Study On Expression Profiling Of CircHIPK3 In Mammary Gland Epithelial Cells And Its Related Function

Circular RNA is a special class of endogenous non-coding RNA molecules and is another class of RNA molecules with great research value after miRNA and lncRNA.In this study,high throughput sequencing results of bovine mammary epithelial cells treated by prolactin were analyzed,preliminary screening of circular RNA with differential expression,verifying its circular structure in the mammary epithelial cells of dairy cows.After preliminary screening,circHIPK3 is selected as the research object,explore the effect of circHIPK3 on the proliferation and differentiation of mammary epithelial cells.STAT5 inhibitor experiments revealed the relationship between circHIPK3 and the STAT5 pathway induced by prolactin and further explore the effect of STAT5 inhibitor treatment on the expression of circHIPK3 and the proliferation of mammary epithelial cells.Finally,the effects of alternative splicing influence factor SRPK1,SRPK2,ADAR,and ILF3 related genes on the formation of circ HIPK3 were explored.The main results of this study are as follows:(1)Two circular RNA libraries of dairy cow mammary epithelial cells under prolactin treatment were successfully constructed.16364 circRNA were identified by findcirc.The number of circRNA that was significantly up-regulated in two different states was 6760,and the number of circRNA with significant downregulation was 5991.It shows that prolactin has an effect on the expression of circRNA.(2)This experiment has successfully verified 10 circular RNA that existence in mammary gland epithelial cells of dairy cows,indicating that circular RNA is abundant in dairy cow mammary epithelial cells.(3)CircHIPK3 was expressed in different tissues(heart,liver,spleen,lung,kidney,stomach,intestine and breast)of cows and C57BL/6J mice and shows that circHIPK3 were highly conserved among different species.(4)The circHIPK3 promoted the proliferation of HC11,inhibited the proliferation of 4T1 cells,and had no effect on the differentiation of HC11.(5)The expression level of circHIPK3 was affected by prolactin and its induced STAT5 signaling pathway.Increased prolactin significantly increased the expression of circHIPK3,and STAT5 inhibitor treatment significantly inhibited the expression of circHIPK3.STAT5 inhibitor treatment also inhibits the proliferation of mammary epithelial cells.(6)Prolactin can alter the expression of alternative splicing factors such as SRPK1,SRPK2,ADAR and ILF3 through the STAT5 signaling pathway,thereby indirectly affecting the formation of circHIPK3.