Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA‐222 (miR‐222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR‐222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR‐222 functions as an anti‐apoptotic factor in pGCs. MiR‐222 mimics in pGCs result in the upregulation of the anti‐apoptotic BCL‐2 gene, down‐regulation of the proapoptotic caspase‐3 gene, and inhibition of apoptosis. MiR‐222 inhibitors reduced BCL‐2 and had no significant effect on caspase‐3. MiR‐222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1‐siRNA reduced the proapoptotic BAX gene. MiR‐222 can directly target the 3′‐untranslated region of the THBS1 gene. MiR‐222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR‐222 inhibitor. Transfection of THBS1‐siRNA resulted in the inhibition of the miR‐222 inhibitor, which suggests that miR‐222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR‐222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.
Normal estrous cycle is crucial for porcine reproduction, and microRNA is closely related to regulation of estrous cycle in porcine ovaries. In this study, we found that the expression of miR-214 in porcine ovaries was higher than in many other tissues, and miR-21 expression in ovaries was significantly higher than in the uterus and pituitary. Meanwhile, miR-21 was upregulated and miR-214 was downregulated in the ovaries of high litter size (YH) pigs compared with low litter size (YL) pigs. Moreover, the lowest expression of miR-21 and miR-214 occurred on Days 14 and 7 of the estrous cycle and was expressed at greater levels in the granulosa cells of subordinate follicles than in dominant follicles on Day 3 of the estrous cycle. Bioinformatics analysis showed that miR-21 and miR-214 might target several genes that involved in the mTOR signaling, apoptosis, and steroid biosynthesis pathways, and they play important roles in maintaining the porcine estrous cycle. The qPCR and western blot analysis indicated that miR-214 inhibited the expression of SCARB1 gene in the transcriptional level, but not affected the SCARB1 gene's protein level. Our research findings indicated that miR-21 and miR-214 played important roles in reproduction regulation during porcine estrous.
Splay leg is frequently observed in newborn piglets and leads to economic loss as well as welfare concerns. However, the etiology and pathogenesis of splay leg syndrome in piglets are still poorly understood. The aims of this paper were to characterize changes in the transcriptome of splay leg piglets and identify candidate genes responsible for this disease. We chose three splay leg piglets and their healthy full sibs, and constructed six RNA libraries using skeletal muscle samples from both groups and identified the differentially expressed genes between the two groups using RNA-seq. A total of 555 differentially expressed genes were identified, of which 216 were up-regulated and 339 genes were down-regulated in the splay leg group relative to the healthy group. In addition, 321 significantly enriched GO terms and 12 significantly enriched KEGG pathways were identified. FBXO32 is one of the ten most differentially expressed genes in our experiment, and it is regulated by the significantly enriched pathway (PI3K-Akt). The overexpression of FBXO32 which leads to the process of muscle atrophy might be responsible for congenital splay leg in piglets. The result of this study could help improve understanding of the molecular mechanism of congenital splay leg syndrome.
Objective: Apoptosis of ovarian granulosa cells (GCs) affects mammalian follicular development and fecundity. This study aimed to explore the regulatory relationship between microRNA-26a (miR-26a) and the 3β-hydroxysteroid-Δ24-reductase gene (<i>DHCR24</i>) gene in porcine follicular granular cells (pGCs), and to provide empirical data for the development of methods to improve the reproductive capacity of pigs.Methods: The pGCs were transfected with miR-26a mimic, miR-26a inhibitor and <i>DHCR24</i>-siRNA <i>in vitro</i>. The cell apoptosis rate of pGCs was detected by the flow cytometry. The secretion levels of estradiol (E2) and progesterone (P) in pGCs were detected by enzymelinked immunosorbent assay. Double luciferase validation system was used to detect the binding sites between miR-26a and <i>DHCR24</i> 3′-UTR region. Qualitative real-time polymerase chain reaction and Western blotting were used to verify the <i>DHCR24</i> mRNA and protein expression in pGCs, respectively, after transfecting with miR-26a mimic and miR-26a inhibitor.Results: Results showed that enhancement of miR-26a promoted apoptosis, and inhibited E2 and P secretion in pGCs. Meanwhile, inhibition of <i>DHCR24</i> also upregulated the Caspase-3 expression, reduced the BCL-2 expression, promoted pGCs apoptosis, and inhibited E2 and P secretion in pGCs. There were the binding sites of miR-26a located within <i>DHCR24</i> 3′-UTR. Up-regulation of miR-26a inhibited <i>DHCR24</i> mRNA and protein expression in pGCs.Conclusion: This study demonstrates that miR-26a can promote cell apoptosis and inhibit E2 and P secretion by inhibiting the expression of <i>DHCR24</i> in pGCs.
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