The scientific community is continuously working to translate the novel biomedical techniques into effective medical treatments.CRISPR-Cas9 system(Clustered Regularly Interspaced Short Palindromic Repeats-9),commonly ...The scientific community is continuously working to translate the novel biomedical techniques into effective medical treatments.CRISPR-Cas9 system(Clustered Regularly Interspaced Short Palindromic Repeats-9),commonly known as the“molecular scissor”,represents a recently developed biotechnology able to improve the quality and the efficacy of traditional treatments,related to several human diseases,such as chronic diseases,neurodegenerative pathologies and,interestingly,oral diseases.Of course,dental medicine has notably increased the use of biotechnologies to ensure modern and conservative approaches:in this landscape,the use of CRISPR-Cas9 system may speed and personalize the traditional therapies,ensuring a good predictability of clinical results.The aim of this critical overview is to provide evidence on CRISPR efficacy,taking into specific account its applications in oral medicine.展开更多
BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AI...BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AIM To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.METHODS The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology.Forty-two paired specimens of ICC and adjacent noncancerous tissues were collected.PSMD6 protein expression was determined by immunohistochemistry.Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level,and its association with ICC patients’various clinicopathological characteristics was investigated.RESULTS The PSMD6 gene was found to be essential for the growth of ICC cell lines.PSMD6 protein was significantly overexpressed in ICC tissues(P<0.001),but showed no significant association with patient age,gender,pathological grade,or tumor-node-metastasis stage(P>0.05).CONCLUSION PSMD6 can promote the growth of ICC cells,thus playing a pro-oncogenic role.展开更多
BACKGROUND In recent years,many studies have shown that proteasome 26S subunit non-ATPase 6(PSMD6)plays an important role in the occurrence and development of malignant tumours.Unfortunately,there are no reports on th...BACKGROUND In recent years,many studies have shown that proteasome 26S subunit non-ATPase 6(PSMD6)plays an important role in the occurrence and development of malignant tumours.Unfortunately,there are no reports on the evaluation of the potential role of PSMD6 in hepatocellular carcinoma(HCC).AIM To comprehensively evaluate the overexpression pattern and clinical significance of PSMD6 in HCC tissues.METHODS This study integrated PSMD6 mRNA expression profiles from 4672 HCC and 3667 non-HCC tissues,along with immunohistochemical scores from 383 HCC and adjacent tissues,to assess PSMD6 overexpression in HCC.Clustered regularly interspaced short palindromic repeats knockout technology evaluated PSMD6’s essential role in HCC cell growth.Functional enrichment analysis explored the molecular mechanism of PSMD6 abnormalities in HCC.Drug sensitivity analysis and molecular docking analysed the effect of abnormal expression of PSMD6 on the drug sensitivity of HCC cells.RESULTS The results of 41 external and two internal datasets showed that PSMD6 mRNA(SMD=0.26,95%CI:0.09-0.42,P<0.05)and protein(SMD=2.85,95%CI:1.19-4.50,P<0.05)were significantly overexpressed in HCC tissues.The integrated analysis results showed that PSMD6 had a significant overexpression pattern in HCC tissues(SMD=0.40,95%CI:0.15-0.66,P<0.05).PSMD6 knockout inhibited HCC cell growth(chronos scores<-1).Functional enrichment implicated ribosome biogenesis and RNA splicing.Significant enrichment of signalling pathways such as RNA degradation,ribosomes,and chemical carcinogenesis—reactive oxygen species.Drug sensitivity analysis and a molecular docking model showed that high expression of PSMD6 was associated with the tolerance of HCC cells to drugs such as ML323,sepantronium bromide,and GDC0810.Overexpressed PSMD6 effectively distinguished HCC tissues(AUC=0.75,95%CI:0.71-0.79).CONCLUSION This study was the first to discover that PSMD6 was overexpressed in HCC tissues.PSMD6 is essential for the growth of HCC cells and may be involved in ribosome biogenesis and RNA splicing.展开更多
