bta-miR-146b对热应激奶牛乳腺上皮细胞乳蛋白合成信号通路的影响

doi:10.3969/j.issn.1004-1524.20240541

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (6): 1212-1220.DOI: 10.3969/j.issn.1004-1524.20240541

bta-miR-146b对热应激奶牛乳腺上皮细胞乳蛋白合成信号通路的影响

张悦宇(), 黄美琦, 张琳, 齐颖, 李秋玲^*()

廊坊师范学院生命科学学院,河北省动物多样性重点实验室,廊坊市细胞工程与应用研究重点实验室,河北廊坊 065000

收稿日期:2024-06-24 出版日期:2025-06-25 发布日期:2025-07-08
作者简介:张悦宇(2001—),女,河北石家庄人,硕士研究生,研究方向为生物工程。E-mail:zhangyueyu2024@163.com
通讯作者: *李秋玲,E-mail:liqiuling2000@126.com
基金资助:
河北省高等学校科学技术重点研究项目(ZD2021091);河北省属高校基本科研业务费项目—优秀创新团队(JYT202301);河北省属高校基本科研业务费项目—优秀青年项目(JYQ202301);大学生创新创业训练计划项目(X202310100024)

Effects of bta-miR-146b on the signaling pathway of milk protein synthesis in heat-stressed bovine mammary epithelial cells

ZHANG Yueyu(), HUANG Meiqi, ZHANG Lin, QI Ying, LI Qiuling^*()

Hebei Key Laboratory of Animal Diversity, Langfang Key Laboratory of Cell Engineering and Application, College of Life Sciences, Langfang Normal University, Langfang 065000, Hebei, China

Received:2024-06-24 Online:2025-06-25 Published:2025-07-08

摘要/Abstract

摘要： 为了探究热应激条件下bta-miR-146b对奶牛乳蛋白合成的影响,在奶牛乳腺上皮细胞中分别转染bta-miR-146b模拟物、bta-miR-146b抑制物、bta-miR-146b模拟物阴性对照物、bta-miR-146b抑制物阴性对照物,然后进行热应激(40 ℃)处理,通过实时荧光定量PCR(qRT-PCR)技术检测各组细胞中的乳蛋白相关基因(CSN2、CSN3)和乳蛋白合成信号通路相关基因(JAK2、STAT5、mTOR、MAPK)的相对表达量,分析过表达bta-miR-146b和抑制表达bta-miR-146b在热应激与非热应激条件下对乳蛋白和乳蛋白合成信号通路的影响。结果显示:转染bta-miR-146b模拟物后乳腺上皮细胞中bta-miR-146b的表达量提高,转染bta-miR-146b抑制物后乳腺上皮细胞中bta-miR-146b的表达量降低;热应激会抑制空白对照组、bta-miR-146b模拟物组和bta-miR-146b抑制物组奶牛乳腺上皮细胞中CSN2、CSN3、JAK2、STAT5、mTOR、MAPK基因的表达;非热应激条件下,过表达bta-miR-146b会使CSN2、CSN3、JAK2、STAT5、mTOR、MAPK基因表达量显著降低,抑制表达bta-miR-146b会使CSN3、JAK2、STAT5、mTOR、MAPK基因表达量显著升高;热应激条件下,过表达bta-miR-146b会使CSN2、CSN3、JAK2、STAT5、mTOR、MAPK基因表达量显著降低,抑制表达bta-miR-146b会使这些基因表达量显著升高。综上所述,热应激处理和过表达bta-miR-146b均会显著抑制乳蛋白和乳蛋白合成信号通路的相关基因的表达,抑制表达bta-miR-146b会促进乳蛋白和乳蛋白合成信号通路相关基因的表达。

关键词: 奶牛, 热应激, 乳蛋白, 信号通路, 表达量

Abstract:

