miR-139对奶牛乳腺上皮细胞泌乳的调节作用
发布时间：2018-09-03 20:45
【摘要】：mi RNAs是一类单链的在转录后调节基因表达的非编码RNA,能参与生物体的多项生理活动。mi R-139在多种恶性肿瘤的发生发展过程中起作用,且与肿瘤的侵袭和转移有关。目前国内外鲜有关于mi R-139调节奶牛乳腺发育及泌乳功能研究的报道。本研究以中国荷斯坦奶牛为实验动物,以泌乳中期奶牛乳腺上皮细胞为模型,确定mi R-139及其靶基因的表达与奶牛乳腺发育和泌乳之间的关系,明确mi R-139在奶牛乳腺发育和泌乳中的调节作用,为人工调节乳产量提供理论依据。本研究先在Target Scan(http://www.targetscan.org)网站上进行靶基因预测,然后选择GHR和IGF-IR作为mi R-139的目标靶基因;之后以中国荷斯坦奶牛作为实验动物,采用实时荧光定量PCR技术检测mi R-139、GHR和IGF-IR在奶牛不同发育阶段及不同乳品质乳腺组织中的表达变化,用Western blotting方法检测GHR在奶牛不同发育阶段及不同乳品质乳腺组织中的表达变化;接下来以体外培养的泌乳中期奶牛乳腺上皮细胞为研究对象,分别转染mi R-139 mimic和inhibitor,采取实时荧光定量PCR技术检测mi R-139、GHR和IGF-IR的表达变化;用Western blotting方法检测IGF-IR及泌乳相关信号通路蛋白的表达变化;分别用MTT法检测细胞活性、Edu细胞增殖试剂盒检测DNA增殖情况;用相关试剂盒分别检测mi R-139过表达和抑制后?-酪蛋白、甘油三酯以及乳糖的含量;最后用mi R-139 inhibitor和GHR si RNA同时转染细胞,进行mi R-139和GHR共抑制实验,并在m RNA或蛋白水平检测GHR、IGF-IR以及泌乳相关信号通路蛋白的表达变化。研究结果表明:(1)干奶期奶牛乳腺组织中的mi R-139及其靶基因GHR和IGF-IR的mRNA表达显著高于泌乳期(P0.05);泌乳期高乳品质奶牛乳腺组织中的mi R-139及GHR的mRNA表达显著高于低乳品质奶牛(P0.05),但是GHR的蛋白表达在泌乳期高、低乳品质奶牛乳腺组织中没有显著差异;泌乳期高乳品质奶牛乳腺组织中的IGF-IR的mRNA表达显著低于低乳品质奶牛(P0.05)。(2)mi R-139过表达能显著下调奶牛乳腺上皮细胞中GHR、IGF-IR、STAT5、p-STAT5、PPAR?、SREBP1、Cyclin D1、p-70S6K、p-p70S6K、AKT1、p-AKT1、m TOR、p-m TOR的表达(P0.05);mi R-139抑制实验则表现出相反结果(P0.05),说明mi R-139能通过调节其靶基因GHR和IGF-IR进而调节泌乳相关信号通路蛋白的表达。(3)mi R-139过表达显著抑制了奶牛乳腺上皮细胞的活力、增殖能力以及?-酪蛋白、甘油三酯和乳糖的合成(P0.05);mi R-139抑制实验则表现出相反结果(P0.05)。(4)mi R-139和GHR共抑制组与GHR沉寂组相比,GHR、IGF-IR、AKT1、p-AKT1以及酪蛋白的表达量显著上调(P0.05);而mi R-139和GHR基因共抑制组与mi R-139抑制组相比,GHR、IGF-IR、AKT1、p-AKT1以及酪蛋白的表达量显著下调(P0.05),这一结果一方面再次验证了GHR是mi R-139的靶基因;另一方面说明mi R-139的另一个靶基因IGF-IR的表达和作用还受到GHR的调节。
[Abstract]:Mi RNAs is a single-stranded non-coding RNA, that regulates the expression of genes after transcription. Mi R-139 plays a role in the occurrence and development of many kinds of malignant tumors and is related to the invasion and metastasis of tumors. At present, there are few reports on the regulation of mammary gland development and lactation function of dairy cows by mi R-139. The relationship between the expression of mi R-139 and its target gene and mammary gland development and lactation was determined by using Chinese Holstein cows as experimental animals and mammary epithelial cells in middle lactation period. The regulatory role of mi R-139 in breast development and lactation of dairy cows was clarified, which provided theoretical basis for manual regulation of milk yield. In this study, target genes were predicted on the Target Scan (http://www.targetscan.org website, then GHR and IGF-IR were selected as target genes of mi R-139, and then Chinese Holstein cows were used as experimental animals. The expression of mi R-139G HR and IGF-IR in different milk quality and development stage of dairy cattle was detected by real-time fluorescence quantitative PCR, and the expression of GHR was detected by Western blotting method in different development stage and different milk quality of dairy cow. Then the expression of mi R-139 mimic and IGF-IR were detected by real-time quantitative PCR with mi R-139 mimic and inhibitor, transfected with bovine mammary epithelial cells cultured in vitro. The expression of IGF-IR and lactation related signaling pathway protein was detected by Western blotting, the proliferation of DNA was detected by MTT assay, and the expression of mi R-139 and casein were detected by mi R-139. Finally, mi R-139 inhibitor and GHR si RNA were used to transfect the cells. The co-inhibition of mi R-139 and GHR was carried out, and the expression of GHR,IGF-IR and lactation related signal pathway proteins were detected at the level of m RNA or protein. The results showed that: (1) the mRNA expression of mi R-139 and its target gene GHR and IGF-IR in breast tissue of dry milk cows was significantly higher than that in lactation period (P0.05), and the mRNA expression of mi R-139 and GHR in breast tissue of high milk quality dairy cow during lactation period was significantly higher than that of low milk product (P0.05). In dairy cows (P0.05), however, the protein expression of GHR was high during lactation. There was no significant difference in breast tissues of low milk quality cows. The expression of IGF-IR mRNA in breast tissues of high lactation quality cows was significantly lower than that in low milk quality cows (P0.05). (2). The overexpression of mi R-139 could significantly down-regulate the expression of GHR,IGF-IR,STAT5,p-STAT5,PPAR?,SREBP1,Cyclin D1P p-70S6KN p-p70S6KT1 p-AKT1 p-AKT1mTOR,p-m TOR in dairy cow breast epithelial cells (P0.05). Mi R-139 inhibition test showed the opposite result. (P0.05), which indicated that mi R-139 could regulate the expression of lactation related signaling pathway protein by regulating its target genes GHR and IGF-IR. (3) mi R-139 overexpression significantly inhibited the activity of bovine mammary epithelial cells. Proliferation and casein, The inhibitory effect of triglyceride and lactose on the synthesis of triglyceride and lactose (P0.05) showed the opposite results (P0.05). (4). The mi R-139 and GHR co-inhibition groups were significantly up-regulated in the expression of GHR-IGF-IRF-AKT1 p-AKT1 and casein compared with the silent GHR group (P0.05), while the mi R-139 and GHR co-inhibition group was significantly inhibited by mi R-139 (P0.05). Compared with the control group, the expression of IGF-IRT1 p-AKT1 and casein was significantly decreased (P0.05), which confirmed that GHR was the target gene of mi R-139. On the other hand, the expression and function of IGF-IR, another target gene of mi R-139, is regulated by GHR.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
