牦牛“海绵吸附”bta-miR-146a的lncRNA筛选及靶向验证

doi:10.16431/j.cnki.1671-7236.2023.03.002

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (3): 859-869.doi: 10.16431/j.cnki.1671-7236.2023.03.002

牦牛“海绵吸附”bta-miR-146a的lncRNA筛选及靶向验证

王运路^1,2, 孟朝轶^1,2, 姚一龙³, 郭敏⁴, 牛家强^1,2, 索朗斯珠¹, 徐业芬^1,2

1. 西藏农牧学院动物科学学院, 林芝 860000;
2. 西藏农牧学院, 西藏高原动物疫病研究自治区高校重点实验室, 林芝 860000;
3. 中国农业科学院深圳农业基因组研究所, 深圳 518000;
4. 中国农业大学动物科技学院, 北京 100193

修回日期:2022-08-20 出版日期:2023-03-05 发布日期:2023-03-02
通讯作者: 徐业芬 E-mail:xzlzxyf@163.com
作者简介:王运路,E-mail:3225254770@qq.com。
基金资助:
国家自然科学基金项目(31960661);西藏农牧学院研究生创新计划项目(YJS2020-18);国家肉牛牦牛产业体系项目(CARS-37)

Screening and Targeting Verification of lncRNA of "Sponge Adsorption" bta-miR-146a in Yak

WANG Yunlu^1,2, MENG Zhaoyi^1,2, YAO Yilong³, GUO Min⁴, NIU Jiaqaing^1,2, SUOLANG Sizhu¹, XU Yefen^1,2

1. Department of Animal Science, Tibet Agricultural and Animal Husbandry College, Nyingchi 860000, China;
2. Provincial Key Laboratory of Tibet Plateau Animal Epidemic Disease Research, Tibet Agricultural and Animal Husbandry College, Nyingchi 860000, China;
3. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China;
4. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Revised:2022-08-20 Online:2023-03-05 Published:2023-03-02

摘要/Abstract

摘要： 【目的】筛选牦牛卵泡发育过程中可能对靶向Smad家族成员4(Smad family member 4,Smad4)基因的bta-miR-146a具有“海绵吸附”作用的长链非编码RNA(long non-coding RNA,lncRNA)。【方法】使用miRanda和RNAhybrid数据库对靶向牦牛bta-miR-146a的lncRNA进行预测;采集牦牛卵巢,分离健康、闭锁卵泡,用Trizol法提取RNA并反转录为cDNA,利用PCR检测所预测lncRNA的表达情况;利用实时荧光定量PCR法检测所筛选lncRNA和bta-miR-146a/Smad4在牦牛健康、闭锁卵泡中的表达情况;构建lncRNA-ENSBGRT00000000387.1的野生型和突变型双荧光素酶载体,将其与bta-miR-146a-mimics、mimics NC共转染至HEK293T细胞,检测双荧光素酶活性。【结果】试验共筛选出7个可能对靶向Smad4基因的bta-miR-146a具有“海绵吸附”作用的lncRNAs,其中的lncRNA-ENSBGRT00000000387.1在牦牛卵泡中表达量极显著高于其他lncRNAs(P＜0.01)。实时荧光定量PCR检测发现,lncRNA-ENSBGRT00000000387.1、bta-miR-146a和Smad4基因mRNA在牦牛健康和闭锁卵泡中共表达,且lncRNA-ENSBGRT00000000387.1和Smad4基因mRNA在牦牛健康卵泡中的表达量均显著高于闭锁卵泡(P＜0.05),bta-miR-146a在牦牛健康卵泡中的表达量极显著低于闭锁卵泡(P＜0.01)。试验成功构建牦牛lncRNA-ENSBGRT00000000387.1野生型及突变型pmirGLO双荧光素酶报告质粒,双荧光素酶活性结果显示,bta-miR-146a mimics对牦牛lncRNA-ENSBGRT00000000387.1-WT具有极显著下调作用(P＜0.01)。【结论】lncRNA-ENSBGRT00000000387.1、bta-miR-146a和Smad4基因mRNA可能在牦牛卵泡发育或闭锁过程中存在调控机制,并在体外初步证实lncRNA-ENSBGRT00000000387.1与bta-miR-146a具有“海绵吸附”作用,这为进一步研究lncRNA-ENSBGRT00000000387.1在牦牛卵泡中的功能机制提供依据。

