/β-catenin pathway and promotes the repair of damaged intestinal mucosa.Hence,circRNA_Maml2 is a potential therapeutic target to promote intestinal mucosal repair.展开更多
基金Supported by National Natural Science Foundation of China,No.81560390the Guizhou Medical University Cultivation Project of the National Natural Science Foundation of China,No.22NSFCP02Basic Research Project of Science and Technology Department of Guizhou Province,No.ZK[2024]General 136.
文摘BACKGROUND Wnt/FZD-mediated signaling pathways are activated in more than 90%of hepatocellular carcinoma(HCC)cell lines.As a well-known secretory glycoprotein,Wnt3 can interact with FZD receptors on the cell surface,thereby activating the Wnt/β-catenin signaling pathway.However,the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.AIM To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.METHODS Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites,actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation,the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy,and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.RESULTS Wnt3 has two N-glycosylation-modified sites(Asn90 and Asn301);when a single site at amino acid 301 is mutated,the stability of Wnt3 is weakened;the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously;and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated.Cell proliferation,migration and invasion are also weakened in the case of single 301 site and double-site mutations.CONCLUSION These results indicate that by inhibiting the N-glycosylation of Wnt3,the proliferation,migration,invasion and colony formation abilities of liver cancer cells can be weakened,which might provide new therapeutic strategies for clinical liver cancer in the future.
基金supported by the National Natural Science Foundation of China(81772082)the Natural Science Foundation of Jiangsu Province(BK20211060)+1 种基金the Natural Science Foundation of Xuzhou(KC20085)the Postgraduate Innovation Program of Jiangsu Province(KYCX20_2484).
文摘Background:Circular RNA(circRNA)plays key regulatory roles in the development of many diseases.However the biological functions and potential molecular mechanisms of circRNA in the injury and repair of intestinal mucosa in mice after severe burns are yet to be elucidated.Methods:Cell counting kit-8(CCK-8),5-ethynyl-2-deoxyuridine(EdU),wound healing and transwell assays were used to detect cell proliferation and migration ability.Real-time quantitative PCR was used to identify the expression of circRNA,microRNA and messenger RNA.Nuclear and cytoplas-mic separation experiments were employed to perceive the location of circRNA_Maml2.Finally,in vitro and in vivo experiments were conducted to study the repairing effect of circRNA_Maml2 on the intestinal mucosa of mice after severe burns.Results:When compared with the control group,the expression of circRNA_Maml2 was sig-nificantly reduced in the severe burn group.Furthermore,overexpression of circRNA_Maml2 promoted the proliferation and migration of CT26.wt cells in vivo and the repair of damaged intestinal mucosa in vitro.CircRNA_Maml2 acted as a sponge adsorption molecule for miR-93-3p to enhance the expression of frizzled class receptor 7 and activate the downstream Wnt/β-catenin pathway,thereby promoting the repair of the intestinal mucosa.Conclusions:Our findings demonstrate that circRNA_Maml2 regulates the miR-93-3p/FZD7/Wnt/β-catenin pathway and promotes the repair of damaged intestinal mucosa.Hence,circRNA_Maml2 is a potential therapeutic target to promote intestinal mucosal repair.
