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CeRNA effect was an important regulation mode of miRNA mediated bio-activities, however, most of the researches of ceRNA were on ncRNAs synergetic with mRNAs, the exploration of ceRNA effect regulated mRNA interaction was still lack of. Besides, C/EBPα was one of the most crucial adipogenic regulators, which has been demonstrated to form a protein complex with FOXO1 to mediate AdipoQ expression. So that, we try to explore whether the ceRNA effect mediated the interaction of C/EBPα and FOXO1, and identified the key miRNAs of their ceRNA effect. In this paper, we found the ceRNA effect of C/EBPα and FOXO1 mediated their protein complex formation, furthermore regulated its transcriptional role for AdipoQ, thereby influencing pre-adipocytes adipogenesis. More importantly, we demonstrated that the miR-144 was the decisive factor that mediated the ceRNA effect of C/EBPα and FOXO1 to influence AdipoQ, thus regulated pre-adipocytes adipogenesis. This research will provide a new supplementary idea of the miRNA role in mediating coding RNA interaction that regulates pre-adipocyte adipogenesis., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

As a centre enzyme in fatty acid activation, acyl-CoA synthetase long-chain family member 1 (ACSL1) plays an important role in body lipid homeostasis. However, the functions of ACSL1 in the subcutaneous adipogenesis of pigs are largely unknown. In the present study, we found that the expression of ACSL1 significantly increased during the process of porcine preadipocyte differentiation. Moreover, silencing of ACSL1 in preadipocytes decreased levels of triglyceride and adipogenic-related markers, including FABP4 , APOE , and FASN ( p < 0.01), and simultaneously increased levels of lipolytic-related markers, such as ATGL and HSL ( p < 0.05). Conversely, overexpression of ACSL1 in preadipocytes increased levels of triglyceride and FABP4 , APOE , and FASN ( p < 0.01), and reduced levels of ATGL and HSL ( p < 0.05). Luciferase reporter assays revealed that ACSL1 is a target of miR-218-5p, which can reduce the mRNA and protein levels of ACSL1 by directly binding the 3' untranslated region of ACSL1 . Furthermore, miR-218-5p has an inhibition role in porcine preadipocyte differentiation by suppressing ACSL1 expression. Taken together, these data provide insights into the mechanism of the miR-218-5p/ ACSL1 axis in regulating subcutaneous fat deposition of pigs.

Sestrin-3, together with the other two members Sestrin-1 and Sestrin-2, belongs to the Sestrin family. The Sestrin protein family has been demonstrated to be involved in antioxidative, metabolic homeostasis, and even the development of nonalcoholic steatohepatitis (NASH). However, the adipogenic regulatory role of SESN3 in adipogenesis still needs to be further explored. In this study, we demonstrated SESN3 inhibited porcine pre-adipocyte proliferation, thus suppressing its adipogenesis. Meanwhile, SESN3 has been demonstrated to inhibit Smad3 thus protecting against NASH. Further, for our previous study, we found mmu-miR-124 involved in 3T3-L1 cell adipogenesis regulation. In this study, we also identified that ssc-miR-124 inhibited porcine pre-adipocyte proliferation, thus suppressing its adipogenesis, and the SMAD3 was an inhibitor of ssc-miR-124 by binding to its promoter. Furthermore, the ssc-miR-124 targeted porcine C/EBPα and GR and thus inhibited pre-adipocyte adipogenesis. In conclusion, SESN3 inhibited SMAD3, thus improving ssc-miR124 , and then suppressed C/EBPα and GR to regulate pre-adipocytes adipogenesis.

Intramuscular fat (IMF) and subcutaneous fat (SCF) are important traits affecting the economics of the pork industry, in which less SCF and more IMF content is desirable. However, the mechanisms that regulate IMF and SCF content are not clear yet. In this study, we demonstrate that KLF3 (Krüppel-like factor 3) was negatively correlated with IMF content in the longissimus dorsi muscle of Erhualian pigs. In addition, the expression level of KLF3 was significantly higher in IMF than SCF. Overexpression and knockdown experiments revealed that KLF3 could suppress adipocyte differentiation in vitro by downregulating adipogenic markers, including PPARG, C/EBPA, and FABP4. Luciferase activity analysis proved that miR-32-5p was able to suppress KLF3. Notably, miR-32-5p level was negatively correlated to KLF3 mRNA level in both IMF and SCF tissues. The same relationship was proved in samples with different IMF content. Further studies showed that miR-32-5p could promote adipocyte differentiation via inhibiting KLF3. Our results suggest that the miR-32-5p-KLF3 pathway is involved in the regulation of differential fat deposition of IMF and SCF tissues., (© 2020 AOCS.)

