From stability to reliability: Unveiling the un-biased reference genes in porcine ovarian granulosa cells under different conditions.

Selection of optimal reference genes (RGs) is fundamental for functional genomics studies and gene expression analysis, which are two main approaches to identify functional genes and their expression patterns. However, no systematic study has identified the suitable RGs in porcine ovarian granulosa cells (GCs) which are essential for follicle fate and sow fertility. In this study, the expression profiles of 12 widely-used RGs (GAPDH, RPLP0, ACTB, TUBA1B, EIF3K, PPIA, ATP5F1, B2M, HPRT1, UBC, RPS3, and EEF1A1) in porcine GCs during follicular development and under different abiotic stresses were systematically investigated. Expression stability of the candidate RGs were comprehensively accessed by five statistical algorithms including ΔCt, NormFinder, BestKeeper, geNorm, and RefFinder, indicating that RPS3 and PPIA are the optimal RGs during follicular development, EEF1A1 and RPLP0 are most stable under oxidative stress and inflammation, while ATP5F1, B2M, and RPS3 have higher stability under starvation and heat stress. Notably, the most commonly used RGs (ACTB, GAPDH, and TUBA1B) exhibited low stability in GCs. Reliability of stable RGs was verified by RT-qPCR and showed that selection of the stable RGs significantly improved the detection accuracy of qPCR, which confirms once again that the stability of RGs should not be taken for granted. Our findings identified optimal RG sets in porcine GCs under different conditions, which is helpful in future studies to accurately identify the key regulators and their expression patterns during follicular development in sows.

miR-423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis.

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.

Nuclear lncRNA NORSF reduces E2 release in granulosa cells by sponging the endogenous small activating RNA miR-339.

BACKGROUND: Functioning as a competing endogenous RNA (ceRNA) is the main action mechanism of most cytoplasmic lncRNAs. However, it is not known whether this mechanism of action also exists in the nucleus. RESULTS: We identified four nuclear lncRNAs that are presented in granulosa cells (GCs) and were differentially expressed during sow follicular atresia. Notably, similar to cytoplasmic lncRNAs, these nuclear lncRNAs also sponge miRNAs in the nucleus of GCs through direct interactions. Furthermore, NORSF (non-coding RNA involved in sow fertility), one of the nuclear lncRNA acts as a ceRNA of miR-339. Thereby, it relieves the regulatory effect of miR-339 on CYP19A1 encoding P450arom, a rate-limiting enzyme for E2 synthesis in GCs. Interestingly, miR-339 acts as a saRNA that activates CYP19A1 transcription and enhances E2 release by GCs through altering histone modifications in the promoter by directly binding to the CYP19A1 promoter. Functionally, NORSF inhibited E2 release by GCs via the miR-339 and CYP19A1 axis. CONCLUSIONS: Our findings highlight an unappreciated mechanism of nuclear lncRNAs and show it acts as a ceRNA, which may be a common lncRNA function in the cytoplasm and nucleus. We also identified a potential endogenous saRNA for improving female fertility and treating female infertility.

SDNOR, a Novel Antioxidative lncRNA, Is Essential for Maintaining the Normal State and Function of Porcine Follicular Granulosa Cells.

Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR's regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs.

AQP4 Aggravates Cognitive Impairment in Sepsis-Associated Encephalopathy through Inhibiting Nav 1.6-Mediated Astrocyte Autophagy.

The pathology of sepsis-associated encephalopathy (SAE) is related to astrocyte-inflammation associated with aquaporin-4 (AQP4). The aim here is to investigate the effects of AQP4 associated with SAE and reveal its underlying mechanism causing cognitive impairment. The in vivo experimental results reveal that AQP4 in peripheral blood of patients with SAE is up-regulated, also the cortical and hippocampal tissue of cecal ligation and perforation (CLP) mouse brain has significant rise in AQP4. Furthermore, the data suggest that AQP4 deletion could attenuate learning and memory impairment, attributing to activation of astrocytic autophagy, inactivation of astrocyte and downregulate the expression of proinflammatory cytokines induced by CLP or lipopolysaccharide (LPS). Furthermore, the activation effect of AQP4 knockout on CLP or LPS-induced PPAR-γ inhibiting in astrocyte is related to intracellular Ca2+ level and sodium channel activity. Learning and memory impairment in SAE mouse model are attenuated by AQP4 knockout through activating autophagy, inhibiting neuroinflammation leading to neuroprotection via down-regulation of Nav 1.6 channels in the astrocytes. This results in the reduction of Ca2+ accumulation in the cell cytosol furthermore activating the inhibition of PPAR-γ signal transduction pathway in astrocytes.

