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This study aimed to investigate the regulatory role of 6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 3 （PFKFB3） in the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes， screen its interacting proteins and provide clues for understanding their regulatory mechanisms. PFKFB3 was overexpressed or interfered in porcine intramuscular preadipocytes. qRT-PCR was used to detect changes in the expression of proliferation marker genes. CCK-8 and EdU assays were employed to measure changes in the number of viable cells and cells in the proliferation phase， respectively. After inducing adipogenic differentiation of porcine intramuscular preadipocytes， qRT-PCR and Oil Red O staining were used to assess its regulatory effect on adipogenic differentiation. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry was utilized to identify and screen proteins interacting with PFKFB3. The results showed that overexpression of PFKFB3 significantly increased the number of EdU-positive cells （P<0.01） and cell viability （P<0.01）， and significantly upregulated the expression of proliferation-related genes CDK1， CCND1， PCNA， and KI67 （P<0.05）. Conversely， interfering with PFKFB3 significantly inhibited cell proliferation （P<0.05）. Regarding adipogenic differentiation， overexpression of PFKFB3 significantly increased the number of lipid droplets （P<0.01） and triglyceride content （P<0.01）， and significantly upregulated the expression of key adipogenic genes PPARγ， CEBPα， CEBPβ， and LPL （P<0.05）. Interfering with PFKFB3 significantly inhibited adipogenic differentiation （P<0.05）. Furthermore， Flag-tag immunoprecipitation combined with mass spectrometry analysis identified 17 specific interacting proteins of PFKFB3. GO analysis indicated that these proteins were primarily involved in processes such as ATP binding and RNA binding. STRING protein-protein interaction network prediction revealed interactions between PFKFB3 and proteins including CHORDC1， ALAS1， ASCC3， and KCNK2. In summary， PFKFB3 positively regulates the proliferation and adipogenic differentiation of porcine intramuscular preadipocytes. Potential interacting proteins of PFKFB3 were preliminarily screened， providing new clues for in-depth analysis of the molecular mechanisms underlying porcine intramuscular fat deposition and improvement of pork quality.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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