N6-methyladenosine(m^(6)A)plays an important role in embryogenesis,nuclear export,transcription splicing,and protein translation control.Herein,we demonstrate a copper-free click chemistry-mediated assembly of single ...N6-methyladenosine(m^(6)A)plays an important role in embryogenesis,nuclear export,transcription splicing,and protein translation control.Herein,we demonstrate a copper-free click chemistry-mediated assembly of single quantum dot(QD)nanosensor for accurately monitoring locus-specific m^(6)A in cancer cells.The m^(6)A-sensitive endoribonuclease MazF can digest the unmethylated A-RNA,and the intact m^(6)A-RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid,initiating the click chemistry to generate probe a–b ligation product via first tandem ligation detection reaction(LDR)cycle.Subsequently,DNA probes c and d can hybridize with the probe a–b ligation product to generate the probe c–d ligation product via second LDR cycle.Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin-/fluorophore-modified probe c–d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer(FRET)between QD and Cy5.This assay can be homogenously performed without the involvement of copper catalyst,m^(6)A-specific antibody,radioactive labeling,ligase enzyme,enzymatic reverse transcription,and next-generation sequencing.Moreover,it can discriminate even 0.01% m^(6)A level in complex samples and accurately measure cellular m^(6)A-RNA expression,providing a promising avenue for clinical diagnostics and biomedical research.展开更多
The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development,whereas improper activation of apoptosis can impair spermatogenesis and cause defects in repr...The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development,whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction.We previously demonstrated that in mice,the makorin-2(Mkrn 2)gene is expressed exclusively in the testis and its deletion leads to male infertility.To understand the potential molecular mechanism,in this study,we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2.To identify specific gene(s)involved,we performed digital gene expression profiling(DGE)and pathway analysis via gene set enrichment analysis(GSEA)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22(PERP)expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels.GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility.Further,Gene Expression Omnibus(GEO)dataset analysis showed that p53,upstream of PERP,was upregulated in oligoasthenoteratozoospermia(OAT).These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.展开更多
The Hedyotis diffusa Willd herbal compounds(HDWHCs)are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China.However,the underlying mechanisms remain to be ...The Hedyotis diffusa Willd herbal compounds(HDWHCs)are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China.However,the underlying mechanisms remain to be elucidated.In this study,we used freeze-dried powder of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment.HDWHCs treatment significantly inhibited vascular endothelial growth factor(VEGF)mRNA levels and VEGF transcriptional activation in cancer cells.HDWHCs also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha(HIF-1alpha).Forced expression of HIF-1αrestored VEGF transcriptional activation inhibited by HDWHCs,indicating that HDWHCs suppressed VEGF expression through decreasing HIF-1alpha expression.Moreover,HDWHCs inhibited cyclooxygenase-2(COX-2)expression,and overexpression of HIF-1alpha restored HDWHCs’inhibitory effect on COX-2 at transcriptional level.These findings may provide better understanding of HDWHCs’anti-cancer mechanism in cancer treatment.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.21735003).
文摘N6-methyladenosine(m^(6)A)plays an important role in embryogenesis,nuclear export,transcription splicing,and protein translation control.Herein,we demonstrate a copper-free click chemistry-mediated assembly of single quantum dot(QD)nanosensor for accurately monitoring locus-specific m^(6)A in cancer cells.The m^(6)A-sensitive endoribonuclease MazF can digest the unmethylated A-RNA,and the intact m^(6)A-RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid,initiating the click chemistry to generate probe a–b ligation product via first tandem ligation detection reaction(LDR)cycle.Subsequently,DNA probes c and d can hybridize with the probe a–b ligation product to generate the probe c–d ligation product via second LDR cycle.Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin-/fluorophore-modified probe c–d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer(FRET)between QD and Cy5.This assay can be homogenously performed without the involvement of copper catalyst,m^(6)A-specific antibody,radioactive labeling,ligase enzyme,enzymatic reverse transcription,and next-generation sequencing.Moreover,it can discriminate even 0.01% m^(6)A level in complex samples and accurately measure cellular m^(6)A-RNA expression,providing a promising avenue for clinical diagnostics and biomedical research.
基金the National Natural Science Foundation of China(No.81270736)the China Postdoctoral Science Foundation(No.2016M600432)the Jiangsu Provincial Natural Science Foundation(No.BK20150996).
文摘The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development,whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction.We previously demonstrated that in mice,the makorin-2(Mkrn 2)gene is expressed exclusively in the testis and its deletion leads to male infertility.To understand the potential molecular mechanism,in this study,we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2.To identify specific gene(s)involved,we performed digital gene expression profiling(DGE)and pathway analysis via gene set enrichment analysis(GSEA)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22(PERP)expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels.GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility.Further,Gene Expression Omnibus(GEO)dataset analysis showed that p53,upstream of PERP,was upregulated in oligoasthenoteratozoospermia(OAT).These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant Nos.30470361,30570962,and 30871296)from Nanjing Medical University(No.08NMUM007).
文摘The Hedyotis diffusa Willd herbal compounds(HDWHCs)are commonly used as Chinese medicine to treat cancer patients with established clinical therapeutic efficacy in China.However,the underlying mechanisms remain to be elucidated.In this study,we used freeze-dried powder of the water extracts of HDWHCs to investigate the potential mechanisms of HDWHCs in cancer treatment.HDWHCs treatment significantly inhibited vascular endothelial growth factor(VEGF)mRNA levels and VEGF transcriptional activation in cancer cells.HDWHCs also had a remarkable inhibitory effect on the expression of hypoxia-inducible factor 1alpha(HIF-1alpha).Forced expression of HIF-1αrestored VEGF transcriptional activation inhibited by HDWHCs,indicating that HDWHCs suppressed VEGF expression through decreasing HIF-1alpha expression.Moreover,HDWHCs inhibited cyclooxygenase-2(COX-2)expression,and overexpression of HIF-1alpha restored HDWHCs’inhibitory effect on COX-2 at transcriptional level.These findings may provide better understanding of HDWHCs’anti-cancer mechanism in cancer treatment.
