Table1_Parabolic relationship between SMAD3 expression level and the reprogramming efficiency of goat induced mammary epithelial cells.DOCX

Mammary epithelial cells are the only cells of mammary glands with lactation capacity. They are closely related to mammary development and milk yield. Our earlier studies showed that the transformation of goat fibroblasts into induced mammary epithelial cells (iMECs) was closely correlated with SMAD3 overexpression. Therefore, we further explored the role of SMAD3 on iMECs reprogramming in this study. The SMAD3 gene was overexpressed in goat ear fibroblasts using the tetracycline-induced expression method. The outcomes demonstrated that goat ear fibroblasts can be converted into iMECs by overexpressing the SMAD3 gene. In contrast, it was discovered that SMAD3 downregulation by RNA interference significantly decrease the reprogramming efficiency of iMECs. These results show that SMAD3 plays a key regulatory role in the reprogramming of iMECs. Surprisingly, we also found a parabolic relationship between SMAD3 expression level and iMECs reprogramming efficiency, and that the reprogramming efficiency was maximum when the addition of doxycycline concentration was 5 μg/ml. In light of this, our findings may offer new perspectives on the regulatory mechanism governing mammary epithelial cell fate in goats as well as a fresh approach to studying mammary development and differentiation in vitro.

乳腺上皮细胞是乳腺中唯一具有泌乳能力的细胞。它们与乳腺发育及乳汁产量密切相关。本研究的前期研究表明，山羊成纤维细胞向诱导性乳腺上皮细胞（iMECs）的转变与SMAD3的过表达密切相关。因此，本研究进一步探讨了SMAD3在iMECs重编程中的作用。通过四环素诱导表达法在山羊耳成纤维细胞中过表达SMAD3基因。研究结果表明，通过过表达SMAD3基因，山羊耳成纤维细胞可以被转化为iMECs。相反，通过RNA干扰下调SMAD3的表达显著降低了iMECs的重编程效率。这些结果表明，SMAD3在iMECs重编程中起着关键的调控作用。令人惊讶的是，我们还发现SMAD3的表达水平与iMECs重编程效率之间存在抛物线关系，当添加的强力霉素浓度为5 μg/ml时，重编程效率达到最大。鉴于这一发现，本研究可能为调控山羊乳腺上皮细胞命运的控制机制提供了新的视角，并为在体外研究乳腺发育和分化提供了新的途径。