SMARCA2, an evolutionarily conserved catalytic ATPase subunit of SWI/SNF complexes, has been implicated in development and diseases; however, its role in mammalian ovarian function and female fertility is unknown. Here, we identified and characterized the 3'-UTR of the porcine SMARCA2 gene and identified a novel adenylate number variation. Notably, this mutation was significantly associated with sow litter size traits and SMARCA2 levels, due to its influence on the stability of SMARCA2 mRNA in ovarian granulosa cells (GCs). Immunohistochemistry and functional analysis showed that SMARCA2 is involved in the regulation of follicular atresia by inhibiting GC apoptosis. In addition, miR-29c, a pro-apoptotic factor, was identified as a functional miRNA that targets SMARCA2 in GCs and mediates regulation of SMARCA2 expression via the NORFA -SMAD4 axis. Although a potential miR-29c-responsive element was identified within NORFA , negative regulation of miR-29c expression by NORFA was not due to activity as a competing endogenous RNA. In conclusion, our findings demonstrate that SMARCA2 is a candidate gene for sow litter size traits, because it regulates follicular atresia and GC apoptosis. Additionally, we have defined a novel candidate pathway for sow fertility, the NORFA -TGFBR2-SMAD4-miR-29c-SMARCA2 pathway.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The mechanism of adipocyte regulation specifically in muscle and the influence of muscle tissue on intramuscular fat deposition are unknown. Our previous studies have shown that myostatin, a myokine, is involved in inhibiting the differentiation of preadipocytes and may be a potential regulator that affects the deposition of intramuscular fat. Myostatin inhibited adipogenesis by downregulating the expression of glucocorticoid receptor (GR) in porcine preadipocytes. However, the mechanism of regulation is not yet clear. In this study, we demonstrate microRNA (miR-124-3p) mediates regulation of GR by myostatin. We found that miR-124-3p can target GR 3'-UTR and negatively regulate GR expression. We demonstrate that overexpression of miR-124-3p can reduce differentiation of 3T3-L1 cells by inhibiting GR, and vice versa. The expression of miR-124-3p was upregulated in 3T3-L1 cells treated with myostatin. Further study revealed that myostatin also promotes the expression of SMAD4 and its transfer and localization to the nucleus. The activated myostatin/SMAD4 signal promotes the expression of miR-124-3p by SMAD4 binding to the promoter region of miR-124-3p. When myostatin or SMAD4 activity is inhibited, the upregulation of miR-124-3p is also inhibited. All of these findings suggested that myostatin could inhibit adipogenic differentiation of 3T3-L1 cells by activating miR-124-3p to inhibit GR. These data may provide an explanation for how myostatin signaling affects intramuscular fat deposition in a tissue-specific manner.

The cytochrome P450 family 19 subfamily A member 1 (CYP19A1) gene, encodes aromatase, a key enzyme in estradiol (E2) synthesis, and is down-regulated during porcine follicular atresia. However, its role in and the mechanism of transcriptional repression in follicular atresia is largely unknown. In the present study, we show that the CYP19A1 gene stimulates E2 release and inhibits cell apoptosis in porcine granulosa cells (GCs). SMAD4, an anti-apoptotic moderator, was identified as a transcription factor of the porcine CYP19A1 gene and enhanced the expression and function of CYP19A1 in porcine GCs through direct binding to a SMAD4-binding element (SBE) within the promoter region of CYP19A1 gene. Moreover, we found that miR-10b, a pro-apoptotic factor, directly interacted with 3'-UTR of the porcine CYP19A1 mRNA, inhibiting its expression and function in porcine GCs. Collectively, we demonstrated that CYP19A1 is an inhibitor of follicular atresia and is regulated by both SMAD4 and miR-10b. These findings provide further insight into the mechanisms of CYP19A1 in steroid hormone synthesis and GC apoptosis and provide molecular targets for exploring methods of treatment for steroid-dependent reproductive disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To investigate the molecular mechanism underlying differential lipid deposition between intramuscular (IM) and subcutaneous (SC) fat tissues of pigs, peroxisome proliferator-activated receptor gamma (PPARG) expression levels were compared in these two types of adipose tissues. The results showed that both mRNA and protein levels of PPARG were significantly higher in SC fat than in IM fat. Correspondingly, the expression levels of miR-128 and miR-130a, which potentially targeting PPARG 3'-untranslated region (3'-UTR), in IM fat were 8.37 and 2.30-fold of those in SC fat respectively. Further dual luciferase activity assay showed that only miR-130a directly targeting PPARG. Moreover, over-expression of miR-130a in preadipocytes significantly inhibited its differentiation by suppressing PPARG expression. In conclusion, tissue variance of miR-130a levels results in the diverse of PPARG, and might be the reason for differential fat deposition between IM and SC fat tissues in pigs. Our study would provide the molecular foundation for IM fat deposition increase and therefore contribute to pork quality improvement., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Follicle-stimulating hormone receptor (FSHR) and its intracellular signaling control mammalian follicular development and female infertility. Our previous study showed that FSHR is downregulated during follicular atresia of porcine ovaries. However, its role and regulation in follicular atresia remain unclear. Here, we showed that FSHR knockdown induced porcine granulosa cell (pGC) apoptosis and follicular atresia, and attenuated the levels of intracellular signaling molecules such as PKA, AKT and p-AKT. FSHR was identified as a target of miR-143, a microRNA that was upregulated during porcine follicular atresia. miR-143 enhanced pGC apoptosis by targeting FSHR, and reduced the levels of intracellular signaling molecules. SMAD4, the final molecule in transforming growth factor (TGF)-β signaling, bound to the promoter and induced significant downregulation of miR-143 in vitro and in vivo. Activated TGF-β signaling rescued miR-143-reduced FSHR and intracellular signaling molecules, and miR-143-induced pGC apoptosis. Overall, our findings offer evidence to explain how TGF-β signaling influences and FSHR signaling for regulation of pGC apoptosis and follicular atresia by a specific microRNA, miR-143.

