胸腺上皮细胞中雌激素诱导lncRNA的鉴定分析及表达载体构建

doi:10.16431/j.cnki.1671-7236.2021.01.007

摘要/Abstract

摘要： 为研究胸腺上皮细胞（thymic epithelial cells，TECs）中雌激素（estradiol，E₂）对长链非编码RNA（long non-coding RNA，lncRNA）表达的调节作用，本研究首先培养小鼠胸腺髓质上皮细胞系1（medullary thymic epithelial cell line 1，MTEC1），经50 nmol/L E₂作用24 h后，观察对细胞表型变化的影响，并用CCK-8试剂盒检测细胞活力；提取细胞总RNA，运用实时荧光定量PCR技术验证E₂对lncRNA-2410006H16Rik表达的调节作用；最后运用RT-PCR技术扩增其目的基因，构建pEGFP-N1-lncRNA-2410006H16Rik重组过表达载体。结果显示，50 nmol/L E₂能够明显抑制MTEC1的增殖，且相较于对照组细胞，50 nmol/L E₂处理组细胞的D_{450 nm}值极显著降低（P<0.01），表明其细胞活力极显著下降。实时荧光定量PCR结果显示，在E₂作用下，lncRNA-2410006H16Rik在MTEC1细胞中的表达极显著上调（P<0.01），约是对照组的2倍，与高通量测序结果一致。经RT-PCR、双酶切及测序结果分析显示，试验成功构建pEGFP-N1-lncRNA-2410006H16Rik表达载体。结果表明，TECs中lncRNA-2410006H16Rik的表达与E₂作用密切相关，为后续在细胞水平上进一步验证lncRNA-2410006H16Rik的调节功能奠定了基础。

关键词: 长链非编码RNA (lncRNA); lncRNA-2410006H16Rik; 雌激素(E₂); 胸腺髓质上皮细胞系1(MTEC1); 表达载体

Abstract: To study the regulation role of estradiol (E₂) on long non-coding RNA (lncRNA) expression in thymic epithelial cells (TEC).The cell phenotypic changes were observed after the 50 nmol/L E₂ was added in cultured mouse epithelial cell line 1 (MTEC1),while the cell viability was also measured with CCK-8 kit.Furthermore,total RNA was extracted and the expression regulatory role of E₂ on lncRNA-2410006H16Rik was verified by Real-time quantitative PCR.Moreover,the target gene was amplified by RT-PCR to construct a recombinant vector of pEGFP-N1-lncRNA-2410006H16Rik.The results showed the proliferation of MTEC1 cells was significantly inhibited by 50 nmol/L E₂,and the D_{450 nm} value was also extremely significantly reduced as compared with control group detected by CCK-8 (P<0.01),indicating that the cell viability was extremely significantly decreased by E₂.Real-time quantitative PCR results indicated that the expression of lncRNA-2410006H16Rik in MTEC1 cells was extremely significantly up-regulated by 50 nmol/L E₂ (P<0.01),and about two fold-changes higher than control group cells,which was consistent with the high-throughput sequencing results.RT-PCR,double enzyme digestion and sequencing results showed that the pEGFP-N1-lncRNA-2410006H16Rik expression vector was successfully constructed.All the above results indicated that the expression of lncRNA-2410006H16Rik in TECs was closely related to the effect of E₂,which laid a foundation for further verification of the regulatory function of lncRNA-2410006H16Rik at the cellular level.

Key words: lncRNA; lncRNA-2410006H16Rik; E₂; MTEC1; expression vector

中图分类号:

Q291

郭东光, 陈明艳, 崔芳微, 李文明, 郭赞莹, 朱艳平, 岳锋, 王选年. 胸腺上皮细胞中雌激素诱导lncRNA的鉴定分析及表达载体构建[J]. 中国畜牧兽医, 2021, 48(1): 64-71.

GUO Dongguang, CHEN Mingyan, CUI Fangwei, LI Wenming, GUO Zanying, ZHU Yanping, YUE Feng, WANG Xuannian. Identification Analysis and Construction of Expression Vector of Estrogen-induced lncRNA in Thymic Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 64-71.

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