NORFA, long intergenic noncoding RNA, maintains sow fertility by inhibiting granulosa cell death

Du, Xiaohong; Liu, Lu; Zhang, Lifan; Pan, Zengxiang

doi:10.1038/s42003-020-0864-x

Cited by 43 publications

(58 citation statements)

References 69 publications

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“…In vitro assays proved that knockdown of NORFA significantly induced porcine GC apoptosis, impaired the normal morphology and cell viability, and dramatically inhibited E2 synthesis. Interestingly, we also noticed that NFIX, a transcription factor targeting the promoter of NORFA ( Du et al, 2020 ), is dramatically down-regulated in NORFA -reduced porcine GCs, indicating that a positive feedback regulatory network between NORFA and NFIX exists. Apart from the well-known genes, 202 function-unknown DEmRNAs were identified and further research will focus on investigating their functions.…”

Section: Discussionmentioning

confidence: 68%

“…The oligonucleotide sequences used in this study were synthesized by GenePharma (Shanghai, China) and listed as follows: NORFA -specific small interfering RNA (siNORFA), 5′-CAG ACA GAU GUG GAU GAA UTT-3′ (Sense), 5′-UGU GGU UGA UGU UGU UGG CTT-3′ (Anti-sense); NC-siRNA (siNC), 5′-UUC UCC GAA CGU GUC ACG UTT-3′ (Sense), 5′-ACG UGA CAC GUU CGG AGA ATT-3′ (Anti-sense). pcDNA3.1- NFIX , the eukaryotic expression vector for porcine NFIX , was constructed in our previous study ( Du et al, 2020 ). For pan-caspase inhibitor treatment, porcine GCs were seeded into six-well cell culture plates for 36 h, and the medium was then replaced with serum-free DMEM/F12 medium for 12 h. Subsequently, Z-VAD-FMK (#MK3270-1MG, MKBio, Shanghai, China), a well-known pan-caspase inhibitor, was added to the medium to a final concentration of 50 μM.…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of each protein sample was detected by BCA method (#P0012, Beyotime Biotechnology). Equal amounts of total protein (15 μg) were loaded and separated on 4–20% SDS-PAGE gel to measure the expression levels of interested proteins; Western blotting assays were performed as described previously ( Du et al, 2020 ). The primary antibodies used in this study were anti-Caspase3/cleaved-Caspase3 (#19677-1-AP, Proteintech, 1:1,000), anti-PCNA (#13110, Cell Signaling Technology, 1:1,000), anti-MCM2 (#4007s, Cell Signaling Technology, 1:1,000), anti-NFIX (#F3250, Affinity, 1:1,000), and anti-GAPDH (#TA802519, ORIGENE, 1:2,000).…”

Section: Methodsmentioning

confidence: 99%

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Transcriptomic Data Analyses Reveal That Sow Fertility-Related lincRNA NORFA Is Essential for the Normal States and Functions of Granulosa Cells

Yang

Zeng

et al. 2021

Front. Cell Dev. Biol.

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NORFA, the first lincRNA associated with sow fertility, has been shown to control granulosa cell (GC) functions and follicular atresia. However, the underlying mechanism is not fully understood. In this study, RNA-seq was performed and we noticed that inhibition of NORFA led to dramatic transcriptomic alterations in porcine GCs. A total of 1,272 differentially expressed transcripts were identified, including 1167 DEmRNAs and 105 DEmiRNAs. Furthermore, protein–protein interaction, gene-pathway function, and TF–miRNA–mRNA regulatory networks were established and yielded four regulatory modules with multiple hub genes, such as AR, ATG5, BAK1, CENPE, NR5A1, NFIX, WNT5B, ssc-miR-27b, and ssc-miR-126. Functional assessment showed that these hub DEGs were mainly enriched in TGF-β, PI3K-Akt, FoxO, Wnt, MAPK, and ubiquitin pathways that are essential for GC states (apoptosis and proliferation) and functions (hormone secretion). In vitro, we also found that knockdown of NORFA in porcine GCs significantly induced cell apoptosis, impaired cell viability, and suppressed 17β-estradiol (E2) synthesis. Notably, four candidate genes for sow reproductive traits (INHBA, NCOA1, TGFβ-1, and TGFBR2) were also identified as potential targets of NORFA. These findings present a panoramic view of the transcriptome in NORFA-reduced GCs, highlighting that NORFA, a candidate lincRNA for sow fertility, is crucial for the normal states and functions of GCs.

