MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells

Abstract

Background: Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear.

Methods: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression.

Results: The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation.

Conclusions: This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Keywords: AGO2; FXR1; LRWD1; miR-320a; testicular cancer.

Figure 1

The effect of miR-320a in regulating LRWD1 expression. (A) The predicted binding sequence of miR-320a on the LRWD1 3′UTR. (B) The effect of the deletion of LRWD1 on regulating endogenous miR-320a expression. (C) The regulatory effect of miR-320a on LRWD1 expression was demonstrated using transfecting cells with the miR-320a mimic or inhibitor by luciferase assay. (D) The effects of the miR-320a mimic and inhibitor on protein expression. (E) Quantitative analysis of LRWD1 protein expression regulated by the miR-320a mimic and inhibitor.

Figure 2

Regulatory effect of miR-320a on NT2D1 cell behaviors. (A) NT2D1 viability was affected by the miR-320a mimic and inhibitor. The treatment groups included the mock, miR-320a mimic, and miR-320a inhibitor groups. (B) Cell migration regulated by the miR-320a mimic/inhibitor and shLRWD1 in the presence or absence of LRWD1 gene expression. (C) The effect of the miR-320a mimic/inhibitor and shLRWD1 in regulating NT2D1 cell invasion ability in the presence or absence of LRWD1 gene expression.

Figure 3

Knockdown of AGO2 and FXR1 was correlated with a reduction in miR-320a-mediated upregulation of LRWD1. The effect of the presence and absence of AGO2 and FXR1 expression in regulating (A) LRWD1 expression by luciferase assay. (B) LRWD1 gene expression by qRT-PCR using AGO2- and FXR1-specific shRNAs (shLuc: negative control; shAGO2: AGO2 shRNA; shFXR1: FXR1 shRNA; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the vector-only control group; Abbreviation: NS, no significance).

Figure 4

Overexpression of AGO2 and FXR1 enhanced the miR-320a-mediated increase in LRWD1 gene expression. (A) The enhancing effect of AGO2 and FXR1 overexpression in promoting LRWD1 expression was investigated by using the pMIR-LRWD1 3′UTR plasmid, miR-320a mimic and FXR1/AGO2 overexpression FLAG-tag plasmid. qRT-PCR results of LRWD1 gene expression regulated by overexpression of (B) FXR1 and (C) AGO2 under treatment with the miR-320a mimic or inhibitor. (D) The potential AGO2 and binding elements gene expression which directly increased miRNA target gene transcription. (^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the vector control or negative control; ^###p < 0.001 compared with the miR-320a mimic-treated group).

Figure 5

Overexpression of AGO2 and FXR1 enhanced the miR-320a-mediated increase in LRWD1 protein expression. Western blotting was performed to determine the protein expression of LRWD1 in cells overexpressing (A) FXR1 and (B) AGO2 in the presence or absence of the miR-320a mimic and inhibitor. Quantitative analysis of LRWD1 protein expression modified by (C) FXR1 and (D) AGO2 overexpression showed the significance of the regulatory effect of the miR-320a mimic and inhibitor (^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the negative control).

Figure 6

LRWD1 and miR-320a expression and their regulatory effect in response to oxidative stress. The gene expression of (A) miR-320a and (B) LRWD1 responding to the oxidative stress OH^- (H₂O₂), NO donor (SNP), and the individual inhibitor treatments. (C) The activity of LRWD1 stimulated by miR-320 mimic and SNP in NT2D1 cells (^***p < 0.001 compared with the vector-only control; ^###p < 0.001 and ^&&&p < 0.001 compared with the indicated groups).

Figure 7

Regulation of miR-320a in the LRWD1 and NRF2 genes and NRF2 protein expression. (A) qRT-PCR results reveal LRWD1 and NRF2 gene expression under stimulation with miR-320a mimic and inhibitor. (B) Western blot images and quantitative results (bottom panel) of NRF2 protein regulated by miR-320a mimic and inhibitor. ^*p < 0.05 compared with individual mock controls, and ^#p < 0.05 compared with individual miR-320a inhibitors.

Figure 8

Schematic diagram of miR-320 targeting human LRWD1 mRNA and influencing biological functions. miR-320a exerts a favorable regulatory effect on the expression of LRWD1 and NRF2 genes and proteins in response to oxidative stress. miR-320a potentially has a significant impact on cell behavior via interacting with the miRNP AGO2/FXR1 complex, eLF4E, and eEF2. These interactions may facilitate binding to LRWD1 and enhance mRNA transcriptional activity. Moreover, there is a positive correlation between miR-320a and LRWD1 and the proliferation of NT2D1 cells. However, they have a detrimental effect on cell migration and invasion. Therefore, under conditions of oxidative stress, elevated levels of miR-320a and LRWD1 promote the proliferation of NT2D1 testicular cancer cells.