Estrogen-induced circFAM171A1 regulates sheep myoblast proliferation through the oar-miR-485-5p/MAPK15/MAPK pathway

Estrogen is an important hormone that affects muscle development in female animals. Previous studies have shown that estrogen can protect muscle cells from apoptosis by inhibiting the MAPK signaling pathway. However, the molecular mechanisms by which estrogen-induced MAPK signaling regulates myoblast growth and development remain unclear. In this study, RNA-seq was performed on ovariectomized small-tailed Han (OR-STH) sheep and sham surgery small-tailed Han (STH) sheep to analyze the effects of estrogen on muscle growth and development in female animals. There were 8,721 differentially expressed circRNAs (DECs), 143 differentially expressed miRNAs (DEMs) and 2,238 differentially expressed mRNAs (DEGs) in the longissimus dorsi between the OR-STH and STH groups. Bioinformatics analysis revealed that the differentially expressed gene MAPK15 was significantly enriched in the MAPK signaling pathway, which is important for muscle development. Therefore, we constructed the ceRNA network circFAM171A1/oar-miR-485-5p/MAPK15 and explored its effect on muscle growth and development. The results of the molecular mechanism experiments indicated that circFAM171A1 can sponge oar-miR-485-5p to regulate MAPK15. The addition of the exogenous hormone estradiol (E2) to sheep myoblasts could induce circFAM171A1, regulate the expression of oar-miR-485-5p and MAPK15, and promote the proliferation of sheep myoblasts. The results showed that MAPK15 and circFAM171A1 significantly promoted the proliferation of myoblasts and inhibited the apoptosis of myoblasts in sheep, whereas oar-miR-485-5p inhibited the expression of MAPK15 and circFAM171A1, inhibited myoblast proliferation and promoted apoptosis. Furthermore, circFAM171A1 attenuated the inhibitory effect of oar-miR-485-5p on myoblasts. In summary, estrogen induced the expression of circFAM171A1 in sheep myoblasts, and circFAM171A1 can act as a sponge for oar-miR-485-5p to promote the expression of the target gene MAPK15 and ultimately regulate the proliferation of sheep myoblasts. This study provides new insights into the molecular mechanism of estrogen regulation of muscle growth and development in female animals. Overall design: RNA-seq was performed on ovariectomized small-tailed Han (OR-STH) sheep and sham surgery small-tailed Han (STH) sheep to analyze the effects of estrogen on muscle growth and development in female animals.

雌激素是调控雌性动物肌肉发育的关键激素。既往研究表明，雌激素可通过抑制丝裂原活化蛋白激酶（MAPK）信号通路，保护肌细胞免于凋亡。然而，雌激素诱导的MAPK信号通路调控成肌细胞生长发育的分子机制仍未阐明。本研究对去卵巢小尾寒羊（OR-STH）与假手术小尾寒羊（STH）开展RNA测序（RNA-seq），以分析雌激素对雌性动物肌肉生长发育的影响。两组动物的背最长肌组织中，共鉴定出8721个差异表达环状RNA（DECs）、143个差异表达微小RNA（DEMs）及2238个差异表达信使RNA（DEGs）。生物信息学分析显示，差异表达基因MAPK15显著富集于与肌肉发育密切相关的MAPK信号通路中。据此，本研究构建了内源竞争RNA（competing endogenous RNA, ceRNA）网络circFAM171A1/oar-miR-485-5p/MAPK15，并探究其对肌肉生长发育的调控作用。分子机制实验结果表明，circFAM171A1可作为分子海绵吸附oar-miR-485-5p，从而调控MAPK15的表达。向绵羊成肌细胞中添加外源激素雌二醇（E2），可诱导circFAM171A1的表达，调控oar-miR-485-5p与MAPK15的表达水平，并促进绵羊成肌细胞的增殖。实验结果显示，MAPK15与circFAM171A1可显著促进绵羊成肌细胞增殖并抑制其凋亡；而oar-miR-485-5p则可抑制MAPK15与circFAM171A1的表达，阻碍成肌细胞增殖并促进其凋亡。此外，circFAM171A1可削弱oar-miR-485-5p对绵羊成肌细胞的抑制作用。综上，雌激素可诱导绵羊成肌细胞中circFAM171A1的表达，而circFAM171A1可作为oar-miR-485-5p的分子海绵，促进靶基因MAPK15的表达，最终调控绵羊成肌细胞的增殖。本研究为阐明雌激素调控雌性动物肌肉生长发育的分子机制提供了新的视角。
实验整体设计：本研究对去卵巢小尾寒羊（OR-STH）与假手术小尾寒羊（STH）开展RNA测序，以分析雌激素对雌性动物肌肉生长发育的影响。