miR-92a-3p和miR-25-3p海绵上调Kiss1并影响雌性小鼠的青春期启动及动情周期

下丘脑Kiss1神经元产生的神经肽kisspeptin通过影响促性腺激素释放激素的分泌，参与青春期的启动、生殖系统的成熟以及排卵等过程的神经内分泌调节。Kiss1基因的表达受到包括多种反式调控因子及表观遗传的调控。预测与前期研究表明，miR-92a-3p、miR-25-3p的种子序列能够与Kiss1的3′-UTR直接结合，抑制Kiss1的表达。为进一步研究miR-92a-3p、miR-25-3p在Kiss1表达调控中的作用，本研究分别构建了对miR-92a-3p、miR-25-3p具有抑制作用的特异性吸附海绵载体：sponge-miR-92a和sponge-miR-25，以实现miRNA的功能缺失。流式细胞术和双荧光素酶报告基因系统分别证实，这2个海绵载体均能够非常有效地吸附外源性或内源性靶miRNA。sponge-miR-92a和sponge-miR-25载体被进一步包装成慢病毒LV-sponge-miR-92a和LV-sponge-miR-25。实时荧光定量PCR结果显示，经LV-sponge-miR-92a和LV-sponge-miR-25感染的下丘脑原代神经元细胞中，Kiss1表达水平均显著上调（P<0.05）；将LV-sponge-miR-92a注射到下丘脑后，雌性小鼠阴门开启时间明显提前（P<0.05）；下丘脑注射LV-sponge-miR-92a和LV-sponge-miR-25扰乱了雌性小鼠的正常动情周期。综上所述，成功构建了能够有效吸附miR-92a-3p、miR-25-3p的海绵载体，证明它们在解除miR-92a-3p、miR-25-3p对Kiss1的抑制中的作用，下丘脑注射海绵对雌性小鼠的阴门开启时间和动情周期均产生一定程度的影响，提示miR-92a-3p、miR-25-3p可能在青春期的启动和生殖成熟过程中发挥重要的作用。

miR-92a-3p and miR-25-3p Sponges Up-regulate Kiss1 and Affect the Onset of Puberty and Estrous Cycle of Female Mice

Kisspeptin, the neuropeptide produced by Kiss1 neurons in the hypothalamus, is involved in the neuroendocrine regulation of puberty initiation, reproductive system maturation, ovulation and other processes by influencing the secretion of gonadotropin-releasing hormone. Kiss1 gene expression is regulated by multiple trans regulatory factors and epigenetics. Prediction and preliminary experiments have shown that the seed sequences of miR-92a-3p and miR-25-3p can directly bind to the 3′-UTR of Kiss1 and inhibit the expression of Kiss1. In order to further study the role of miR-92a-3p and miR-25-3p in the regulation of Kiss1, specific absorptive sponge vectors (sponge-miR-92a and sponge-miR-25) with inhibitory effects on miR-92a-3p and miR-25-3p were constructed to realize the functional loss of miRNA. Flow cytometry and dual luciferase reporter assays both confirmed that both sponge vectors could adsorb exogenous or endogenous target miRNAs very effectively. The sponge-miR-92a and sponge-miR-25 vectors are further packaged into the lentivirus LV-sponge-miR-92a and LV-sponge-miR-25. The results of real-time fluorescence quantitative PCR showed that the expression level of Kiss1 in the hypothalamic primary neurons infected by LV-sponge-miR-92a and LV-sponge-miR-25 was significantly up-regulated (P<0.05). After injecting LV-sponge-miR-92a into the hypothalamus, the time of female mouse vulva opening was significantly earlier (P<0.05). The normal oestrus cycle of female mice with was disrupted by injections of LV-sponge--miR-92a and LV-sponge-miR-25 in the hypothalamus. In conclusion, we successfully constructed sponge vectors capable of effectively adsorbingmiR-92a-3pand miR-25-3p, and demonstrated their role in removing the inhibition ofmiR-92a-3pand miR-25-3p on Kiss1. Hypothalamic sponge injection had a certain effect on both the time of vulva opening and the estrus cycle of female mice, suggesting thatmiR-92a-3pand miR-25-3p may play an important role in the initiation of puberty and reproductive maturity.