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Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x.

ABSTRACT: 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.

SUBMITTER: Che Q

PROVIDER: S-EPMC6683552 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x.

Che Qi Q Wang Wei W Duan Peng P Fang Fang F Liu Chunyan C Zhou Ting T Li Honggang H Xiong Chengling C Zhao Kai K

Reproductive biology and endocrinology : RB&E 20190805 1

<h4>Background</h4>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 ...[more]

PMID: 31382975

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Project description:A microarray analysis of an animal model with experimental sepsis induced by caecal ligation and puncture revealed that the level of microRNA?195 (miR?195) was upregulated. However, to the best of our knowledge, the role of miR?195 in sepsis remains unknown. The present study investigated the effect of miR?195 on apoptosis in sepsis and investigated the underlying mechanism. The level of miR?195 was measured in human intestinal epithelial cells following exposure to lipopolysaccharide (LPS). Cell viability and apoptosis were detected using Cell Counting kit?8 and flow cytometry assays. The expression levels of apoptosis?associated proteins were determined using western blot analysis. In addition, a dual?luciferase reporter assay was employed to verify the association between miR?195 and sirtuin 1 (SIRT1). Furthermore, the SIRT1 inhibitor EX527 was applied to further confirm the regulatory network of miR?195/SIRT1 in LPS?induced apoptosis. It was demonstrated that LPS significantly inhibited cell viability and promoted cell apoptosis in NCM460 cells in a dose?dependent manner. In addition, miR?195 was significantly upregulated following LPS treatment. The present results revealed that silencing miR?195 prevented apoptosis and alleviated cell injury in LPS?induced NCM460 cells. Further investigation demonstrated that miR?195 bound directly to and negatively regulated SIRT1. Inhibition of SIRT1 reversed the protective effects of miR?195?silencing on the apoptosis and viability of NCM460 cells. Furthermore, silencing miR?195 prevented endoplasmic reticulum (ER) stress?induced apoptosis via a downregulation of SIRT1 and its downstream effectors, including activating transcription factor 4, C/EBP homologous protein, glucose?regulated protein 78 and growth arrest and DNA?damage protein 34, as well as the phosphorylation of eukaryotic translation initiation factor 2A. In conclusion, the present study revealed a novel mechanism by which miR?195 regulates SIRT1?mediated downstream effectors in ER stress?induced apoptosis in sepsis.

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Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x.

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Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x.

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