MicroRNAs (miRNAs) are important modulators of skeletal muscle development in multiple mammalian species, but their role in skeletal muscle growth in castrated male pigs has not been well studied. The aim of the present study was to determine the role of miRNAs in longissimus dorsi muscle under castration. Our results showed that castration caused a significant decrease in serum testosterone levels as well as carcass lean mass, but led to an increase in carcass fat mass. Moreover, miRNA expression profiles in skeletal muscle were significantly altered by castration, and seven differentially expressed miRNAs were discovered. More importantly, functional analysis suggested that these differentially expressed miRNAs and their targets are involved in the regulation of skeletal muscle contractile function and fat metabolism. Taken together, these results demonstrate altered miRNA expression in skeletal muscle of castrated male pigs, and suggest a potential mechanism underlying the effects of castration on porcine skeletal muscle growth., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are class of molecular regulators found to participate in numerous biological processes, such as adipogenesis and obesity in mammals. To determine the roles of miRNAs involved in castration-induced body fatness, we investigated the different miRNA expression patterns in subcutaneous adipose tissue between intact and castrated male pigs. Our results showed that castration led to decrease serum testosterone but increase serum Leptin levels (P < 0.01). Moreover, castration also increased adipocyte size, body fat content and backfat thickness in male pigs (P < 0.01). Meanwhile, miRNA expression profiles in adipose tissue were changed by castration, and 18 miRNAs were considered as the differentially expressed candidates between intact and castrated male pigs. Furthermore, functional analysis indicated that the differential expressed miRNAs and their target genes are involved in the regulation of fatty acid metabolism. In brief, our present study provides a comprehensive view on how miRNAs works in subcutaneous adipose tissue with castration. These results suggested that miRNAs might play an important role in the castration-induced fat deposition in male pigs.

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play important roles in gene expressions at transcriptional and post-transcriptional stages in mammalian brain. So far, a growing number of porcine miRNAs and their function have been identified, but little is known regarding the porcine developing hypothalamus and pituitary. In the present study, Solexa sequencing analysis showed 14,129,397 yielded reads, 6,680,678 of which were related to 674 unique miRNAs. After a microarray assay, we detected 175 unique miRNAs in the hypothalamus, including 136 previously known miRNAs and 39 novel candidates, while a total of 140 miRNAs, including 104 known and 36 new candidate miRNAs, were discovered in pituitary. More importantly, 37 and 30 differentially expressed miRNAs from several developmental stages of hypothalamus and pituitary were revealed, respectively. The 37 differentially expressed miRNAs in hypothalamus represented 6 different expression patterns, while the 30 differentially expressed miRNAs in pituitary represented 7 different expression patterns. To clarify potential target genes and specific functions of these differentially expressed miRNAs in hypothalamus and pituitary, TargetScan and Gorilla prediction tools were then applied. The current functional analysis showed that the differentially expressed miRNAs in hypothalamus and pituitary shared many biological processes, with the main differences being found in tissue-specific processes including: CDP-diacylglycerol biosynthetic/metabolic process; phosphatidic acid biosynthetic/metabolic process; energy reserve metabolic process for hypothalamus; adult behavior; sterol transport/homeostasis; and cholesterol/reverse cholesterol transport for pituitary. Overall, this study identified miRNA profiles and differentially expressed miRNAs among various developmental stages in hypothalamus and pituitary and indicated miRNA profiles change with age and brain location, enhancing our knowledge about spatial and temporal expressions of miRNAs in the porcine developing brain.

Lung cancer is one of the main causes of cancer death worldwide. The cortactin gene, CTTN, may play a pivotal role in the proliferation and invasion of tumors. A microRNA (miR-182) was cloned and used to study the expression of CTTN and its regulatory effects on the proliferation and invasion of the lung cancer cell line, A549. Cortactin protein and CTTN mRNA expression decreased in A549 cells that were transfected with the miR-182 expression plasmid. A cell proliferation assay indicated that miR-182 expression affected cell cycle regulation and suppressed proliferation of lung cancer cells in vitro. In addition, xenograft experiments confirmed the suppression of tumor growth in vivo, which was due to the promotion of apoptosis. In conclusion, endogenous mature miR-182 expression may have an important role in the pathogenesis of lung cancer through its interference with the target gene CTTN by epigenetic modification.