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

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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.

Electronic supplementary material

The online version of this article (10.1186/s12958-019-0506-7) contains supplementary material, which is available to authorized users.

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Most cited references 26

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Roles for microRNAs in conferring robustness to biological processes.

Margaret S. Ebert, Phillip A. Sharp (2012)

Biological systems use a variety of mechanisms to maintain their functions in the face of environmental and genetic perturbations. Increasing evidence suggests that, among their roles as posttranscriptional repressors of gene expression, microRNAs (miRNAs) help to confer robustness to biological processes by reinforcing transcriptional programs and attenuating aberrant transcripts, and they may in some network contexts help suppress random fluctuations in transcript copy number. These activities have important consequences for normal development and physiology, disease, and evolution. Here, we will discuss examples and principles of miRNAs that contribute to robustness in animal systems. Copyright © 2012 Elsevier Inc. All rights reserved.

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The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.

Parviz Gharagozloo, R. Alan Aitken (2011)

Oxidative stress in the male germ line is thought to affect male fertility and impact upon normal embryonic development. Accordingly, fertility specialists are actively exploring the diagnosis of such stress in spermatozoa and evaluating the possible use of antioxidants to ameliorate this condition. In this review, evidence for the presence of oxidative stress in human spermatozoa, the origins of this phenomenon, its clinical significance in the aetiology of male infertility and recent advances in methods for its diagnosis and treatment are re-examined. Moreover, an extensive review of the results presented in published clinical studies has been conducted to evaluate the overall impact of oral antioxidants on measures of sperm oxidative stress and DNA damage. Administration of antioxidants to infertile men has been assessed in numerous clinical studies with at least 20 reports highlighting its effect on measures of oxidative stress in human spermatozoa. A qualitative but detailed review of the results revealed that 19 of the 20 studies conclusively showed a significant reduction relating to some measure of oxidative stress in these cells. Strong evidence also supports improved motility, particularly in asthenospermic patients. However, of these studies, only 10 reported pregnancy-related outcomes, with 6 reporting positive associations. Adequately powered, placebo-controlled comprehensive clinical trials are now required to establish a clear role for antioxidants in the prevention of oxidative stress in the male germ line, such that the clinical utility of this form of therapy becomes established once and for all.

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Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

Guus M Bol, Farhad Vesuna, Min Xie … (2015)

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3–β-catenin axis and inhibited non-homologous end joining—the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

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Author and article information

Contributors

Kai Zhao: zk443@163.com

Journal

Journal ID (nlm-ta): Reprod Biol Endocrinol

Journal ID (iso-abbrev): Reprod. Biol. Endocrinol

Title: Reproductive Biology and Endocrinology : RB&E

Publisher: BioMed Central (London )

ISSN (Electronic): 1477-7827

Publication date (Electronic): 5 August 2019

Publication date PMC-release: 5 August 2019

Publication date Collection: 2019

Volume: 17

Electronic Location Identifier: 63

Affiliations

[1 ]ISNI 0000 0004 0368 7223, GRID grid.33199.31, Family Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College, , Huazhong University of Science and Technology, ; Wuhan, 430030 China

[2 ]Wuhan Tongji Reproductive Medicine Hospital, Wuhan, 430014 China

Article

Publisher ID: 506

DOI: 10.1186/s12958-019-0506-7

PMC ID: 6683552

PubMed ID: 31382975

SO-VID: ec8a82fb-3aaf-414c-aea1-06071d280368

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 3 March 2019

Date accepted : 21 July 2019

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;

Award ID: 81571496

Award Recipient : Kai Zhao

Funded by: National Natural Science Foundation of China

Award ID: 81701539

Award Recipient : Kai Zhao

Custom metadata

ScienceOpen disciplines: Human biology

Keywords: mir-322,ddx3x,gc-2 cell,apoptosis

Data availability:

ScienceOpen disciplines: Human biology

Keywords: mir-322, ddx3x, gc-2 cell, apoptosis

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

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Abstract

Background

Methods

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Conclusion

Electronic supplementary material

Related collections

Genome Engineering using CRISPR

Most cited references 26

Roles for microRNAs in conferring robustness to biological processes.

The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy

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Cited by 13

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