Background:Pancreatic adenocarcinoma(PAAD)is an extremely lethal malignancy.Identification of the functional genes and genetic variants related to PAAD prognosis is important and challenging.Previously identified prog...Background:Pancreatic adenocarcinoma(PAAD)is an extremely lethal malignancy.Identification of the functional genes and genetic variants related to PAAD prognosis is important and challenging.Previously identified prognostic genes from several expression profile analyses were inconsistent.The regulatory genetic variants that affect PAAD prognosis were largely unknown.Methods:Firstly,a meta-analysis was performed with seven published datasets to systematically explore the candidate prognostic genes for PAAD.Next,to identify the regulatory variants for those candidate genes,expression quantitative trait loci analysis was implemented with PAAD data resources from The Cancer Genome Atlas.Then,a two-stage association study in a total of 893 PAAD patients was conducted to interrogate the regulatory variants and find the prognostic locus.Finally,a series of biochemical experiments and phenotype assays were carried out to demonstrate the biological function of variation and genes in PAAD progression process.Results:A total of 128 genes were identified associated with the PAAD prognosis in the meta-analysis.Fourteen regulatory loci in 12 of the 128 genes were discovered,among which,only rs4887783,the functional variant in the promoter of Ring Finger and WD Repeat Domain 3(RFWD3),presented significant association with PAAD prognosis in both stages of the population study.Dualluciferase reporter and electrophoretic mobility shift assays demonstrated that rs4887783-G allele,which predicts the worse prognosis,enhanced the binding of transcript factor REST,thus elevating RFWD3 expression.Further phenotypic assays revealed that excess expression of RFWD3 promoted tumor cell migration without affecting their proliferation rate.RFWD3 was highly expressed in PAAD and might orchestrate the genes in the DNA repair process.Conclusions:RFWD3 and its regulatory variant are novel genetic factors for PAAD prognosis.展开更多
Background:Findings on the association of genetic factors and colorectal cancer(CRC)survival are limited and inconsistent,and revealing the mechanism underlying their prognostic roles is of great importance.This study...Background:Findings on the association of genetic factors and colorectal cancer(CRC)survival are limited and inconsistent,and revealing the mechanism underlying their prognostic roles is of great importance.This study aimed to explore the relationship between functional genetic variations and the prognosis of CRC and further reveal the possible mechanism.Methods:We first systematically performed expression quantitative trait locus(eQTL)analysis using The Cancer Genome Atlas(TCGA)dataset.Then,the Kaplan-Meier analysis was used to filter out the survival-related eQTL target genes of CRC patients in two public datasets(TCGA and GSE39582 dataset from the Gene Expression Omnibus database).The seven most potentially functional eQTL single nucleotide polymorphisms(SNPs)associated with six survival-related eQTL target genes were genotyped in 907 Chinese CRC patients with clinical prognosis data.The regulatory mechanism of the survival-related SNP was further confirmed by functional experiments.Results:The rs71630754 regulating the expression of endoplasmic reticulum aminopeptidase 1(ERAP1)was significantly associated with the prognosis of CRC(additive model,hazard ratio[HR]:1.43,95%confidence interval[CI]:1.08-1.88,P=0.012).The results of dual-luciferase reporter assay and electrophoretic mobility shift assay showed that the A allele of the rs71630754 could increase the binding of transcription factor 3(TCF3)and subsequently reduce the expression of ERAP1.The results of bioinformatic analysis showed that lower expression of ERAP1 could affect the tumor immune microenvironment and was significantly associated with severe survival outcomes.Conclusion:The rs71630754 could influence the prognosis of CRC patients by regulating the expression of the immune-related gene ERAP1.Trial Registration:No.NCT00454519(https://clinicaltrials.gov/)展开更多
