lncRNA-MSTRG.7889.1竞争性结合bta-miR-146a靶向Smad4调控牦牛颗粒细胞的凋亡

doi:10.3864/j.issn.0578-1752.2024.04.013

中国农业科学 ›› 2024, Vol. 57 ›› Issue (4): 797-809.doi: 10.3864/j.issn.0578-1752.2024.04.013

lncRNA-MSTRG.7889.1竞争性结合bta-miR-146a靶向Smad4调控牦牛颗粒细胞的凋亡

孟朝轶¹(), 王运路¹(), 姚一龙², 席广银³, 牛家强¹, 索朗斯珠¹, 郭敏³, 徐业芬¹()

¹ 西藏农牧学院动物科学学院，西藏林芝 860000
² 中国农业科学院深圳农业基因组研究所，广东深圳 518000
³ 中国农业大学动物科学技术学院，北京 100193

收稿日期:2023-06-27 接受日期:2023-09-10 出版日期:2024-02-16 发布日期:2024-02-20
通信作者:
徐业芬，E-mail：xzlzxyf@163.com。
联系方式: 孟朝轶，E-mail：392472987@qq.com。王运路，E-mail：3225254770@qq.com。孟朝轶和王运路为同等贡献作者。
基金资助:
国家自然科学基金(31960661); 西藏自治区科技厅区域科技协同创新专项(QYXTZX-NQ2022-04); 国家肉牛牦牛产业体系项目(CARS-37)

lncRNA-MSTRG.7889.1 Competitively Binds to bta-miR-146a Targeting Smad4 to Regulate Apoptosis of Yak Granulosa Cells

MENG ZhaoYi¹(), WANG YunLu¹(), YAO YiLong², XI GuangYin³, NIU JiaQiang¹, SOLANGSIZHU¹, GUO Min³, XU YeFen¹()

¹ College of Animal Sciences, Tibet Agriculture and Animal Husbandry College, Nyingchi 860000, Tibet
² Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, Guangdong
³ College of Animal Science and Technology, China Agricultural University, Beijing 100193

Received:2023-06-27 Accepted:2023-09-10 Published:2024-02-16 Online:2024-02-20

摘要/Abstract

摘要：

【背景】卵泡颗粒细胞（Granulosa cells，GCs）凋亡是导致卵泡闭锁的重要原因，竞争性内源RNA（competing endogenous RNAs，ceRNA）机制已被证明参与调控GCs的凋亡过程。前期牦牛卵巢转录组测序发现lncRNA-MSTRG.7889.1与bta-miR-146a具有结合位点，而bta-miR-146a和Smad4存在靶向关系。【目的】通过探究影响牦牛卵泡GCs凋亡的ceRNA分子调控机制，为揭示牦牛生殖调控的奥秘奠定基础。【方法】采集和分离成年母牦牛的健康和闭锁卵泡，利用RT-qPCR法检测lncRNA-MSTRG.7889.1、bta-miR-146a和Smad4在卵泡中的表达。制作牦牛健康和闭锁卵泡组织切片，TUNEL检测健康卵泡和闭锁卵泡中GCs的凋亡情况;荧光原位杂交分析lncRNA-MSTRG.7889.1、bta-miR-146a和Smad4在卵泡中的定位。体外分离和培养牦牛GCs，分别转染Smad4过表达载体和bta-miR-146a mimics，流式细胞术检测GCs细胞凋亡率，Western blot检测促凋亡蛋白CASPASE3和BAX，抑凋亡蛋白BCL-2的表达;GCs共转染Smad4过表达载体和bta-miR-146a mimics后，探究bta-miR-146a是否靶向Smad4影响牦牛GCs的凋亡。将lncRNA-MSTRG.7889.1过表达慢病毒载体感染GCs，流式细胞术和Western blot检测lncRNA-MSTRG.7889.1对GCs凋亡的影响;lncRNA-MSTRG.7889.1载体和bta-miR-146a mimics共转染GCs，进一步分析lncRNA-MSTRG.7889.1是否竞争性结合bta-miR-146a靶向Smad4影响牦牛GCs的凋亡。【结果】lncRNA-MSTRG.7889.1和Smad4 mRNA在牦牛健康卵泡中的表达极显著高于闭锁卵泡（P<0.01），而bta-miR-146a的表达则相反（P<0.01）。TUNEL检测结果显示，在闭锁卵泡中GCs的荧光强度极显著高于健康卵泡（P<0.01），说明闭锁卵泡中GCs凋亡显著高于健康卵泡。荧光原位杂交结果显示Smad4、bta-miR-146a和lncRNA-MSTRG.7889.1在牦牛卵泡中共表达，其在健康和闭锁卵泡中的表达与RT-qPCR结果基本一致，提示可能存在ceRNA机制参与牦牛健康卵泡发育和卵泡闭锁过程。在牦牛GCs过表达Smad4使细胞凋亡率显著降低（P<0.01），CASPASE3和BAX蛋白的表达显著降低（P<0.01），BCL-2蛋白的表达显著升高（P<0.01）;过表达bta-miR-146a使GCs凋亡率显著升高（P<0.01），CASPASE3和BAX蛋白的表达显著升高（P<0.01），BCL-2蛋白的表达显著降低（P<0.01）;bta-miR-146a靶向抑制Smad4的表达（P<0.01）;Smad4和bta-miR-146a共转染GCs，结果显示bta-miR-146a靶向抑制Smad4降低前者对GCs凋亡的促进作用（P<0.01）。过表达lncRNA-MSTRG.7889.1使牦牛GCs凋亡率显著降低（P<0.01），CASPASE3和BAX蛋白的表达显著降低（P<0.01），BCL-2蛋白的表达显著升高（P<0.01）;lncRNA-MSTRG.7889.1显著抑制bta-miR-146a的表达（P<0.01）。将lncRNA-MSTRG.7889.1和bta-miR-146a共转染GCs，结果表明lncRNA-MSTRG.7889.1靶向bta-miR-146a降低后者对GCs凋亡的促进作用（P<0.01）;而且RT-qPCR法和Western blot检测结果表明lncRNA-MSTRG7889.1竞争性结合bta-miR-146a促进Smad4 mRNA和蛋白水平的表达（P<0.01）。【结论】lncRNA-MSTRG.7889.1竞争性结合bta-miR-146a促进Smad4的表达抑制牦牛GCs的凋亡。

