NORHA,a long non-coding RNA(lncRNA),serves as a key inducer of follicular atresia in sows by triggering granulosa cells(GCs)apoptosis.However,its regulation by N6-methyladenosine(m6A)-the most abundant RNA modificatio...NORHA,a long non-coding RNA(lncRNA),serves as a key inducer of follicular atresia in sows by triggering granulosa cells(GCs)apoptosis.However,its regulation by N6-methyladenosine(m6A)-the most abundant RNA modification-remains unresolved.This study identified NORHA as a functional target of the m6A reader HNRNPA2B1 in sow GCs(sGCs).Transcriptome-wide mapping of RNA modification sites revealed extensive m6A enrichment on NORHA,with HNRNPA2B1 binding directly to the transcript and enhancing its stability via modification of multiple m6A sites,including A261,A441,and A919.HNRNPA2B1 suppressed 17β-estradiol(E2)biosynthesis and promoted sGC apoptosis by activating the NORHA-FoxO1 axis.FoxO1 subsequently repressed expression of cytochrome P450 family 19 subfamily A member 1(CYP19A1),which encodes the enzyme essential for E2 biosynthesis.Additionally,HNRNPA2B1 functioned as a critical mediator of METTL3-dependent m6A modification,modulating NORHA expression and activity in sGCs.This study highlights an important m6Adependent regulatory mechanism governing NORHA expression in sGCs.展开更多
Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,th...Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.展开更多
Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class...Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class of noncoding RNAs(ncRNAs).However,little is known about long noncoding RNAs(lncRNAs),which constitute another ncRNA family that regulate follicular atresia.Results:A total of 77 differentially expressed lncRNAs,including 67 upregulated and 10 downregulated lncRNAs,were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing.We characterized a noncoding RNA that was highly expressed in atretic follicles(NORHA).As an intergenic lncRNA,NORHA was one of the upregulated lncRNAs identified in the atretic follicles.To determine NORHA function,RT-PCR,flow cytometry and western blotting were performed,and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress.To determine the mechanism of action,bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation assay were performed,and the results showed that NORHA acted as a‘sponge’,that directly bound to the miR-183-96-182 cluster,and thus prevented its targeted inhibition of FoxO1,a major sensor and effector of oxidative stress.Conclusions:We provide a comprehensive perspective of lncRNA regulation of follicular atresia,and demonstrate that NORHA,a novel lncRNA related to follicular atresia,induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.展开更多
基金supported by the National Natural Science Foundation of China(32072693)Fundamental Research Funds for the Central Universities(KYLH2025010)Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX24-0994)。
文摘NORHA,a long non-coding RNA(lncRNA),serves as a key inducer of follicular atresia in sows by triggering granulosa cells(GCs)apoptosis.However,its regulation by N6-methyladenosine(m6A)-the most abundant RNA modification-remains unresolved.This study identified NORHA as a functional target of the m6A reader HNRNPA2B1 in sow GCs(sGCs).Transcriptome-wide mapping of RNA modification sites revealed extensive m6A enrichment on NORHA,with HNRNPA2B1 binding directly to the transcript and enhancing its stability via modification of multiple m6A sites,including A261,A441,and A919.HNRNPA2B1 suppressed 17β-estradiol(E2)biosynthesis and promoted sGC apoptosis by activating the NORHA-FoxO1 axis.FoxO1 subsequently repressed expression of cytochrome P450 family 19 subfamily A member 1(CYP19A1),which encodes the enzyme essential for E2 biosynthesis.Additionally,HNRNPA2B1 functioned as a critical mediator of METTL3-dependent m6A modification,modulating NORHA expression and activity in sGCs.This study highlights an important m6Adependent regulatory mechanism governing NORHA expression in sGCs.
基金supported by the National Key R&D Program of China(2022YFD1600903)the National Natural Science Foundation of China(32072693)the College Students’Innovative Entrepreneurial Training Plan Program(202110307028).
文摘Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.
基金This work was supported by the National Natural Science Foundation of China(32072693 and 31630072)the Qing Lan Project of Jiangsu Province(2020).
文摘Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class of noncoding RNAs(ncRNAs).However,little is known about long noncoding RNAs(lncRNAs),which constitute another ncRNA family that regulate follicular atresia.Results:A total of 77 differentially expressed lncRNAs,including 67 upregulated and 10 downregulated lncRNAs,were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing.We characterized a noncoding RNA that was highly expressed in atretic follicles(NORHA).As an intergenic lncRNA,NORHA was one of the upregulated lncRNAs identified in the atretic follicles.To determine NORHA function,RT-PCR,flow cytometry and western blotting were performed,and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress.To determine the mechanism of action,bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation assay were performed,and the results showed that NORHA acted as a‘sponge’,that directly bound to the miR-183-96-182 cluster,and thus prevented its targeted inhibition of FoxO1,a major sensor and effector of oxidative stress.Conclusions:We provide a comprehensive perspective of lncRNA regulation of follicular atresia,and demonstrate that NORHA,a novel lncRNA related to follicular atresia,induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.
