Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yiel...Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows,indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis.Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B(Cyclin B), cell cycle protein D(Cyclin D), cell cycle protein E(Cyclin E), and cyclin-dependent kinase 4(CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and steroidogenic acute regulatory protein(StAR)(i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.Conclusions: Our findings suggest that miR-214-3 p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.展开更多
Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study...Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.展开更多
基金supported by grants from the National Natural Science Foundation (No.31802047)the National Science and Technology Major Project of China (No. 2016ZX08006003)Shaanxi Provincial Key Research and Development Project (CN)(No. 2018ZDXM-NY-035)。
文摘Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows,indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis.Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B(Cyclin B), cell cycle protein D(Cyclin D), cell cycle protein E(Cyclin E), and cyclin-dependent kinase 4(CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and steroidogenic acute regulatory protein(StAR)(i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.Conclusions: Our findings suggest that miR-214-3 p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.
基金supported by grants from the Natural Science Fund (ZR2010HM035) of Shandong Provincethe Shandong Provincial Health Development Project Fund (2011WSB19002) in China
文摘Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