目的在妊娠期糖尿病(GDM)中探讨miR-372-3p表达及其与胰岛素抵抗(IR)的相互关系并初步探究其分子机制;方法选取2018年1月至2019年1月于兰州大学第二附属医院正规产检并住院分娩的56例GDM孕妇为GDM组,选取同期在本院分娩的60例正常孕妇...目的在妊娠期糖尿病(GDM)中探讨miR-372-3p表达及其与胰岛素抵抗(IR)的相互关系并初步探究其分子机制;方法选取2018年1月至2019年1月于兰州大学第二附属医院正规产检并住院分娩的56例GDM孕妇为GDM组,选取同期在本院分娩的60例正常孕妇作为正常对照组(NC组)。观察对比两组孕妇一般临床资料(如年龄、产前BMI、OGTT孕周等)和代谢相关生化指标(总胆固醇(TC)、三酰甘油(TG)空腹血糖值(FPG)、餐后2 h血糖(2 h PG)、空腹胰岛素(FINS)等),并计算胰岛β细胞功能指数(HOMA-β)及胰岛素抵抗指数(HOMA-IR);RT-PCR检测两组受试者胎盘组织中的miR-372-3p的表达水平;Pearson相关系数分析GDM组中miR-372-3p表达量与代谢相关指标的相关性;生物信息学选取GLUT-4作为miR-372-3p的目标靶基因,并采用双荧光素酶基因报告实验进行验证两者的靶向关系;在HTR-8细胞中转染miR-372-3p mimics或miR-372-3p inhibitor进行上调或抑制miR-372-3p表达,RT-PCR检测转染效率后,Western blot实验检测miR-372-3p对GLUT-4蛋白表达的影响。结果与NC组相比,GDM组孕产妇仅产前BMI显著增加(P<0.01),其余临床资料差异均无统计学意义(P>0.05);代谢相关指标相比,GDM组的TC、TG、FPG、2 h PG、FINS及HOMA-IR均较NC组显著增加(P<0.01),而HOMA-β显著低于NC组(P<0.01),其余指标差异均无统计学意义(P>0.05);GDM组的胎盘miR-372-3p表达较NC组显著升高(P<0.05)且其表达水平与FPG、FINS、HOMA-IR均呈正相关关系(P<0.05);双荧光素酶基因报告实验证实miR-372-3p可靶向调控GLUT-4表达且在HTR-8细胞中miR-372-3p可负向调控GLU-4蛋白表达;结论在GDM中miR-372-3p可能通过负向调控GLUT-4表达参与胰岛素抵抗。展开更多
Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially ...Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.展开更多
Transcriptome deep sequencing(RNA‐seq)hasbecome a routine method for global geneexpression profiling.However,its application tolarge‐scale experiments remains limited by costand labor constraints.Here we describe am...Transcriptome deep sequencing(RNA‐seq)hasbecome a routine method for global geneexpression profiling.However,its application tolarge‐scale experiments remains limited by costand labor constraints.Here we describe amassively parallel 3′end RNA‐seq(MP3RNA‐seq)method that introduces unique samplebarcodes during reverse transcription to permitsample pooling immediately following this initialstep.MP3RNA‐seq allows for handling of hun-dreds of samples in a single experiment,at acost of about$6 per sample for libraryconstruction and sequencing.MP3RNA‐seq iseffective for not only high‐throughput geneexpression profiling,but also genotyping.Todemonstrate its utility,we applied MP3RNA‐seqto 477 double haploid lines of maize.We iden-tified 19,429 genes expressed in at least 50%ofthe lines and 35,836 high‐quality singlenucleotide polymorphisms for genotypinganalysis.Armed with these data,we performedexpression and agronomic trait quantitativetrait locus(QTL)mapping and identified 25,797expression QTLs for 15,335 genes and 21 QTLsfor plant height,ear height,and relative earheight.We conclude that MP3RNA‐seq is highlyreproducible,accurate,and sensitive forhigh‐throughput gene expression profiling andgenotyping,and should be generally applicableto most eukaryotic species.展开更多
基金This work was supported by grants from the Youth Program of National Natural Science Foundation of China (No. NSFC-82003547) , China Postdoctoral Science Foundation (No. 2019M662644) , and the Fundamental Research Funds for the Central Universities (No. WHU 2042022kf1031) for Ying Zhu, National Science Fund for Distinguished Young Scholars of China (No. NSFC-81925032) and Natural Science Foundation of Hubei Province (No. 2019CFA009) for Xiaoping Miao, National Program for Support of Top-notch Young Professionals and the Young Elite Scientist Sponsorship Program by CAST (No. 2018QNRC001) for Jiang Chang, Youth Program of National Natural Science Foundation of China (No. NSFC-82103929) and the Fundamental Research Funds for the Central Universities (No. WHU 2042022kf1205) for Jianbo Tian.
