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

Identification Analysis and Construction of Expression Vector of Estrogen-induced lncRNA in Thymic Epithelial Cells

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D450 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。

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 D450 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.