A regulatory network controlling ovarian granulosa cell death.

Follicular atresia triggered by granulosa cell (GC) apoptosis severely reduces female fertility and accelerates reproductive aging. GC apoptosis is a complex process regulated by multiple factors, regulatory axes, and signaling pathways. Here, we report a novel, small regulatory network involved in GC apoptosis and follicular atresia. miR-187, a miRNA down-regulated during follicular atresia in sows, maintains TGFBR2 mRNA stability in sow GCs by directly binding to its 5'-UTR. miR-187 activates the transforming growth factor-ß (TGF-ß) signaling pathway and suppresses GC apoptosis via TGFBR2 activation. NORHA, a pro-apoptotic lncRNA expressed in sow GCs, inhibits TGFBR2-mediated activation of the TGF-ß signaling pathway by sponging miR-187. In contrast, NORFA, a functional lncRNA associated with sow follicular atresia and GC apoptosis, enhances miR-187 and TGFBR2 expression by inhibiting NORHA and activating NFIX. Our findings define a simple regulatory network that controls GC apoptosis and follicular atresia, providing new insights into the mechanisms of GC apoptosis, follicular atresia, and female fertility.

The super-enhancer repertoire in porcine liver.

The transcriptional initiation of genes is inextricably bound with the functions of cis-regulatory sequences. The pig is one of the most important livestock species and an ideal animal model for biomedical studies. At the same time, the liver is a critical organ with diverse and complex metabolic functions. Here, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with high-throughput sequencing to profile the chromatin landscape of histone H3 lysine 27 acetylation (H3K27ac), histone H3 lysine 4 monomethylation (H3K4me1), and CCAAT enhancer-binding protein ß (C-EBPß) in the 70-d-old porcine liver, compared the different profiles among the three markers and their associated stitched-enhancers by stitching and sorting the peaks within 12.5 kb (Pott and Lieb, 2015) and generated the porcine liver-specific super-enhancers (SEs) by the combination of three markers. Compared to typical enhancers (TEs) and other stitched-enhancers, liver-specific SEs showed a higher density of cis-motifs and SNPs, which may recruit more tissue-specific vital TFs. The expression profiles in fetal and 70-d-old pigs proved that a large proportion of SE-associated genes were up-regulated and were more related to hepatic metabolisms and detoxification pathways. Our results illustrated the difference and connection among promoter and enhancer markers, identified the features of liver SEs and their associated genes, and provided novel insight into cis-element identification, function, and liver transcriptional regulation.

The cis-regulatory elements including promoters, enhancers, and newly identified super-enhancers (SEs), which were reported to function both promoter and enhancer capabilities, play critical roles in selective gene expression during development and disease. To reveal and compare the characteristics of these cis-elements in liver, we first performed a genome-wide profile of H3K27ac, H3K4me1, and C-EBPß, then constructed their associated stitched-enhancers respectively. Porcine liver-specific SEs were generated by overlapping the three stitched-enhancers. The genomic and genic location, TF binding sites and SNP distribution patterns were compared among these cis-elements. We found that stitched-enhancers gather in regions with higher gene densities and locate closer to the transcription starting sites. Additionally, SEs showed higher density of TF binding sites and SNPs. To access the transcriptional consequences of liver SEs, we first analyzed the genes locationally associated with SEs. The KEGG results suggested that these genes are significantly involved in metabolisms, detoxification, and autophagy pathways. We also detected the liver gene expression profiles using RNA-seq and noticed that SE-associated genes are more likely to be up-regulated. Our results provided novel information on the identification, function, and transcriptional regulation of cis-elements in the liver.

