Integrative analysis links autophagy to intrauterine adhesion and establishes autophagy-related circRNA-miRNA-mRNA regulatory network

Abstract

Background: Intrauterine adhesion (IUA) is a troublesome complication characterized with endometrial fibrosis after endometrial trauma. Increasing number of investigations focused on autophagy and non-coding RNA in the pathogenesis of uterine adhesion, but the underlying mechanism needs to be further studied.

Methods: mRNA expression profile and miRNA expression profile were obtained from Gene Expression Omnibus database. The autophagy related genes were low. Venn diagram was used to set the intersection of autophagy genes and DEGs to obtain ARDEGs. Circbank was used to select hub autophagy-related circRNAs based on ARDEMs. Then, the differentially expressed autophagy-related genes, miRNAs and circRNAs were analyzed by functional enrichment analysis, and protein-protein interaction network analysis. Finally, the expression levels of hub circRNAs and hub miRNAs were validated through RT-PCR of clinical intrauterine adhesion samples. In vitro experiments were investigated to explore the effect of hub ARCs on cell autophagy, myofibroblast transformation and collagen deposition.

Results: 11 autophagy-related differentially expressed genes (ARDEGs) and 41 differentially expressed miRNA (ARDEMs) compared between normal tissues and IUA were identified. Subsequently, the autophagy-related miRNA-mRNA network was constructed and hub ARDEMs were selected. Furthermore, the autophagy-related circRNA-miRNA-mRNA network was established. According to the ranking of number of regulated ARDEMs, hsa-circ-0047959, hsa-circ-0032438, hsa-circ-0047301 were regarded as the hub ARCs. In comparison of normal endometrial tissue, all three hub ARCs were upregulated in IUA tissue. All hub ARDEMs were downregulated except has-miR-320c.

Conclusions: In the current study, we firstly constructed autophagy-related circRNA-miRNA-mRNA regulatory network and identified hub ARCs and ARDEMs had not been reported in IUA.

Keywords: autophagy; bioinformatics; circRNA-miRNA-mRNA; intrauterine adhesion; regulatory network.

Figure 1

(A) Identification of 11 ARDEGs between DEGs and autophagy related genes. (B) Heatmaps of ARDEGs based on Log2FC. (C) Interaction between ARDEGs by PPI network (D) Location of ARDEGs on chromosomes.

Figure 2

GO and KEGG analysis of ARDEGs in IUA to reveal the function of ARDEGS. (A) BP aspect; (B) CC aspect; (C) MF aspect; (D) KEGG analysis.

Figure 3

Drug-ARDEGs network including 8 ARDEGs and 47 drugs. Blue circles, potential target drugs; Red circles, upregulated ARDEGs; Blue-green circles, downregulated ARDEGs.

Figure 4

Homology modeling and molecular docking. The crystal structure and evaluation of (A) HIF1A and (B) PQKCQ. The ramachandran plot of (C) HIF1A structure. The ramachandran plot of (D) PQKCQ structure. The LDDT score of (E) HIF1A structure. The LDDT score of (F) PQKCQ structure. The assessment of protein conservation of HIF1A and PQKCQ was exhibited in (G, H) respectively. The best docking position between (I, J) ML228 and HIF1A or (K, L) staurosporine and PRKCQ were indicated. The absolute value of affinity between predicated small molecules and (M) HIF1A or (N) PQKCQ was shown.

Figure 5

(A) Venn diagram of the intersection of DEMs and target miRNAs to obtain 41 ARDEMs. (B) Heatmaps of ARDEMs including 3 upregulated and 38 downregulated ARDEMs. (C) Construction of the autophagy related miRNA-mRNA regulatory network.

Figure 6

(A) Location of ARDEMs on chromosomes. GO and KEGG analysis of hsa-miR-320c, hsa-miR-449a, hsa-miR-449c-5p and hsa-miR-345-5p. (B) BP-terms, (C) CC-terms, (D) MF-terms and (E) KEGG pathways.

Figure 7

(A) Construction of the autophagy related circRNA-miRNA-mRNA regulatory network. (B) Location of ARDECs on chromosomes. GO and KEGG analysis of hsa-circ-0047959, hsa-circ-0032438, hsa-circ-0047301. (C) BP-terms, (D) CC-terms, (E) MF-terms and (F) KEGG pathways.

Figure 8

(A) B-ultrasound examination, hysteroscopy, Hematoxylin-Eosin Staining of intrauterine adhesion and normal uterine cavity. Validation of 3 ARDECs and 4 ARDEGs expression of RT-PCR. Relative expression level of (B) hsa-circ-0047301, (C) hsa-circ-0032438, (D) hsacirc-0047959 were obviously upregulated. Relative expression level of (E) hsa-miR-320c showed no significant statistical differences. Relative expression level of (F) hsa-miR-345-5p, (G) hsa-miR-449a and (H) hsa-miR-449c-5p were downregulated in IUA tissue. Data are presented as mean ± SD. *P < 0.05 compared to the normal tissues.

Figure 9

The siRNA interference efficiency of hsa-circ-0047959 (A), hsa-circ-0032438 (B), hsa-circ-0047301 (C).

Figure 10

The effect of hub ARCs on cell autophagy, myofibroblast transformation and collagen deposition. The mRNA expression of Beclin1 (A) LC3B (B) α-SMA (C) FN1 (D) Col III (E) was detected through RT-PCR. The percentage of α-SMA positive cell was calculated in immunofluorescence results (F). The phosphorylation level of Beclin1 (G) protein expression of LC3B (H) and p62 (I) and release of Col III (J) and FN1 (K) was evaluated through ELISA. The immunofluorescence images of α-SMA in ESCs (L). *p <0.05, compared with Control; **p <0.05, compared with TGF-β+siRNA-NC.