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

【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JAK2、STAT3和TYK2基因外,过表达载体pcDNA3.1-SOCS1转染组中的SOCS2、SOCS3、JAK1、STAT2、STAT5A、IL-6、PRLR、MCL1和PIAS3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。

【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 de-velopment,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 prolifera-tion 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 signifi-cantly 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.【Con-clusion】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 impor-tant role in the regulation mechanism of mammary gland development in pigs.