MEIS1 Is a Common Transcription Repressor of the miR-23a and NORHA Axis in Granulosa Cells.

MicroRNA-23a (miR-23a) is an endogenous small activating RNA (saRNA) involved in ovarian granulosa cell (GC) apoptosis and sow fertility by activating lncRNA NORHA transcription. Here, we reported that both miR-23a and NORHA were repressed by a common transcription factor MEIS1, which forms a small network regulating sow GC apoptosis. We characterized the pig miR-23a core promoter, and the putative binding sites of 26 common transcription factors were detected in the core promoters of both miR-23a and NORHA. Of them, transcription factor MEIS1 expression was the highest in the ovary, and widely distributed in various ovarian cells, including GCs. Functionally, MEIS1 is involved in follicular atresia by inhibiting GC apoptosis. Luciferase reporter and ChIP assays showed that transcription factor MEIS1 represses the transcription activity of miR-23a and NORHA through direct binding to their core promoters. Furthermore, MEIS1 represses miR-23a and NORHA expression in GCs. Additionally, MEIS1 inhibits the expression of FoxO1, a downstream of the miR-23a/NORHA axis, and GC apoptosis by repressing the miR-23a/NORHA axis. Overall, our findings point to MEIS1 as a common transcription repressor of miR-23a and NORHA, and develop the miR-23a/NORHA axis into a small regulatory network regulating GC apoptosis and female fertility.

Two single nucleotide variants in the miR-23a promoter affect granulosa cell apoptosis.

microRNAs (miRNAs) are well known to be important in mammalian female fertility. However, the genetic regulation of miRNAs associated with female fertility remains largely unknown. Here, we report that two single-nucleotide variants (SNVs) in the miR-23a promoter strongly influence miR-23a transcription and function in granulosa cell (GC) apoptosis. Two novel SNVs, g.-283G>C and g.-271C>T, were detected in the porcine miR-23a promoter by pooled-DNA sequencing. Furthermore, SNVs in the promoter region influenced miR-23a transcription in porcine GCs by altering its promoter activity. Functionally, SNVs in the promoter strongly influenced miR-23a regulation of early apoptosis in porcine GCs cultured in vitro. In addition, a preliminary association analysis showed that the combined genotypes of the two SNVs, rather than a single SNV, were tentatively associated with sow fertility traits in a Large White population. Overall, our findings suggest that the SNVs g.-283G>C and g.-271C>T in the miR-23a promoter are causal variants affecting GC apoptosis and miR-23a may be a potential small-molecule nonhormonal drug for regulating female fertility.

TGF-ß1 regulates the lncRNA transcriptome of ovarian granulosa cells in a transcription activity-dependent manner.

OBJECTIVES: Transforming growth factor ß1 (TGF-ß1), an essential cytokine belongs to TGF-ß superfamily, is crucial for female fertility. Increasing evidence show that long noncoding RNAs (lncRNAs) influence the state of granulosa cells (GCs). This study aimed to detect the effects of TGF-ß1 on the lncRNA transcriptome, and investigate whether lncRNAs mediate the functions of TGF-ß1 in GCs. MATERIAL AND METHODS: RNA-seq and bioinformatics analyses were performed to identify and characterize the differentially expressed lncRNAs (DElncRNAs). The regulatory mechanism of TGF-ß1 to lncRNA transcriptome was analyzed by chromatin immunoprecipitation. The effects of lncRNAs on the antiapoptotic and proproliferative functions of TGF-ß1 were examined by morphological analysis, fluorescence-activated cell sorting, Cell Counting Kit-8, and Western blot. RESULTS: A total of 72 DElncRNAs highly sensitive to TGF-ß1 were identified with the criteria of |log2 (fold chage)| ≥ 3 and false discovery rate < 0.05. Functional assessment showed that DElncRNAs were enriched in TGF-ß, nuclear factor kappa B, p53, and Hippo pathways which are crucial for the normal state and function of GCs. Importantly, SMAD4 is essential for the regulation of TGF-ß1 to lncRNA transcriptome. In vitro studies confirmed that TGF-ß1 induced TEX14-IT1 transcription in a SMAD4-dependent manner, and TEX14-IT1 mediated the antiapoptotic and proproliferative effects of TGF-ß1 in GCs. CONCLUSIONS: Our findings demonstrate that TGF-ß1 alters lncRNA transcriptome in a SMAD4-dependent manner, and highlight that lncRNAs mediate the functions of TGF-ß1 in GCs, which contribute to a better understanding of the epigenetic regulation of female fertility.

