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

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.