SOCS1基因过表达对猪乳腺上皮细胞增殖和凋亡的影响

Effects of SOCS1 gene overexpression on proliferation and apoptosis of porcine mammary epithelial cells

  • 摘要: 【目的】明确细胞因子信号转导抑制因子1基因(SOCS1)过表达对乳腺发育关键信号通路(JAK/STAT)相关基因表达的影响,为揭示SOCS1基因调节猪乳腺上皮细胞增殖和凋亡的作用机制提供参考依据。【方法】构建SOCS1基因过表达载体pcDNA3.1-SOCS1和阴性对照载体pcDNA3.1-NC,分别转染猪乳腺上皮细胞,然后通过CCK-8检测细胞增殖情况,流式细胞术检测细胞周期及细胞凋亡情况,实时荧光定量PCR检测转染后JAK/STAT信号通路相关基因的表达变化。【结果】以过表达载体pcDNA3.1-SOCS1和阴性对照载体pcDNA3.1-NC分别转染猪乳腺上皮细胞,过表达载体pcDNA3.1-SOCS1转染组的SOCS1基因相对表达量极显著高于阴性对照载体pcDNA3.1-NC转染组(P<0.01,下同);转染48和72 h后,过表达载体pcDNA3.1-SOCS1转染组猪乳腺上皮细胞的OD值极显著低于阴性对照载体pcDNA3.1-NC转染组,说明过表达载体pcDNA3.1-SOCS1能有效抑制猪乳腺上皮细胞的增殖能力;过表达载体pcDNA3.1-SOCS1转染组的细胞凋亡率(36.15±0.92)%显著高于阴性对照载体pcDNA3.1-NC转染组(30.55±0.35)%(P<0.05,下同),且过表达载体pcDNA3.1-SOCS1转染组中的G1期细胞比例极显著降低,而S期和G2期细胞比例显著升高。与阴性对照载体pcDNA3.1-NC转染组相比,除JAK2STAT3TYK2基因外,过表达载体pcDNA3.1-SOCS1转染组中的SOCS2SOCS3JAK1STAT2STAT5AIL-6PRLR、MCL1PIAS3等9个JAK/STAT信号通路相关基因的相对表达量均极显著升高。【结论】SOCS1基因过表达可抑制猪乳腺上皮细胞增殖及促进细胞凋亡,并影响JAK/STAT信号通路相关基因的表达变化,从而在猪的乳腺发生发育调控机制中发挥重要作用。

     

    Abstract: 【Objective】The purpose of the study was to clarify the effect of overexpressed cytokine signal transducer suppressor 1(SOCS1) gene on the expression of key signaling pathway(JAK/STAT) related genes in mammary gland development, and to provide reference for revealing the mechanism of SOCS1 gene regulating proliferation and apoptosis of porcine mammary epithelial cells. 【Method】The overexpressed vector pcDNA3.1-SOCS1 and the negative control vector pcDNA3.1-NC were constructed and transfected into porcine mammary epithelial cells, respectively. Then, cell proliferation was detected by CCK-8, cell cycle and apoptosis were detected by flow cytometry, and the expression changes of JAK/STAT signaling pathway related genes after transfection were detected by real-time fluorescence quantitative PCR.【Result】Porcine mammary epithelial cells were transfected with overexpressed vector pcDNA3.1-SOCS1 and negative control vector pcDNA3.1-NC, respectively. The relative expression of SOCS1 gene in the overexpressed vector pcDNA3.1-SOCS1 transfection group was extremely significantly higher than that in the negative control vector pcDNA3.1-NC transfection group(P<0.01, the same below). After transfection for 48 and 72 h, the OD value of porcine mammary epithelial cells in the overexpressed vector pcDNA3.1-SOCS1 transfection group was extremely significantly lower than that in the negative control vector pcDNA3.1-NC transfection group, indicating that the overexpressed vector pcDNA3.1-SOCS1 could effectively inhibit the proliferation ability of porcine mammary epithelial cells. The apoptosis rate of cells in the overexpressed vector pcDNA3.1-SOCS1 transfection group (36.15±0.92)% was significantly higher than that of the negative control vector pcDNA3.1-NC transfection group (30.55±0.35)%(P<0.05, the same below), and the rate of G1 phase cells in the overexpressed vector pcDNA3.1-SOCS1 transfection group was extremely significantly decreased, while the rates of S and G2 phase cells were significantly increased. Compared with the negative control vector pcDNA3.1-NC transfection group, except for JAK2, STAT3 and TYK2 genes, the relative expressions of 9 JAK/STAT signaling pathway related genes, including SOCS2, SOCS3, JAK1, STAT2, STAT5A, IL-6, PRLR, MCL1 and PIAS3, in the overexpressed vector pcDNA3.1-SOCS1 transfection group were extremely significantly increased. 【Conclusion】Overexpression of SOCS1 gene can inhibit the proliferation of porcine mammary epithelial cells and promote the apoptosis of cells and affect the expression changes of JAK/STAT signaling pathway related genes, thus playing an important role in the regulation mechanism of mammary gland development in pigs.

     

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