miR-139對奶牛乳腺上皮細(xì)胞泌乳的調(diào)節(jié)作用
發(fā)布時間：2018-09-03 20:45
【摘要】：mi RNAs是一類單鏈的在轉(zhuǎn)錄后調(diào)節(jié)基因表達(dá)的非編碼RNA,能參與生物體的多項生理活動。mi R-139在多種惡性腫瘤的發(fā)生發(fā)展過程中起作用,且與腫瘤的侵襲和轉(zhuǎn)移有關(guān)。目前國內(nèi)外鮮有關(guān)于mi R-139調(diào)節(jié)奶牛乳腺發(fā)育及泌乳功能研究的報道。本研究以中國荷斯坦奶牛為實驗動物,以泌乳中期奶牛乳腺上皮細(xì)胞為模型,確定mi R-139及其靶基因的表達(dá)與奶牛乳腺發(fā)育和泌乳之間的關(guān)系,明確mi R-139在奶牛乳腺發(fā)育和泌乳中的調(diào)節(jié)作用,為人工調(diào)節(jié)乳產(chǎn)量提供理論依據(jù)。本研究先在Target Scan(http://www.targetscan.org)網(wǎng)站上進(jìn)行靶基因預(yù)測,然后選擇GHR和IGF-IR作為mi R-139的目標(biāo)靶基因;之后以中國荷斯坦奶牛作為實驗動物,采用實時熒光定量PCR技術(shù)檢測mi R-139、GHR和IGF-IR在奶牛不同發(fā)育階段及不同乳品質(zhì)乳腺組織中的表達(dá)變化,用Western blotting方法檢測GHR在奶牛不同發(fā)育階段及不同乳品質(zhì)乳腺組織中的表達(dá)變化;接下來以體外培養(yǎng)的泌乳中期奶牛乳腺上皮細(xì)胞為研究對象,分別轉(zhuǎn)染mi R-139 mimic和inhibitor,采取實時熒光定量PCR技術(shù)檢測mi R-139、GHR和IGF-IR的表達(dá)變化;用Western blotting方法檢測IGF-IR及泌乳相關(guān)信號通路蛋白的表達(dá)變化;分別用MTT法檢測細(xì)胞活性、Edu細(xì)胞增殖試劑盒檢測DNA增殖情況;用相關(guān)試劑盒分別檢測mi R-139過表達(dá)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同時轉(zhuǎn)染細(xì)胞,進(jìn)行mi R-139和GHR共抑制實驗,并在m RNA或蛋白水平檢測GHR、IGF-IR以及泌乳相關(guān)信號通路蛋白的表達(dá)變化。研究結(jié)果表明:(1)干奶期奶牛乳腺組織中的mi R-139及其靶基因GHR和IGF-IR的mRNA表達(dá)顯著高于泌乳期(P0.05);泌乳期高乳品質(zhì)奶牛乳腺組織中的mi R-139及GHR的mRNA表達(dá)顯著高于低乳品質(zhì)奶牛(P0.05),但是GHR的蛋白表達(dá)在泌乳期高、低乳品質(zhì)奶牛乳腺組織中沒有顯著差異;泌乳期高乳品質(zhì)奶牛乳腺組織中的IGF-IR的mRNA表達(dá)顯著低于低乳品質(zhì)奶牛(P0.05)。(2)mi R-139過表達(dá)能顯著下調(diào)奶牛乳腺上皮細(xì)胞中GHR、IGF-IR、STAT5、p-STAT5、PPAR?、SREBP1、Cyclin D1、p-70S6K、p-p70S6K、AKT1、p-AKT1、m TOR、p-m TOR的表達(dá)(P0.05);mi R-139抑制實驗則表現(xiàn)出相反結(jié)果(P0.05),說明mi R-139能通過調(diào)節(jié)其靶基因GHR和IGF-IR進(jìn)而調(diào)節(jié)泌乳相關(guān)信號通路蛋白的表達(dá)。(3)mi R-139過表達(dá)顯著抑制了奶牛乳腺上皮細(xì)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抑制實驗則表現(xiàn)出相反結(jié)果(P0.05)。(4)mi R-139和GHR共抑制組與GHR沉寂組相比,GHR、IGF-IR、AKT1、p-AKT1以及酪蛋白的表達(dá)量顯著上調(diào)(P0.05);而mi R-139和GHR基因共抑制組與mi R-139抑制組相比,GHR、IGF-IR、AKT1、p-AKT1以及酪蛋白的表達(dá)量顯著下調(diào)(P0.05),這一結(jié)果一方面再次驗證了GHR是mi R-139的靶基因;另一方面說明mi R-139的另一個靶基因IGF-IR的表達(dá)和作用還受到GHR的調(diào)節(jié)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823
本文編號：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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 孫遜,朱尚權(quán);生長激素的結(jié)構(gòu)與功能[J];國外醫(yī)學(xué)(生理、病理科學(xué)與臨床分冊);1999年01期
2 李真;李慶章;;奶山羊乳腺發(fā)育過程中生長激素、胰島素及其受體的變化規(guī)律研究[J];中國農(nóng)業(yè)科學(xué);2010年08期
，本文編號：2221118
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