Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x
Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x
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下调miR-322通过靶向Ddx3x促进GC-2细胞凋亡
DOI：
10.1186/s12958-019-0506-7
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发表时间：
2019-08-05
影响因子：
4.4
通讯作者：
Zhao, Kai
中科院分区：
文献类型：
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作者：
Che, Qi;Wang, Wei;Zhao, Kai
研究背景生殖细胞DNA异常损伤是导致男性不育的重要因素之一。microRNAs（miRNAs）在精子发生过程中参与多个基因的表达调控。我们之前的研究发现，与生育组相比，具有高DFI（DNA片段化指数）的不育患者精浆中的miR-424（小鼠同源miR-322）水平要低得多。然而，miR-322在精子发生过程中调节生殖细胞的机制仍然未知。方法建立miR-322下调导致精子发生障碍的GC-2细胞模型。并且使用细胞计数试剂盒-8（CCK-8; Dojindo，日本）和MTT（Sigma Aldrich，美国）测量细胞活力。免疫荧光法检测细胞损伤情况，实时荧光定量PCR和Western blot法检测凋亡相关蛋白的表达。通过在线数据库检索和双荧光素酶报告基因检测对靶基因进行预测和验证。结果miR-322抑制后，GC-2细胞存活率沿着下降，凋亡率明显增加。在miR-322下调后，胃癌相关基因Bax和caspase 3、9和8的表达显著增加，而抗凋亡基因Bcl-2的表达降低。发现Ddx 3x是miR-322的直接靶点。然后在具有高DFI（DNA片段化指数）的不育患者的精浆中检测到miR-424;该miRNA在不育组中下调，但Ddx 3x在不育组中上调。结论miR-322通过直接调控Ddx 3x的表达，促进胃癌细胞凋亡。不育男性中MiR-424表达下调可能通过直接作用于靶基因位点Ddx 3x，诱导生精细胞凋亡和精子DNA损伤，导致男性不育。
Background Aberrant DNA damage of germ cells, which impairs spermatogenesis and lowers fertility, is an important factor contributing to male infertility. MicroRNAs (miRNAs) play a significant role in the expression and regulation of multiple genes during spermatogenesis. Our previous study found much lower miR-424 (murine homologue miR-322) levels in the seminal plasma of infertile patients with high DFI(DNA Fragmentation Index)than in the fertile group. However, the mechanism by which miR-322 regulates germ cells during spermatogenesis remains unknown. Methods In this study, we successfully established a GC-2 cell model of miR-322 downregulation resulting in impaired spermatogenesis. And the cell viability were measured using Cell Counting Kit-8 (CCK-8; Dojindo, Japan) and MTT (Sigma Aldrich, USA). Immunofluorescence assay was used to detect cell damage and the expression of apoptosis-related proteins were measured using real-time quantitative PCR and Western blot analysis. Target genes were predicted and verified by online database retrieval and Dual-luciferase reporter gene assay. Results We observed evident decreases in the cell viability of GC-2 cells along with remarkable increases in apoptosis after miR-322 inhibition. While the expression of apoptosis-related genes, including Bax and caspases 3, 9, and 8 greatly increased in GC-2 cells after miR-322 downregulation, that of the anti-apoptotic Bcl-2 gene decreased. Ddx3x was found to be the direct target of miR-322. MiR-424 was then detected in the seminal plasma of infertile patients with high DFI(DNA Fragmentation Index); this miRNA was down-regulated but Ddx3x was upregulated in the infertile group. Conclusion MiR-322 plays a key role in promoting GC-2 cell apoptosis by directly regulating Ddx3x expression. MiR-424 downregulation in infertile men may induce spermatogenic cell apoptosis and sperm DNA damage by directly acting on the target gene locus Ddx3x, resulting in male infertility.