Lnc2300 is a cis-acting long noncoding RNA of CYP11A1 in ovarian granulosa cells.

The high level of progesterone and 17ß-estradiol ratio (P4/E2) in follicular fluid has been considered as a biomarker of follicular atresia. CYP11A1, the crucial gene encoding the rate-limiting enzyme for steroid hormone synthesis, has been reported differently expressed in the ovary during follicular atresia. However, the regulation mechanism of CYP11A1 expression during follicular atresia still remains unclear. Here, we have demonstrated that lnc2300, a novel pig ovary-specific highly expressed cis-acting long noncoding RNA (lncRNA) transcribed from chromosome 7, has the ability to induce the expression of CYP11A1 and inhibit the apoptosis of porcine granulosa cells (GCs). Mechanistically, lnc2300, mainly located in the cytoplasm of porcine GCs, sponges and suppresses the expression of miR-365-3p through acting as a competing endogenous RNA (ceRNA), which further relieves the inhibitory effects of miR-365-3p on the expression of CYP11A1. Besides, CYP11A1 is validated as a direct functional target of miR-365-3p in porcine GCs. Functionally, lnc2300 is an antiapoptotic lncRNA that reduces porcine GC apoptosis by inhibiting the proapoptotic function of miR-365-3p. In summary, our findings reveal a cis-acting regulation mechanism of CYP11A1 through lncRNA, and define a novel signaling pathway, lnc2300/miR-365-3p/CYP11A1 axis, which is involved in the regulation of GC apoptosis and follicular atresia.

Betaine Promotes Fat Accumulation and Reduces Injury in Landes Goose Hepatocytes by Regulating Multiple Lipid Metabolism Pathways.

Betaine is a well-established supplement used in livestock feeding. In our previous study, betaine was shown to result in the redistribution of body fat, a healthier steatosis phenotype, and an increased liver weight and triglyceride storage of the Landes goose liver, which is used for foie-gras production. However, these effects are not found in other species and strains, and the underlying mechanism is unclear. Here, we studied the underpinning molecular mechanisms by developing an in vitro fatty liver cell model using primary Landes goose hepatocytes and a high-glucose culture medium. Oil red-O staining, a mitochondrial membrane potential assay, and a qRT-PCR were used to quantify lipid droplet characteristics, mitochondrial ß-oxidation, and fatty acid metabolism-related gene expression, respectively. Our in vitro model successfully simulated steatosis caused by overfeeding. Betaine supplementation resulted in small, well-distributed lipid droplets, consistent with previous experiments in vivo. In addition, mitochondrial membrane potential was restored, and gene expression of fatty acid synthesis genes (e.g., sterol regulatory-element binding protein, diacylglycerol acyltransferase 1 and 2) was lower after betaine supplementation. By contrast, the expression of lipid hydrolysis transfer genes (mitochondrial transfer protein and lipoprotein lipase) was higher. Overall, the results provide a scientific basis and theoretical support for the use of betaine in animal production.

A Mutation in Endogenous saRNA miR-23a Influences Granulosa Cells Response to Oxidative Stress.