关键词: lncRNA, bta-miR-146a, 凋亡, 颗粒细胞, 牦牛

Abstract:

【Background】 Apoptosis of granulosa cells (GCs) is an important cause of follicular atresia, and competing endogenous RNA mechanism has been proven to be involved in regulating the apoptosis process of GCs. The previous transcriptome sequencing study of yak ovary showed that lncRNA-MSTRG.7889.1 had binding sites with bta-miR-146a, while bta-miR-146a had a targeting relationship with Smad4. 【Objective】The aim of this experiment was to explore the molecular regulatory mechanisms of ceRNA that affected the apoptosis of follicular GCs in yak, so as to lay a foundation for revealing the mystery of the reproductive regulation of the yak.【Method】 Healthy and atretic follicles of adult female yaks were collected and isolated, and the expressions of lncRNA-MSTRG.7889.1, bta-miR-146a and Smad4 in follicles were detected by RT-qPCR. Healthy and atretic follicles were sliced, and GCs apoptosis in healthy and atretic follicles was detected by TUNEL. The localizations of lncRNA-MSTRG.7889.1, bta-miR-146a and Smad4 in follicles were analyzed by fluorescence in situ hybridization. The yak GCs were isolated and cultured in vitro, and transfected with Smad4 overexpression vector and bta-miR-146a mimics, respectively. The apoptosis rate of GCs cells was detected by flow cytometry, and the expressions of pro-apoptotic proteins CASPASE3 and BAX and anti-apoptotic proteins BCL-2 were detected by Western blot. After co-transfecting Smad4 overexpression vector and bta-miR-146a mimics with GCs, it was investigated whether bta-miR-146a targeting Smad4 affects GCs apoptosis in yaks. lncRNA-MSTRG.7889.1 overexpression lentiviral vector was used to infect GCs, and the effect of lncRNA-MSTRG.7889.1 on GCs apoptosis was detected by flow cytometry and Western blot. lncRNA-MSTRG.7889.1 vector and bta-miR-146a mimics were co-transfected with GCs to further analyze whether lncRNA-MSTRG.7889.1 competitive binding of bta-miR-146a targeting Smad4 affected the apoptosis of GCs in yaks. 【Result】 The expression of lncRNA-MSTRG.7889.1 and Smad4 gene mRNA in healthy yak follicles was significantly higher than that in atresia follicles (P<0.01), while the expression of bta-miR-146a was opposite (P<0.01). TUNEL test results showed that the fluorescence intensity of GCs in atretic follicles was significantly higher than that in healthy follicles (P<0.01), indicating that the apoptosis of GCs in atretic follicles was significantly higher than that in healthy follicles. The results of fluorescence in situ hybridization showed that Smad4, bta-miR-146a and lncRNA-MSTRG.7889.1 were co-expressed in the follicles of yaks, and their expressions in healthy and atretic follicles were basically consistent with the results of RT-qPCR, indicating that ceRNA mechanism might be involved in the development of healthy follicles and follicle atresia in yaks. Overexpression of Smad4 in yaks GCs significantly decreased the apoptosis rate of GCs (P<0.01), and the expressions of CASPASE3 and BAX protein were significantly decreased (P<0.01), while the expression of BCL-2 was significantly increased (P<0.01). Overexpression of bta-miR-146a significantly increased the apoptosis rate of GCs (P<0.01), increased the expressions of CASPASE3 and BAX proteins (P<0.01), and decreased the expression of BCL-2 (P<0.01). bta-miR-146a targeted inhibition of Smad4 expression (P<0.01); Smad4 and bta-miR-146a were co-transfected into GCs, and the results showed that bta-miR-146a targeted inhibition of Smad4 reduced the former's promoting effect on GCs apoptosis (P<0.01). Overexpression of lncRNA-MSTRG.7889.1 significantly decreased the apoptosis rate of GCs in yaks (P<0.01), the expressions of CASPASE3 and BAX protein were significantly decreased (P<0.01), and the expression of BCL-2 was significantly increased (P<0.01). lncRNA-MSTRG.7889.1 significantly inhibited the expression of bta-miR-146a (P<0.01). lncRNA-MSTRG.7889.1 and bta-miR-146a were co-transfected into GCs, and lncRNA-MSTRG.7889.1 targeting bta-miR-146a decreased the promotion effect of the latter on GCs apoptosis (P<0.01), and the results of RT-qPCR and Western blot showed that lncRNA-MSTRG7889.1 competitively bound bta-miR-146a to promote the expression of Smad4 gene at mRNA and protein levels (P<0.01). 【Conclusion】 lncRNA-MSTRG.7889.1 competitively binds to bta-miR-146a, promotes the expression of Smad4 and inhibits the apoptosis of yak GCs.