文摘Background:Pancreatic adenocarcinoma(PAAD)is an extremely lethal malignancy.Identification of the functional genes and genetic variants related to PAAD prognosis is important and challenging.Previously identified prognostic genes from several expression profile analyses were inconsistent.The regulatory genetic variants that affect PAAD prognosis were largely unknown.Methods:Firstly,a meta-analysis was performed with seven published datasets to systematically explore the candidate prognostic genes for PAAD.Next,to identify the regulatory variants for those candidate genes,expression quantitative trait loci analysis was implemented with PAAD data resources from The Cancer Genome Atlas.Then,a two-stage association study in a total of 893 PAAD patients was conducted to interrogate the regulatory variants and find the prognostic locus.Finally,a series of biochemical experiments and phenotype assays were carried out to demonstrate the biological function of variation and genes in PAAD progression process.Results:A total of 128 genes were identified associated with the PAAD prognosis in the meta-analysis.Fourteen regulatory loci in 12 of the 128 genes were discovered,among which,only rs4887783,the functional variant in the promoter of Ring Finger and WD Repeat Domain 3(RFWD3),presented significant association with PAAD prognosis in both stages of the population study.Dualluciferase reporter and electrophoretic mobility shift assays demonstrated that rs4887783-G allele,which predicts the worse prognosis,enhanced the binding of transcript factor REST,thus elevating RFWD3 expression.Further phenotypic assays revealed that excess expression of RFWD3 promoted tumor cell migration without affecting their proliferation rate.RFWD3 was highly expressed in PAAD and might orchestrate the genes in the DNA repair process.Conclusions:RFWD3 and its regulatory variant are novel genetic factors for PAAD prognosis.
基金supported by grants from the National Natural Science Foundation of China(Nos.82273713 and 82103929)the Young Elite Scientists Sponsorship Program by CAST(No.2022QNRC001)+6 种基金the Fundamental Research Funds for the Central Universities(No.WHU:2042022kf1205)the Knowledge Innovation Program of Wuhan(No.whkxjsj011)the Distinguished Young Scholars of China(No.81925032)the Key Program of National Natural Science Foundation of China(No.82130098)the Hubei Provincial Natural Science Foundation of China(No.2019CFA009)the Youth Program of National Natural Science Foundation of China(No.82003547)the Fundamental Research Funds for the Central Universities(No.WHU:2042022kf 1031)
文摘Background:Findings on the association of genetic factors and colorectal cancer(CRC)survival are limited and inconsistent,and revealing the mechanism underlying their prognostic roles is of great importance.This study aimed to explore the relationship between functional genetic variations and the prognosis of CRC and further reveal the possible mechanism.Methods:We first systematically performed expression quantitative trait locus(eQTL)analysis using The Cancer Genome Atlas(TCGA)dataset.Then,the Kaplan-Meier analysis was used to filter out the survival-related eQTL target genes of CRC patients in two public datasets(TCGA and GSE39582 dataset from the Gene Expression Omnibus database).The seven most potentially functional eQTL single nucleotide polymorphisms(SNPs)associated with six survival-related eQTL target genes were genotyped in 907 Chinese CRC patients with clinical prognosis data.The regulatory mechanism of the survival-related SNP was further confirmed by functional experiments.Results:The rs71630754 regulating the expression of endoplasmic reticulum aminopeptidase 1(ERAP1)was significantly associated with