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

doi:10.3969/j.issn.1004-1524.20240541

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (6): 1212-1220.DOI: 10.3969/j.issn.1004-1524.20240541

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S823
Q78

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.

Figures/Tables 8

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

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

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

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).

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.

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.

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.

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

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