Key words: lncRNA, bta-miR-146a, apoptosis, granulosa cells, yak

孟朝轶, 王运路, 姚一龙, 席广银, 牛家强, 索朗斯珠, 郭敏, 徐业芬. lncRNA-MSTRG.7889.1竞争性结合bta-miR-146a靶向Smad4调控牦牛颗粒细胞的凋亡[J]. 中国农业科学, 2024, 57(4): 797-809.

MENG ZhaoYi, WANG YunLu, YAO YiLong, XI GuangYin, NIU JiaQiang, SOLANGSIZHU, GUO Min, XU YeFen. lncRNA-MSTRG.7889.1 Competitively Binds to bta-miR-146a Targeting Smad4 to Regulate Apoptosis of Yak Granulosa Cells[J]. Scientia Agricultura Sinica, 2024, 57(4): 797-809.

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引物名称 Primer	引物序列 Primer sequence (5′ to 3′)		片段大小 Fragment size (bp)
lnc-MSTRG.7889探针Probes	5′-Cy3-CATTGAGAAGGAAATGGTAACCCACTCTAGTGTTCTTGCCTGCAG-3′
Smad4探针Probes	5′-Cy3-CTCCAGGTGCACGCCCAGCTTCTCTGTCTAAGTAGTAGCTCTGTA-3′
bta-miR-146a探针Probes	5′-FAM- ACAACCTATGGAATTCAGTTCTCA-3′
Smad4 酶切引物Primer	F	GAATTCCAATATGTCTATTACGAATACACCAACAAGTAATGATGCCTGTCT	1162
Smad4 酶切引物Primer	R	AGATCTCGATGAAGTCCTTCACACCATGCCTATTGCCGACCCACAGCCT	1162
Smad 4	F	TACCAGAACAAAGGAGTATCGTT	148
Smad 4	R	CGGTAAGATATTAGCTGGAGTGAC	148
bta-miR-146a	F	TGAGAACTGAATTCCATACCTTCT	98
bta-miR-146a	R	GCGAGCACAGAATTAATACGAC	98
lnc-MSTRG.7889	F	TAGCAGGTGGGCAGTG	118
lnc-MSTRG.7889	R	TTTCAAATACCGCCAC	118
U6	F	TCGCTTCGGCAGCACATATAC	102
U6	R	GCGAGCACAGAATTAATACGAC	102
β-actin	F	CCAACTGGGACGACATGGA	146
β-actin	R	GTCTCGAACATGATCTGGGTCAT	146

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lncRNA-MSTRG.7889.1竞争性结合bta-miR-146a靶向Smad4调控牦牛颗粒细胞的凋亡

lncRNA-MSTRG.7889.1 Competitively Binds to bta-miR-146a Targeting Smad4 to Regulate Apoptosis of Yak Granulosa Cells

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