The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into ...The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.展开更多
Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulator...Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.展开更多
In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a smal...In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents.Thus,there has been a sustained search for novel molecular targets for iCCA.The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6(PSMD6),a 19S regulatory subunit of the proteasome,in human iCCA cells and specimens.The authors employed clustered regularly interspaced short palindromic repeat(CRISPR)knockout screening technology integrated with the computational CERES algorithm,and analyzed the human protein atlas(THPA)database and tissue microarrays.The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines,and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters.The authors conclude that PSMD6 may play a promoting role in iCCA.The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening,in vivo experiments,and a discussion of plausible mechanistic cues,which,therefore,dampen the significance of the results.Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA.In addition,the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA,development of proteasome inhibitors for cancer therapy,and advantages of CRISPR screening technology,computational methods,and THPA database as experimental tools for fighting cancer.We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.展开更多
目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6...目的:利用成簇规律性间隔短回文重复序列(clustered regularly interspaced short palindromic repeat,CRISPR)相关蛋白9(CRISP associated protein 9,Cas9)技术构建微小核糖核酸-551b(miR-551b)基因敲除小鼠模型。方法:选择健康C57BL/6J小鼠,针对miR-551b外显子1区域,设计导向RNA(guide RNA,gRNA),构建Cas9载体质粒,将体外转录的Cas9 RNA及gRNA显微注射入小鼠的受精卵并体外培养。将培养合格的胚胎移植到代孕小鼠的输卵管中,待小鼠生育后得到F0代小鼠,使用基因测序确定基因敲除情况,与野生型小鼠繁育后,得到F1代杂合小鼠,F1代小鼠经自交繁育获得F2代小鼠,F3代小鼠由F2代纯合小鼠自交获得,采用电泳鉴定小鼠基因型,RT-PCR检测F3代小鼠组织miR-551b的表达。结果:利用CRISPR/Cas9技术构建模型小鼠得到F0代小鼠,通过测序筛选出缺失目标序列的F0代杂合子小鼠。与WT小鼠繁育后,琼脂糖凝胶电泳及测序筛选出F1代杂合小鼠,同样的方法鉴定并获得F2、F3代基因敲除小鼠,获取F3代纯合小鼠的心脏及下腔静脉样本,RT-PCR结果证实F3代纯合小鼠miR551b表达明显低于WT小鼠(P<0.05),成功敲除miR-551b基因。结论:通过CRISPR-Cas9技术成功构建miR⁃155基因敲除小鼠模型并稳定遗传,为进一步研究提供了有利条件。展开更多
文摘The scientific community is continuously working to translate the novel biomedical techniques into effective medical treatments.CRISPR-Cas9 system(Clustered Regularly Interspaced Short Palindromic Repeats-9),commonly known as the“molecular scissor”,represents a recently developed biotechnology able to improve the quality and the efficacy of traditional treatments,related to several human diseases,such as chronic diseases,neurodegenerative pathologies and,interestingly,oral diseases.Of course,dental medicine has notably increased the use of biotechnologies to ensure modern and conservative approaches:in this landscape,the use of CRISPR-Cas9 system may speed and personalize the traditional therapies,ensuring a good predictability of clinical results.The aim of this critical overview is to provide evidence on CRISPR efficacy,taking into specific account its applications in oral medicine.
文摘BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AIM To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.METHODS The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology.Forty-two paired specimens of ICC and adjacent noncancerous tissues were collected.PSMD6 protein expression was determined by immunohistochemistry.Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level,and its association with ICC patients’various clinicopathological characteristics was investigated.RESULTS The PSMD6 gene was found to be essential for the growth of ICC cell lines.PSMD6 protein was significantly overexpressed in ICC tissues(P<0.001),but showed no significant association with patient age,gender,pathological grade,or tumor-node-metastasis stage(P>0.05).CONCLUSION PSMD6 can promote the growth of ICC cells,thus playing a pro-oncogenic role.
