鸡circSFMBT2克隆及对DF-1细胞增殖的影响

焦振海; 王府建; 张丽; 李国; 宾乘锋; 张为露; 郭东雪; 王晓彤; 林树带

doi:10.7671/j.issn.1001-411X.202209022

鸡circSFMBT2克隆及对DF-1细胞增殖的影响

Cloning of chicken circSFMBT2 and its effect on proliferation of DF-1 cells

摘要

摘要:
目的验证鸡circSFMBT2的环形结构，探究其表达规律及功能。
方法以麒麟鸡和DF-1细胞为研究对象，根据反向剪切位点序列特征设计引物，通过PCR和测序验证circSFMBT2的环形结构。通过RNase R和反转录试验分析circSFMBT2的基本特性，利用qRT-PCR探究不同组织、不同时期circSFMBT2的表达水平。通过构建过表达载体，结合qRT-PCR、Edu和CCK-8等试验，探究circSFMBT2对鸡DF-1细胞增殖的影响。
结果鸡circSFMBT2全长827 nt，由SFMBT2基因外显子12~18环化形成。RNase R耐受性试验表明，circSFMBT2不易被RNase R降解，随机引物对circSFMBT2的反转录效率是Oligo-d(T)₁₈引物的8倍。组织表达谱表明，circSFMBT2在麒麟鸡14胚龄以及1周龄的肝脏和脾脏中高表达、在胸肌和腿肌中低表达。circSFMBT2在胸肌和腿肌的时序表达谱表明，circSFMBT2在胚胎时期表达量较高，出生后表达量迅速下降。在DF-1细胞中circSFMBT2过表达48 h后，增殖标记基因PCNA和CCND1的表达量分别较对照组上调85%和92%。Edu和CCK-8细胞增殖试验表明，鸡circSFMBT2可以促进细胞增殖进程。
结论 circSFMBT2是鸡SFMBT2基因的一个环状转录本，可以促进DF-1细胞的增殖，研究结果为深入探究鸡circSFMBT2的生物学功能和作用机制奠定了基础。

Abstract:
Objective In order to verify the circular structure of chicken circSFMBT2 and explore its expression and functions.
Method Kirin chicken and DF-1 cells were used in this study. Primers were designed according to the reverse splicing site, and the circular structure of circSFMBT2 was verified by PCR and sequencing. The characteristics of circSFMBT2 were analyzed by RNase R and reverse transcription experiments. The expression levels of circSFMBT2 in different tissues and periods were explored by qRT-PCR. The effect of circSFMBT2 on proliferation of chicken DF-1 cells was investigated by qRT-PCR, Edu and CCK-8 methods.
Result The full length of chicken circSFMBT2 was 827 nt and it was formed by cyclization of exons 12−18 of SFMBT2 gene. RNase R tolerance experiments indicated that circSFMBT2 was not easily degraded by RNase R. The reverse transcription efficiency of random primers on circSFMBT2 was eight times of that of oligo d(T)₁₈ primer. The tissue expression profile exhibited that circSFMBT2 expression was high in liver and spleen of Kirin chickens at the age of 14 embryonic and one week old, and low in the pectoral and leg muscles. The temporal expression profile of circSFMBT2 in the pectoral and leg muscles indicated that circSFMBT2 expression was high in embryonic period and decreased rapidly after birth. When circSFMBT2 was overexpressed in DF-1 cells for 48 h, the expressions of PCNA and CCND1 were up-regulated by 85% and 92% respectively compared with control group. The Edu and CCK-8 cell proliferation tests showed that circSFMBT2 promoted the cell proliferation process.
Conclusion This study confirms the existence of circSFMBT2 as a circular transcript of chicken SFMBT2 and circSFMBT2 can promote DF-1 cell proliferation, which lays a foundation for further exploring the biological function and working mechanism of circSFMBT2 in chicken.

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