the prognosis of CRC(additive model,hazard ratio[HR]:1.43,95%confidence interval[CI]:1.08-1.88,P=0.012).The results of dual-luciferase reporter assay and electrophoretic mobility shift assay showed that the A allele of the rs71630754 could increase the binding of transcription factor 3(TCF3)and subsequently reduce the expression of ERAP1.The results of bioinformatic analysis showed that lower expression of ERAP1 could affect the tumor immune microenvironment and was significantly associated with severe survival outcomes.Conclusion:The rs71630754 could influence the prognosis of CRC patients by regulating the expression of the immune-related gene ERAP1.Trial Registration:No.NCT00454519(https://clinicaltrials.gov/)
文摘目的在妊娠期糖尿病(GDM)中探讨miR-372-3p表达及其与胰岛素抵抗(IR)的相互关系并初步探究其分子机制;方法选取2018年1月至2019年1月于兰州大学第二附属医院正规产检并住院分娩的56例GDM孕妇为GDM组,选取同期在本院分娩的60例正常孕妇作为正常对照组(NC组)。观察对比两组孕妇一般临床资料(如年龄、产前BMI、OGTT孕周等)和代谢相关生化指标(总胆固醇(TC)、三酰甘油(TG)空腹血糖值(FPG)、餐后2 h血糖(2 h PG)、空腹胰岛素(FINS)等),并计算胰岛β细胞功能指数(HOMA-β)及胰岛素抵抗指数(HOMA-IR);RT-PCR检测两组受试者胎盘组织中的miR-372-3p的表达水平;Pearson相关系数分析GDM组中miR-372-3p表达量与代谢相关指标的相关性;生物信息学选取GLUT-4作为miR-372-3p的目标靶基因,并采用双荧光素酶基因报告实验进行验证两者的靶向关系;在HTR-8细胞中转染miR-372-3p mimics或miR-372-3p inhibitor进行上调或抑制miR-372-3p表达,RT-PCR检测转染效率后,Western blot实验检测miR-372-3p对GLUT-4蛋白表达的影响。结果与NC组相比,GDM组孕产妇仅产前BMI显著增加(P<0.01),其余临床资料差异均无统计学意义(P>0.05);代谢相关指标相比,GDM组的TC、TG、FPG、2 h PG、FINS及HOMA-IR均较NC组显著增加(P<0.01),而HOMA-β显著低于NC组(P<0.01),其余指标差异均无统计学意义(P>0.05);GDM组的胎盘miR-372-3p表达较NC组显著升高(P<0.05)且其表达水平与FPG、FINS、HOMA-IR均呈正相关关系(P<0.05);双荧光素酶基因报告实验证实miR-372-3p可靶向调控GLUT-4表达且在HTR-8细胞中miR-372-3p可负向调控GLU-4蛋白表达;结论在GDM中miR-372-3p可能通过负向调控GLUT-4表达参与胰岛素抵抗。
基金partly supported by the Genome Tagging Project,Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation and grants from the Chinese Academy of Sciences(XDB19010204,OYZDJ-SSW-SMC023 and Facility-based Open Research Program)the National Natural Science Foundation of China(31530048,81672117,31730062,31821004,and31601163)+1 种基金the Ministry of Science and Technology of China(2019YFA0109900)Shanghai Municipal Commission for Science and Technology(16JC1420500,17JC1420102,17JC1400900,and17411954900)
文摘Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.
基金supported by grants from National NaturalScience Foundation of China(31421005)National Key Re-search and Development Program(2016YFD0100404,2016YFD0101804)+1 种基金National Postdoctoral Program for Innovative Talents(BX201600149)China PostdoctoralScience Foundation(2017M611049)。
文摘Transcriptome deep sequencing(RNA‐seq)hasbecome a routine method for global geneexpression profiling.However,its application tolarge‐scale experiments remains limited by costand labor constraints.Here we describe amassively parallel 3′end RNA‐seq(MP3RNA‐seq)method that introduces unique samplebarcodes during reverse transcription to permitsample pooling immediately following this initialstep.MP3RNA‐seq allows for handling of hun-dreds of samples in a single experiment,at acost of about$6 per sample for libraryconstruction and sequencing.MP3RNA‐seq iseffective for not only high‐throughput geneexpression profiling,but also genotyping.Todemonstrate its utility,we applied MP3RNA‐seqto 477 double haploid lines of maize.We iden-tified 19,429 genes expressed in at least 50%ofthe lines and 35,836 high‐quality singlenucleotide polymorphisms for genotypinganalysis.Armed with these data,we performedexpression and agronomic trait quantitativetrait locus(QTL)mapping and identified 25,797expression QTLs for 15,335 genes and 21 QTLsfor plant height,ear height,and relative earheight.We conclude that MP3RNA‐seq is highlyreproducible,accurate,and sensitive forhigh‐throughput gene expression profiling andgenotyping,and should be generally applicableto most eukaryotic species.
