香猪睾丸间质细胞 TAS 1R3基因干扰后对自噬相关因子的影响

Effect of TAS 1R3 Gene Interference on Autophagy Related Factors in Leydig Cells of Xiang Pig

旨在探究 TAS1 R3基因通过mTOR对从江香猪自噬相关基因表达的影响。本试验选择30日龄健康雄性从江香猪3头,采集睾丸组织并培养出睾丸间质细胞。将构建成功的干扰载体和空载体转染至细胞中,利用qRT-PCR检测其对 TAS 1R3基因表达影响,运用qRT-PCR和Western blot检测 TAS1 R3基因干扰后对味觉受体第一家族一号受体(TAS1R1)、细胞外信号调节激酶1(ERK1 ) 、细胞外信号调节激酶2(ERK2)、雷帕霉素靶蛋白(mTOR)、 BECN1 和微管相关蛋白1轻链3β(MAP1LC3B)表达的影响。结果表明,当 TAS 1R3基因被干扰后,相较于shRNA-NC对照组, TAS 1R1、 ERK 1、 ERK 2、 mTOR 基因mRNA表达量均极显著降低( P <0.01),自噬因子 BECN1 和 MAP 1LC3B表达量极显著升高( P <0.01)。Western blot结果表明当 TAS 1R3基因被干扰后,与 shRNA-NC 对照组相比,蛋白T1R3、T1R1、p-mTOR、p-S6K1、p-ERK12均极显著降低( P <0.01),而自噬蛋白 Beclin 1 表达量极显著升高( P <0.01)。本试验通过将 TAS 1R3基因干扰载体转染至睾丸间质细胞,发现当 TAS 1R3基因被干扰后可通过影响 mTOR 基因的表达,调控其下游自噬因子的表达,提示 TAS 1R3基因与睾丸间质细胞的自噬密切相关,这为进一步研究 TAS 1R3基因通过调节自噬影响香猪生殖活动提供了借鉴。

The purpose of this study was to explore the effect of TAS 1R3 gene on the expression of autophagy-related genes in Congjiang Xiang pigs through mTOR. In this experiment, three 30-day-old healthy male Congjiang Xiang pigs were selected, testis tissue was collected and Leydig cells were cultured. The interference vector and empty vector were transfected into the cells, and their effects on the expression of TAS1 R3 gene were detected by qRT-PCR. The effects of TAS 1R3 gene interference on the expression of taste receptor family 1 receptor (TAS1R1), extracellular signal-regulated kinase 1 (ERK1), extracellular signal-regulated kinase 2 (ERK2), rapamycin target protein (mTOR), BECN1 and microtubule-associated protein 1 light chain 3 β (MAP1LC3B) were detected by qRT-PCR and Western blot. The results showed that when TAS 1R3 gene was interfered, compared with shRNA-NC control group, the mRNA expression of TAS 1R1, ERK1 , ERK 2 and mTOR genes decreased significantly( P <0.01), while the expression of autophagy factors BECN 1 and MAP 1LC3B increased significantly ( P <0.01). The results of Western blot showed that when TAS1R3 gene was silenced, compared with shRNA-NC control group, the expression of T1R3, T1R1, p-mTOR, p-S6K1 and p-ERK12 decreased significantly( P <0.01), while the expression of autophagy protein Beclin 1 increased significantly( P <0.01). In this experiment, by transfecting TAS 1R3 gene interference vector into Leydig cells, it was found that when TAS 1R3 gene was interfered, it could affect the expression of mTOR gene, and then regulate the expression of downstream autophagy factors, suggesting that TAS 1R3 gene is closely related to autophagy of Leydig cells, which provides a reference for further study on the regulation mechanism of TAS 1R3 gene affecting the reproduction of Xiang pigs by regulating autophagy.