FGF12 regulates cell cycle gene expression and promotes follicular granulosa cell proliferation through ERK phosphorylation in geese.

The granulosa cells play an important role in the fate of follicular development or atresia in poultry. Fibroblast growth factor 12 (FGF12) is downregulated in atretic follicles and may be involved in regulating granulosa cell survival in previous studies, but its molecular mechanism remains unclear. In this study, FGF12 overexpression and knockdown models of goose granulosa cells were constructed to investigate its function. The downstream expression of the cell cycle pathway was analyzed by qPCR. Granulosa cell proliferative activity and apoptosis were detected by CCK8 and TUNEL. Protein phosphorylation levels of ERK and AKT were measured using Western blotting to analyze the key pathway of FGF12 regulation of granulosa cell proliferation. ERK protein phosphorylation inhibitor was added for further verification. After overexpression of FGF12, cell proliferation activity was increased, the expressions of cell cycle pathway genes CCND1, CCNA2, MAD2, and CHK1 were upregulated, the apoptosis of granulosa cell was decreased, and Caspase 3 gene and protein expression were downregulated. After the knockdown of FGF12, cell proliferation activity decreased, the expression of downstream genes in the cell cycle pathway was downregulated, the apoptosis of granulosa cells was increased, and the Bcl-2 gene and protein were downregulated. Overexpression of FGF12 promoted the synthesis of P4 and upregulates the expression of the STAR gene. Overexpression of FGF12 promoted ERK protein phosphorylation but did not affect AKT phosphorylation. The addition of ERK phosphorylation inhibitors resulted in the elimination of the increase in cell proliferative activity caused by FGF12 overexpression. In conclusion, FGF12 could promote proliferation and inhibit apoptosis of goose granulosa cells by increasing ERK phosphorylation.

Tumor suppressive functions of hsa­miR­34a on cell cycle, migration and protective autophagy in bladder cancer.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa­microRNA­34a (miR­34a) presents anti­tumor function in several types of cancer. However, the functional mechanism of miR­34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR­34a mimic exhibited LC3­II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome­lysosome fusion, was downregulated upon miR­34a mimic treatment. Mechanistically, miR­34a reduced the expression of STX17 proteins that directly bind on STX17 3'­untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR­34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR­34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR­34a suppressed cell motility through the downregulation of epithelial­mesenchymal transition. In summary, miR­34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Egg Yolk Fat Deposition Is Regulated by Diacylglycerol and Ceramide Enriched by Adipocytokine Signaling Pathway in Laying Hens.

The mechanism which regulates differential fat deposition in egg yolk from the indigenous breeds and commercial laying hens is still unclear. In this research, Chinese indigenous Huainan Partridge chickens and Nongda III commercial laying hens were used for egg collection and liver sampling. The weight of eggs and yolk were recorded. Yolk fatty acids were determined by gas chromatography-mass spectrometry. Lipid metabolites in the liver were detected by liquid chromatography-mass spectrometry. Yolk weight, yolk ratio and yolk fat ratio exhibited higher in the Huainan Partridge chicken than that of the Nongda III. Compared to the Nongda III, the content of total saturated fatty acid was lower, while the unsaturated fatty acid was higher in the yolk of the Huainan Partridge chicken. Metabolites of phosphatidylinositol and phosphatidylserine from glycerolphospholipids, and metabolites of diacylglycerol from glycerolipids showed higher enrichment in the Huainan Partridge chicken than that of the Nongda III, which promoted the activation of the adipocytokine signaling pathway. However, metabolites of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine from glycerol phospholipids, and metabolites of triacylglycerol from glycerolipids showed lower enrichment in the Huainan Partridge chicken than that of the Nongda III. The high level of yolk fat deposition in the Huainan Partridge chicken is regulated by the activation of the adipocytokine signaling pathway which can promote the accumulation of diacylglycerol and ceramide in the liver.

BMP4/SMAD8 signaling pathway regulated granular cell proliferation to promote follicle development in Wanxi white goose.

Granular cells proliferation in goose regulated by bone morphogenetic proteins (BMPs) signaling pathway is still unknown. In this experiment, BMPs and their receptor, and receptor activated mothers against decapentaplegic homologs (SMADs) were quantitatively expressed in granular cell layer of pre-hierarchycal and hierarchycal follicles in Wanxi White goose. The screened BMP was then used for construction of overexpressed and knockdown vectors and transfected into granular cells of goose to assess the cell proliferation and apoptosis. Granular cells with BMP-overexpressed were then used for ChIP-Seq analysis to elucidate the molecular mechanism of BMP affecting granular cell proliferation. The results showed that the mRNA expression of BMP4 was significantly expressed in pre-hierarchical follicles, and also highly expressed in hierarchical follicles than other BMPs, while the Ⅰ and Ⅱ type of BMP receptors were expressed in basic level. The mRNA expression of SMAD8 was significantly elevated in pre-hierarchical follicles. Overexpression of BMP4 could promote the proliferation of granular cells and inhibited the expression of BMP4 caused a higher cell apoptosis. ChIP-Seq identified multiple regulatory targets of SMAD4, which were mostly related to cell cycle and lipid metabolism according to the GO and KEGG pathway enrichment. From the five most significant binding motif and quantitative expression verification, the activin membrane binding inhibitor (BAMBI) was down regulated in BMP4 overexpressed granular cells. In conclusion, the BMP4 was highly expressed in granular cells and phosphorylates SMAD8, the activated SMAD8 combined with SMAD4 transfers into nucleus to regulate the expression of BAMBI to promote lipid synthesis.