本文编号：2221118
[Abstract]:Mi RNAs is a single-stranded non-coding RNA, that regulates the expression of genes after transcription. Mi R-139 plays a role in the occurrence and development of many kinds of malignant tumors and is related to the invasion and metastasis of tumors. At present, there are few reports on the regulation of mammary gland development and lactation function of dairy cows by mi R-139. The relationship between the expression of mi R-139 and its target gene and mammary gland development and lactation was determined by using Chinese Holstein cows as experimental animals and mammary epithelial cells in middle lactation period. The regulatory role of mi R-139 in breast development and lactation of dairy cows was clarified, which provided theoretical basis for manual regulation of milk yield. In this study, target genes were predicted on the Target Scan (http://www.targetscan.org website, then GHR and IGF-IR were selected as target genes of mi R-139, and then Chinese Holstein cows were used as experimental animals. The expression of mi R-139G HR and IGF-IR in different milk quality and development stage of dairy cattle was detected by real-time fluorescence quantitative PCR, and the expression of GHR was detected by Western blotting method in different development stage and different milk quality of dairy cow. Then the expression of mi R-139 mimic and IGF-IR were detected by real-time quantitative PCR with mi R-139 mimic and inhibitor, transfected with bovine mammary epithelial cells cultured in vitro. The expression of IGF-IR and lactation related signaling pathway protein was detected by Western blotting, the proliferation of DNA was detected by MTT assay, and the expression of mi R-139 and casein were detected by mi R-139. Finally, mi R-139 inhibitor and GHR si RNA were used to transfect the cells. The co-inhibition of mi R-139 and GHR was carried out, and the expression of GHR,IGF-IR and lactation related signal pathway proteins were detected at the level of m RNA or protein. The results showed that: (1) the mRNA expression of mi R-139 and its target gene GHR and IGF-IR in breast tissue of dry milk cows was significantly higher than that in lactation period (P0.05), and the mRNA expression of mi R-139 and GHR in breast tissue of high milk quality dairy cow during lactation period was significantly higher than that of low milk product (P0.05). In dairy cows (P0.05), however, the protein expression of GHR was high during lactation. There was no significant difference in breast tissues of low milk quality cows. The expression of IGF-IR mRNA in breast tissues of high lactation quality cows was significantly lower than that in low milk quality cows (P0.05). (2). The overexpression of mi R-139 could significantly down-regulate the expression of GHR,IGF-IR,STAT5,p-STAT5,PPAR?,SREBP1,Cyclin D1P p-70S6KN p-p70S6KT1 p-AKT1 p-AKT1mTOR,p-m TOR in dairy cow breast epithelial cells (P0.05). Mi R-139 inhibition test showed the opposite result. (P0.05), which indicated that mi R-139 could regulate the expression of lactation related signaling pathway protein by regulating its target genes GHR and IGF-IR. (3) mi R-139 overexpression significantly inhibited the activity of bovine mammary epithelial cells. Proliferation and casein, The inhibitory effect of triglyceride and lactose on the synthesis of triglyceride and lactose (P0.05) showed the opposite results (P0.05). (4). The mi R-139 and GHR co-inhibition groups were significantly up-regulated in the expression of GHR-IGF-IRF-AKT1 p-AKT1 and casein compared with the silent GHR group (P0.05), while the mi R-139 and GHR co-inhibition group was significantly inhibited by mi R-139 (P0.05). Compared with the control group, the expression of IGF-IRT1 p-AKT1 and casein was significantly decreased (P0.05), which confirmed that GHR was the target gene of mi R-139. On the other hand, the expression and function of IGF-IR, another target gene of mi R-139, is regulated by GHR.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
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相关期刊论文 前2条
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2 李真;李庆章;;奶山羊乳腺发育过程中生长激素、胰岛素及其受体的变化规律研究[J];中国农业科学;2010年08期
，本文编号：2221118
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