To investigate the impact of bta-miR-146b on milk protein synthesis in dairy cows under heat stress, bovine mammary epithelial cells (BMECs) were transfected with bta-miR-146b mimics, bta-miR-146b inhibitor, bta-miR-146b mimics negative control (NC) and bta-miR-146b inhibitor NC. Following transfection, the cells were subjected to heat stress (40 ℃). The relative expression levels of milk protein-related genes (CSN2, CSN3) and genes associated with milk protein synthesis signaling pathways (JAK2, STAT5, mTOR, MAPK) in each group were detected using quantitative real-time PCR (qRT-PCR). This allowed for analysis of the effects of bta-miR-146b overexpression and inhibition on milk proteins and their synthesis signaling pathways under both heat stress and non-heat stress conditions. The results showed that: Transfection with the bta-miR-146b mimics significantly increased bta-miR-146b expression in BMECs, while transfection with the inhibitor significantly decreased its expression; Heat stress significantly suppressed the expression of CSN2, CSN3, JAK2, STAT5, mTOR and MAPK genes in the blank control group, the bta-miR-146b mimic group, and the bta-miR-146b inhibitor group; Under non-heat stress conditions, overexpression of bta-miR-146b significantly decreased the expression levels of CSN2, CSN3, JAK2, STAT5, mTOR and MAPK genes, while inhibition of bta-miR-146b significantly increased the expression of CSN3, JAK2, STAT5, mTOR and MAPK genes. Under heat stress conditions, overexpression of bta-miR-146b significantly decreased the expression levels of CSN2, CSN3, JAK2, STAT5, mTOR and MAPK genes, while inhibition of bta-miR-146b significantly increased the expression of these genes. In summary, both heat stress treatment and overexpression of bta-miR-146b significantly inhibit the expression of genes related to milk proteins and their synthesis signaling pathways. Conversely, inhibition of bta-miR-146b expression promotes the expression of these genes.

Key words: dairy cow, heat stress, milk protein, signaling pathway, expression level

中图分类号:

S823
Q78

张悦宇, 黄美琦, 张琳, 齐颖, 李秋玲. bta-miR-146b对热应激奶牛乳腺上皮细胞乳蛋白合成信号通路的影响[J]. 浙江农业学报, 2025, 37(6): 1212-1220.

ZHANG Yueyu, HUANG Meiqi, ZHANG Lin, QI Ying, LI Qiuling. Effects of bta-miR-146b on the signaling pathway of milk protein synthesis in heat-stressed bovine mammary epithelial cells[J]. Acta Agriculturae Zhejiangensis, 2025, 37(6): 1212-1220.

图/表 8

表1 转染物序列

Table 1 The sequences of the transfection

名称 Name	序列 Sequence
名称 Name	正义链Sense(5'→3')	反义链Antisense(5'→3')
bta-miR-146b模拟物	UGAGAACUGAAUUCCAUAGGCUGU	ACAGCCUAUGGAAUUCAGUUCUCA
bta-miR-146b mimics
bta-miR-146b 抑制物	ACAGCCUAUGGAAUUCAGUUCUCA
bta-miR-146b inhibitor
bta-miR-146b模拟物阴性对照物	UUGUACUACACAAAAGUACUG	GUACUUUUGUGUAGUACAAUU
bta-miR-146b mimics NC
bta-miR-146b抑制物阴性对照物	CAGUACUUUUGUGUAGUACAA
bta-miR-146b inhibitor NC

表2 奶牛乳腺上皮细胞处理与分组情况

Table 2 The treatments and groups of bovine mammary epithelial cells

处理 Treatment	分组 Group	转染物 Transfections
非热应激组NHS group	非转染组Non-transfected group	无Blank
	转染组Transfected group	bta-miR-146b模拟物bta-miR-146b mimics
		bta-miR-146b 模拟物阴性对照物bta-miR-146b mimics NC
		bta-miR-146b抑制物bta-miR-146b inhibitor
		bta-miR-146b 抑制物阴性对照物bta-miR-146b inhibitor NC
热应激组HS group	非转染组Non-transfected group	无Blank
	转染组Transfected group	bta-miR-146b模拟物bta-miR-146b mimics
		bta-miR-146b 模拟物阴性对照物bta-miR-146b mimics NC
		bta-miR-146b抑制物btabta-miR-146b inhibitor
		bta-miR-146b 抑制物阴性对照物bta-miR-146b inhibitor NC