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Section: Discussionmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Transcriptomic Data Analyses Reveal That Sow Fertility-Related lincRNA NORFA Is Essential for the Normal States and Functions of Granulosa Cells

Yang

Zeng

et al. 2021

Front. Cell Dev. Biol.

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“…In ovary, SMAD4 and SMAD4-dependent TGF-β signaling pathway have shown to be associated with follicular development, especially follicular atresia, the ultimate fate of most follicular development 27,28 . Indeed, recent reports demonstrated that SMAD4 contributes to follicular atresia through inhibiting granulosa cell (GC) apoptosis, which is the main inducement of follicular atresia 29 . However, the mechanism of SMAD4 regulating GC apoptosis is not completely understood.…”

Section: Introductionmentioning

confidence: 99%

SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis

Yang

Liu

et al. 2020

Cell Death Dis

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The TGF-β and Wnt signaling pathways are interrelated in many cell types and tissues, and control cell functions in coordination. Here, we report that SMAD4, a downstream effector of the TGF-β signaling pathway, induces FZD4, a receptor of the Wnt signaling pathway, establishing a novel route of communication between these two pathways in granulosa cells (GCs). We found that SMAD4 is a strong inducer of FZD4, not only initiating FZD4 transcription but also activating FZD4-dependent Wnt signaling and GC apoptosis. Furthermore, we identified the direct and indirect mechanisms by which SMAD4 promotes expression of FZD4 in GCs. First, SMAD4 functions as a transcription factor to directly bind to the FZD4 promoter region to increase its transcriptional activity. Second, SMAD4 promotes production of SDNOR, a novel lncRNA that acts as a sponge for miR-29c, providing another mean to block miR-29c from degenerating FZD4 mRNA. Overall, our findings not only reveal a new channel of crosstalk between the TGF-β and Wnt signaling pathways, SMAD4-FZD4 axis, but also provide new insights into the regulatory network of GC apoptosis and follicular atresia. These RNA molecules, such as miR-29c and lnc-SDNOR, represent potential targets for treatment of reproductive diseases and improvement of female fertility.

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“…Several lncRNAs have been identified as regulators of GC apoptosis in mammals. For example, the lncRNA NORFA inhibited granulosa cell apoptosis through regulating miR-126/transforming growth factor- β (TGF- β ) axis [ 12 ]. Knockdown of PVT1 promoted cell proliferation, as well as inhibition of apoptosis of ovarian granulosa cells by regulating miR-17-5p and PTEN [ 28 ].…”

Section: Discussionmentioning

confidence: 99%

lncRNA MALAT1 Regulates Mouse Granulosa Cell Apoptosis and 17β-Estradiol Synthesis via Regulating miR-205/CREB1 Axis

Sun

Zhang

2021

BioMed Research International

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Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a known long noncoding RNA, was reported to play a crucial role in follicular growth and ovarian disease. However, the physiological function of MALAT1 in mouse granulosa cells (mGCs) remains largely unclear. The aims of this study were to determine the biological function and molecular mechanism of MALAT1 in mGCs. We knocked down MALAT1 in mGCs by using siRNA against MALAT1. We found that knockdown of MALAT1 promoted apoptosis and caspase-3/9 activities in mGCs. Enzyme-linked immunosorbent assay demonstrated that knockdown of MALAT1 significantly decreased the production of estradiol (E2) and progesterone (P4) in mGCs. Mechanistically, MALAT1 serves as a competing endogenous RNA (ceRNA) to sponge microRNA-205 (miR-205), thereby facilitating its downstream target of cyclic AMP response element- (CRE-) binding protein 1 (CREB1). Furthermore, CREB1 overexpression or miR-205 downregulation partially recovered the effect of MALAT1 depletion in mGCs. In summary, these findings suggested that MALAT1 regulated apoptosis and estradiol synthesis of mGCs through the miR-205/CREB1 axis.

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NORFA, long intergenic noncoding RNA, maintains sow fertility by inhibiting granulosa cell death

Cited by 43 publications

References 69 publications

Transcriptomic Data Analyses Reveal That Sow Fertility-Related lincRNA NORFA Is Essential for the Normal States and Functions of Granulosa Cells

Transcriptomic Data Analyses Reveal That Sow Fertility-Related lincRNA NORFA Is Essential for the Normal States and Functions of Granulosa Cells

SMAD4 activates Wnt signaling pathway to inhibit granulosa cell apoptosis

lncRNA MALAT1 Regulates Mouse Granulosa Cell Apoptosis and 17β-Estradiol Synthesis via Regulating miR-205/CREB1 Axis

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