Identification of ITPR1 gene as a novel target for hsa-miR-34b-5p in non-obstructive azoospermia: a Ca2+/apoptosis pathway cross-talk

Abstract

MiR-34b-5p has been reported as a non-invasive diagnostic biomarker for infertility. However, no gene targets regulating the mechanism of cation of this miRNA are known. In this study, using gene set enrichment analysis the Inositol 1,4,5-Trisphosphate Receptor Type 1 (ITPR1) gene was identified as the sole target for hsa-miR-34b-5p, and found significantly overexpressed in non-obstructive azoospermia (NOA) patients. This finding was confirmed by qRT-PCR on fresh testicular tissues from NOA patients. Then, pathway enrichment analysis as well as the diagnostic value analysis of hsa-miR-34b-5p/ITPR1 indicated ITPR1 as a hub gene in the calcium (Ca²⁺)-apoptosis pathway, and a valuable predictive biomarker for NOA. Moreover, gene expression and histological assays showed the association of the effects of ITPR1's increased expression on spermatogenesis failure through induction of apoptosis in NOA patients. These data suggested that the hsa-miR-34b-5p/ITPR1 axis could serve as a potential regulatory predictive biomarker for human spermatogenesis through the Ca²⁺-apoptosis pathway cross-talk.

Figure 1

The relative expression level of hsa-miR-34b-5p, Bcl-2, Bax, and ITPR1 in NOA and OA patients. The expression of hsa-miR-34b-5p, Bcl-2, Bax, and ITPR1 was investigated in NOA (n = 45) compared to OA (n = 18) patients using qRT-PCR. The expression results were shown as fold change mean, considering OA samples as control (Ctrl) for all the genes tested. The data were depicted as the standard error of the mean with SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2

Principal component analysis (PCA) plot. PCA plots illustrate the clustering pattern of normalized samples before (a) and after (b) batch effect adjustment. The datasets and NOA and OA samples were shown.

Figure 3

Volcano plot of differentially expressed genes (DEGs). The cut-off for screening the statistical significance DEGs was considered P-value < 0.01, adj.P-value < e−10 and |logFC| > 1. The red dot points present more significant genes such as the ITPR1 gene with logFC: 1.3, P-value: 3.37E−12, and adj. P-value: 3.12E−11 as statistical significance.

Figure 4

Protein–protein interaction (PPI) network and heatmap of DEGs. (a) Protein–protein interaction (PPI) network of azoospermia dysregulated genes associated with Ca²⁺/apoptosis signaling pathway based on KEGG biological pathways. The protein–protein association network was retrieved from the STRING enrichment web service (https://string-db.org/). (b) Heatmaps of four differentially expressed genes (CALML3, ITPKA, ITPR1, and PRKACG) between NOA and OA samples. Yellow and purple represent high and low expressions, respectively.

Figure 5

Histological feature of the human testicular tissues. (a–c) H&E stained testis section from adult OA and NOA. (a) OA testicle (Control). (b) NOA testicle. (c) NC. Magnification: × 40. H&E Hematoxylin and eosin, NOA non-obstructive azoospermia, OA obstructive azoospermia, NC negative control. Arrows: Irregularity in NOA patients’ seminiferous. (d–f) TUNEL-stained sections of the human testicular tissue for the detection of apoptosis. (d) OA, control group. A few TUNEL-stained nuclei were observed in the OA (Arrowheads). (e) In the NOA, the increase in the apoptotic cells was observed compared to the OA group (Arrows). (f) NC negative control. Green and Red fluorescent stained nuclei indicate apoptotic and viable cells, respectively. Magnification, × 40. TUNEL deoxy-UTP-digoxigenin nick end labeling, NC negative control. (g) Quantification of staining intensity reveals 2.7-fold increases in apoptosis rate in NOA compared with OA (control group).

Figure 6

Multiple comparisons of correlated areas under the ROC curves (AUC) for DEGs and studied markers. ROC curves were performed using an easyROC web-tool-based R language environment (http://www.biosoft.hacettepe.edu.tr/easyROC/) to assess the predictive accuracy of studied markers for male infertility. The AUC values were ≥ 0.9 demonstrating the predictive power of the identified markers.

Figure 7

A schematic diagram for the function and mechanisms of the hsa-miR-34b-5p/ITPR1 axis in male infertility in human. The Figure depicts hsa-miR-34b-5p acts as a silencer on 3ʹ UTR of ITPR1 expression. Therefore, down-regulation of hsa-miR-34b-5p will follow overexpression of ITPR1 mRNA in human testes. Subsequently, Ca²⁺ influx occurs from ER to cytoplasm in spermatogenic cells in the teste. Finally, this influx creates a cross-talk between Ca²⁺ and apoptosis pathways through unknown upstream factors.