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...
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| Format: | Article |
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BMC
2019-08-01
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| Series: | Reproductive Biology and Endocrinology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12958-019-0506-7 |
| _version_ | 1818279635348094976 |
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| author | Qi Che Wei Wang Peng Duan Fang Fang Chunyan Liu Ting Zhou Honggang Li Chengling Xiong Kai Zhao |
| author_facet | Qi Che Wei Wang Peng Duan Fang Fang Chunyan Liu Ting Zhou Honggang Li Chengling Xiong Kai Zhao |
| author_sort | Qi Che |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T23:36:28Z |
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| id | doaj.art-b0c1260c0a55453ab5a4985f300ee77b |
| institution | Directory Open Access Journal |
| issn | 1477-7827 |
| language | English |
| last_indexed | 2024-12-12T23:36:28Z |
| publishDate | 2019-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Reproductive Biology and Endocrinology |
| spelling | doaj.art-b0c1260c0a55453ab5a4985f300ee77b2022-12-22T00:07:28ZengBMCReproductive Biology and Endocrinology1477-78272019-08-011711910.1186/s12958-019-0506-7Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3xQi Che0Wei Wang1Peng Duan2Fang Fang3Chunyan Liu4Ting Zhou5Honggang Li6Chengling Xiong7Kai Zhao8Family Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyFamily Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and TechnologyAbstract 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.http://link.springer.com/article/10.1186/s12958-019-0506-7miR-322Ddx3xGC-2 cellApoptosis |
| spellingShingle | Qi Che Wei Wang Peng Duan Fang Fang Chunyan Liu Ting Zhou Honggang Li Chengling Xiong Kai Zhao Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x Reproductive Biology and Endocrinology miR-322 Ddx3x GC-2 cell Apoptosis |
| title | Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x |
| title_full | Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x |
| title_fullStr | Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x |
| title_full_unstemmed | Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x |
| title_short | Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x |
| title_sort | downregulation of mir 322 promotes apoptosis of gc 2 cell by targeting ddx3x |
| topic | miR-322 Ddx3x GC-2 cell Apoptosis |
| url | http://link.springer.com/article/10.1186/s12958-019-0506-7 |
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