表3 qRT-PCR引物信息

Table 3 Primer information for qRT-PCR

基因 Gene	正向引物序列 Forward primer sequence (5'→3')	反向引物序列 Reverse primer sequence (5'→3')
β-actin	CTGTTAGCTGCGTTACACCCTT	TGCTGTCACCTTCACCGTTC
bta-miR-146b	GGGTGAGAACTGAATTCCATA	GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACAGCC
U6	GCTTCGGCAGCACATATACATAAAT	CGCTTCACGAATTTGCGTGTCAT
CSN2	TGAGGAACAGCAGCAAACAGAG	TGGGAGGCTGTTATGGATGG
CSN3	ACAACCAATACGCTGTGAGAA	TTGGCAGGCACAGTATTTG
JAK2	CCTGGCAACAGACAAATGGA	ACCCTTGCCAAGTTGCTGTA
STAT5	CTTCCCGTGGTTGTCATCGT	CGGCCTTGAATTTCATGTTG
mTOR	GTGATGAACTTTGAGGCTGTGC	CAAGGAGATGGAACGGAAGAA
MAPK	AAGTCCGAGTCGCCATCAAG	TGGTGTTGAGCAGCAGGTTG

图1 转染后乳腺上皮细胞中bta-miR-146b的表达情况柱上无相同小写字母表示差异显著(P<0.05)。

Fig.1 Expression level of bta-miR-146b in transfected mammary epithelial cells Bars marked without the same lowercase letter indicated significant differences(P<0.05).

图2 未转染组中各基因在非热应激与热应激下的相对表达量 NHS,非热应激;HS,热应激。柱上无相同大写字母表示同一基因在不同处理下差异极显著(P<0.01)。下同。

Fig.2 Relative expression levels of each gene in the non-transfected group under non-heat stress and heat stress conditions NHS, Non-heat stress condition; HS, Heat stress condition. Bars marked without the same uppercase letter indicated the same gene had significant differences (P<0.01) under different treatments. The same as below.

图3 转染组乳腺上皮细胞中各基因在热应激和非热应激下的表达情况 (A)转染bta-miR-146b 模拟物后乳腺上皮细胞中各基因在热应激与非热应激下的表达情况;(B)转染bta-miR-146b 抑制物后乳腺上皮细胞中各基因在热应激与非热应激下的表达情况。

Fig.3 The expression of each gene in transfected mammary epithelial cells under heat stress and non-heat stress condition (A)The expression of each gene in mammary epithelial cells after transfection of bta-miR-146b mimics under the heat stress and non-heat stress condition. (B)The expression of each gene in mammary epithelial cells after transfection of bta-miR-146b inhibitor under heat stress and non-heat stress condition.

图4 非热应激条件下乳腺上皮细胞中各基因的表达情况 (A)非热应激条件下转染bta-miR-146b模拟物后各基因的相对表达情况;(B)非热应激条件下转染bta-miR-146b抑制物后各基因的相对表达情况。无相同大写字母表示同一基因在不同处理下具有极显著差异(P<0.01);无相同小写字母表示同一基因在不同处理下具有显著差异(P<0.05)。下同。

Fig.4 The expression of each gene in mammary epithelial cells under non-heat stress condition (A) The relative expression of each gene after transfection of bta-miR-146b mimics under non-heat stress condition. (B) The relative expression of each gene after transfection of bta-miR-146b inhibitor under non-heat stress condition. Bars marked without the same uppercase letter indicate that the same gene had significant differences at P<0.01 under different treatments; Bars marked without the same lowercase letter indicate that the same gene had significant differences at P<0.05 under different treatments. The same as belw.

图5 热应激条件下转染组乳腺上皮细胞中各基因的表达情况 (A)热应激条件下转染bta-miR-146b 模拟物后各基因的相对表达情况;(B)热应激条件下转染bta-miR-146b 抑制物后各基因的相对表达情况。

Fig.5 The expression of each gene in transfected mammary epithelial cells under heat stress condition (A)The relative expression of each gene after transfection of bta-miR-146b mimics under heat stress condition; (B)The relative expression of each gene after transfection of bta-miR-146b inhibitor under heat stress condition.

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bta-miR-146b对热应激奶牛乳腺上皮细胞乳蛋白合成信号通路的影响

Effects of bta-miR-146b on the signaling pathway of milk protein synthesis in heat-stressed bovine mammary epithelial cells

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