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

Abstract

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

CLC Number:

S823.8⁺5

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

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