关键词: 牦牛; 海绵吸附; lncRNA; bta-miR-146a

Abstract: 【Objective】 The aim of this study was to screen long non-coding RNA (lncRNA) that might had a "sponge adsorption" effect on bta-miR-146a targeting Smad family member 4 (Smad4) gene during follicle development of yak.【Method】 The miRanda and RNAhybrid databases were used to predict lncRNA targeting bta-miR-146a in yak.The ovary in yak was used to isolate healthy and atretic follicles,and the total RNA was extracted by Trizol method and reverse transcribed into cDNA.The expression of predicted lncRNA was detected by PCR,and the expression of lncRNA and bta-miR-146a/Smad4 in healthy and atretic follicles of yak were detected by Real-time quantitative PCR.The wild-type and mutant dual luciferase vectors of lncRNA-ENSBGRT00000000387.1 were constructed and cotransfected with bta-miR-146a mimics and mimics NC into HEK293T cells to detect the double luciferase activity.【Result】 The results showed that 7 lncRNAs were predicted to have "sponge adsorption" effect on bta-miR-146a targeting Smad4 gene,and the expression of lncRNA-ENSBGRT00000000387.1 in follicles of yak was extremely significantly higher than other lncRNAs (P＜0.01).Real-time quantitative PCR results showed that lncRNA-ENSBGRT00000000387.1,bta-miR-146a and Smad4 gene mRNA were co-expressed in healthy and atretic follicles of yak,and the expression of lncRNA-ENSBGRT00000000387.1 and Smad4 gene mRNA in healthy follicles of yak were significantly higher than that in atretic follicles (P＜0.05),but the expression of bta-miR-146a was extremely significantly lower in healthy follicles of yak than that in atretic follicles (P＜0.01).lncRNA-ENSBGRT00000000387.1 wild-type and mutant pmirGLO dual luciferase reporter plasmids of yak were successfully constructed,and the dual luciferase activity results indicated that bta-miR-146a mimics had an extremely significant down-regulation effect on lncRNA-ENSBGRT00000000387.1-WT of yak (P＜0.01).【Conclusion】lncRNA-ENSBGRT00000000387.1,bta-miR-146a and Smad4 gene mRNA might have regulatory mechanisms in follicle development or atresia process of yak,and it was tentatively confirmed in vitro that lncRNA-ENSBGRT00000000387.1 and bta-miR-146a had "sponge adsorption" effect,which provided the reference for further study of the functional mechanism of lncRNA-ENSBGRT00000000387.1 in follicles of yak.

Key words: yak; sponge adsorption; lncRNA; bta-miR-146a

中图分类号:

S823.8⁺5

王运路, 孟朝轶, 姚一龙, 郭敏, 牛家强, 索朗斯珠, 徐业芬. 牦牛“海绵吸附”bta-miR-146a的lncRNA筛选及靶向验证[J]. 中国畜牧兽医, 2023, 50(3): 859-869.

WANG Yunlu, MENG Zhaoyi, YAO Yilong, GUO Min, NIU Jiaqaing, SUOLANG Sizhu, XU Yefen. Screening and Targeting Verification of lncRNA of "Sponge Adsorption" bta-miR-146a in Yak[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 859-869.

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牦牛“海绵吸附”bta-miR-146a的lncRNA筛选及靶向验证

Screening and Targeting Verification of lncRNA of "Sponge Adsorption" bta-miR-146a in Yak

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