Phenotypes are the result of the interaction between the gene and the environment, so the response of individuals with different genotypes to an environment is variable. Here, we reported that a mutation in miR-23a influences granulosa cells (GCs) response to oxidative stress, a common mechanism of environmental factors affecting female reproduction. We showed that nuclear miR-23a is a pro-apoptotic miRNA in porcine GCs through the activation of the transcription and function of NORHA, a long non-coding RNA (lncRNA) induces GC apoptosis and responses to oxidative stress. Mechanistically, miR-23a acts as an endogenous small activating RNA (saRNA) to alter histone modifications of the NORHA promoter through the direct binding to its core promoter. A C > T mutation was identified at −398 nt of the miR-23a core promoter, which created a novel binding site for the transcription factor SMAD4 and recruited the transcription repressor SMAD4 to inhibit miR-23a transcription and function in GCs. Notably, g.−398C > T mutation in the miR-23a promoter reduced GCs response to oxidative stress. In addition, g.−398C > T mutation was significantly associated with sow fertility traits. In short, our findings preliminarily revealed the genetic basis of individual differences in the response to oxidative stress from the perspective of a single mutation and identified miR-23a as a candidate gene for the environmental adaptation to oxidative stress.

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells.

The ubiquitin-specific peptidase 9 X-linked (USP9X) is one of the highly conserved members belonging to the ubiquitin-specific proteases (USPs) family, which has been reported to control substrates-mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF-ß) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin-proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR-143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR-143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3'-untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF-ß signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro-regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR-143) and the TGF-ß signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility.

Reducing Nav1.6 expression attenuates the pathogenesis of Alzheimer's disease by suppressing BACE1 transcription.

Aberrant increases in neuronal network excitability may contribute to cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability of neurons are not fully understood. Voltage-gated sodium channels (VGSC or Nav), which are involved in the formation of excitable cell's action potential and can directly influence the excitability of neural networks, have been implicated in AD-related abnormal neuronal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we have shown that the reduction of VGSC α-subunit Nav1.6 (by injecting adeno-associated virus (AAV) with short hairpin RNA (shRNA) into the hippocampus) rescues cognitive impairments and attenuates synaptic deficits in APP/PS1 transgenic mice. Concurrently, amyloid plaques in the hippocampus and levels of soluble Aß are significantly reduced. Interfering with Nav1.6 reduces the transcription level of ß-site APP-cleaving enzyme 1 (BACE1), which is Aß-dependent. In the presence of Aß oligomers, knockdown of Nav1.6 reduces intracellular calcium overload by suppressing reverse sodium-calcium exchange channel, consequently increasing inactive NFAT1 (the nuclear factor of activated T cells) levels and thus reducing BACE1 transcription. This mechanism leads to a reduction in the levels of Aß in APP/PS1 transgenic mice, alleviates synaptic loss, improves learning and memory disorders in APP/PS1 mice after downregulating Nav1.6 in the hippocampus. Our study offers a new potential therapeutic strategy to counteract hippocampal hyperexcitability and subsequently rescue cognitive deficits in AD by selective blockade of Nav1.6 overexpression and/or hyperactivity.

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging.

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

SMAD4 Inhibits Granulosa Cell Apoptosis via the miR-183-96-182 Cluster and FoxO1 Axis.

The miR-183-96-182 cluster is a polycistronic miRNA cluster necessary for ovarian functions in mammals. However, its transcriptional regulation in the ovary is largely unclear. In this study, we characterized the promoter region of the porcine miR-183-96-182 cluster, and showed that SMAD4 may function as a transcriptional activator of the miR-183-96-182 cluster in GCs through direct binding to SBE motifs in its promoter. SMAD4 may inhibit GC apoptosis via suppression of FoxO1, an effector of GC apoptosis and a direct target of the miR-183-96-182 cluster, by inducing the miR-183-96-182 cluster, and this process may be regulated by the TGF-ß/SMAD signaling pathway. Our findings uncovered the regulatory mechanism of miR-183-96-182 cluster expression in GCs and demonstrated that TGF-ß1/SMAD4/miR-183-96-182 cluster/FoxO1 may be a potential pathway for regulating follicular atresia and female reproduction.

Corrigendum: Scorpion Venom Heat-Resistant Peptide Attenuates Microglia Activation and Neuroinflammation.

[This corrects the article DOI: 10.3389/fphar.2021.704715.].