基金Supported by National Natural Science Foundation of China,No.82160762Guangxi Zhuang Autonomous Region Administration of Traditional Chinese Medicine Scientific Research Project,No.GXZYA20230267+2 种基金China Undergraduate Innovation and Entrepreneurship Training Program,No.S202410598060XChina Undergraduate Innovation and Entrepreneurship Training Program,No.X202410598360Future Academic Star of Guangxi Medical University,No.WLXSZX24074.
文摘BACKGROUND In recent years,many studies have shown that proteasome 26S subunit non-ATPase 6(PSMD6)plays an important role in the occurrence and development of malignant tumours.Unfortunately,there are no reports on the evaluation of the potential role of PSMD6 in hepatocellular carcinoma(HCC).AIM To comprehensively evaluate the overexpression pattern and clinical significance of PSMD6 in HCC tissues.METHODS This study integrated PSMD6 mRNA expression profiles from 4672 HCC and 3667 non-HCC tissues,along with immunohistochemical scores from 383 HCC and adjacent tissues,to assess PSMD6 overexpression in HCC.Clustered regularly interspaced short palindromic repeats knockout technology evaluated PSMD6’s essential role in HCC cell growth.Functional enrichment analysis explored the molecular mechanism of PSMD6 abnormalities in HCC.Drug sensitivity analysis and molecular docking analysed the effect of abnormal expression of PSMD6 on the drug sensitivity of HCC cells.RESULTS The results of 41 external and two internal datasets showed that PSMD6 mRNA(SMD=0.26,95%CI:0.09-0.42,P<0.05)and protein(SMD=2.85,95%CI:1.19-4.50,P<0.05)were significantly overexpressed in HCC tissues.The integrated analysis results showed that PSMD6 had a significant overexpression pattern in HCC tissues(SMD=0.40,95%CI:0.15-0.66,P<0.05).PSMD6 knockout inhibited HCC cell growth(chronos scores<-1).Functional enrichment implicated ribosome biogenesis and RNA splicing.Significant enrichment of signalling pathways such as RNA degradation,ribosomes,and chemical carcinogenesis—reactive oxygen species.Drug sensitivity analysis and a molecular docking model showed that high expression of PSMD6 was associated with the tolerance of HCC cells to drugs such as ML323,sepantronium bromide,and GDC0810.Overexpressed PSMD6 effectively distinguished HCC tissues(AUC=0.75,95%CI:0.71-0.79).CONCLUSION This study was the first to discover that PSMD6 was overexpressed in HCC tissues.PSMD6 is essential for the growth of HCC cells and may be involved in ribosome biogenesis and RNA splicing.
文摘The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.
基金the European Structural and Investment Funded Grant"Cardio Metabolic"(#KK.01.2.1.02.0321)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010)+2 种基金the European Regional Development Fund Grant,project"CRISPR/Cas9-CasMouse"(#KK.01.1.1.04.0085)the European Structural and Investment Funded Project of Centre of Competence in Molecular Diagnostics(#KK.01.2.2.03.0006)the Croatian National Centre of Research Excellence in Personalized Healthcare Grant(#KK.01.1.1.01.0010).
文摘Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.
基金Supported by The National Key Research and Development Program of China,No.2017YFC1308602The Research Funds by the Fifth Affiliated Hospital of Harbin Medical University,No.2022-002 and No.2023-001.
文摘In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents.Thus,there has been a sustained search for novel molecular targets for iCCA.The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6(PSMD6),a 19S regulatory subunit of the proteasome,in human iCCA cells and specimens.The authors employed clustered regularly interspaced short palindromic repeat(CRISPR)knockout screening technology integrated with the computational CERES algorithm,and analyzed the human protein atlas(THPA)database and tissue microarrays.The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines,and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters.The authors conclude that PSMD6 may play a promoting role in iCCA.The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening,in vivo experiments,and a discussion of plausible mechanistic cues,which,therefore,dampen the significance of the results.Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA.In addition,the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA,development of proteasome inhibitors for cancer therapy,and advantages of CRISPR screening technology,computational methods,and THPA database as experimental tools for fighting cancer.We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.
