Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture, Fisheries College, Jimei University, Xiamen 361021, China; Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, China.
Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture, Fisheries College, Jimei University, Xiamen 361021, China; School of Life Sciences, Ningde Normal University, Ningde 352100, China.
Comp Biochem Physiol B Biochem Mol Biol. 2021 Jun-Jul;254:110567. doi: 10.1016/j.cbpb.2021.110567. Epub 2021 Feb 4.
Scylla paramamosain is an economically important cultured crab species in China. Cyclins and cyclin-dependent kinases (CDKs) play important roles in regulations of cell cycle and ovarian development. MiRNAs can negatively regulate gene expression at the post-transcriptional level through base-complementary pairing with the 3'-untranslated region (3-UTR) of the target gene. In this study, bioinformatics prediction showed that miR-9c and miR-263a identified from our group's gonad miRNAome of S. paramamosain may bind to the 3' UTR region of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. Furthermore, the results of double luciferase reporter gene assay showed that the luciferase activities of HEK293T cells co-transfected with miR-9c mimics/miR-9c inhibitor and the 3'-UTR plasmid vectors of the five genes (cyclin A, cyclin B, cyclin H, CDK1, and CDK2) were significantly decreased/increased compared with those in the NC (negative control) and BC (blank control) groups. The results in miR-263a were similar to miR-9c, but all of the six genes could be regulated by miR-263a. In in vivo experiments, agomiR-9c (miR-9c enhancer) injection resulted in decreases of cyclin A and CDK1 expression level, and reverse effects were observed by injecting antagomiR-9c. AgomiR-263a decreased the expression of cyclin A, cyclin B, cyclin H, CDK1, and CDK2, but antagomiR-263a increased their expression. Both the in vitro and in vivo experiments confirmed functions of miR-9c and miR-263a in cell cycle progress of ovarian development by expression regulation of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. The findings provide new insights into the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates.
拟穴青蟹是中国重要的经济养殖蟹种。细胞周期蛋白和细胞周期蛋白依赖性激酶（CDKs）在细胞周期和卵巢发育的调控中发挥重要作用。miRNAs 可以通过与靶基因 3'-非翻译区（3'-UTR）的碱基互补配对，在转录后水平负调控基因表达。在本研究中，生物信息学预测表明，我们课题组从拟穴青蟹性腺 miRNAome 中鉴定出的 miR-9c 和 miR-263a 可能与细胞周期蛋白 A、B、E、H、CDK1 和 CDK2 的 3'UTR 区域结合。此外，双荧光素酶报告基因检测结果显示，与 NC（阴性对照）和 BC（空白对照）组相比，共转染 miR-9c 模拟物/miR-9c 抑制剂和五个基因（细胞周期蛋白 A、B、H、CDK1 和 CDK2）3'-UTR 质粒载体的 HEK293T 细胞的荧光素酶活性显著降低/升高。miR-263a 的结果与 miR-9c 相似，但这六个基因都可以被 miR-263a 调控。在体内实验中，agomiR-9c（miR-9c 增强子）注射导致细胞周期蛋白 A 和 CDK1 表达水平降低，而注射 antagomiR-9c 则观察到相反的效果。agomiR-263a 降低了细胞周期蛋白 A、B、H、CDK1 和 CDK2 的表达，但 antagomiR-263a 增加了它们的表达。体外和体内实验均证实了 miR-9c 和 miR-263a 通过对细胞周期蛋白 A、B、E、H、CDK1 和 CDK2 的表达调控，在卵巢发育的细胞周期进程中发挥作用。这些发现为泥蟹的生殖调控机制提供了新的见解，并进一步丰富了无脊椎动物细胞周期和卵巢发育调控的知识。
