Image1_Construction of ceRNA network and identification of hub genes in aniridia-associated keratopathy using bioinformatics analysis.JPEG

Aniridia-associated keratopathy (AAK) is characteristic at ocular surface of aniridia caused by haploinsufficiency of PAX6. Competing endogenous RNA (ceRNA) has been reported to play an important role in various diseases, whereas its function on AAK is unclear. The microarray data of 20 AAK patients and 20 healthy people were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed lncRNAs, miRNAs, and mRNAs were analyzed using “limma” packages and weighted gene co-expression network analysis (WGCNA). A ceRNA network was constructed by Cytoscape 3.9.1, and miR-224-5p, miR-30a-5p, and miR-204-5p were at the center of the network. CIBERSORTx algorithm and ssGSEA analyses revealed that AAK was associated with immune cell infiltration, showing that activated Mast cells increased while resting Mast cells decreased and NK cells decreased in AAK. Type II INF Response, CCR, parainflammation, T cell co-stimulation, and APC co-stimulation of AAK patients differed from healthy individuals. Additionally, the ROC curve of five genes, MITF(AUC = 0.988), RHOB(AUC = 0.973), JUN(AUC = 0.953), PLAUR (AUC = 0.925), and ARG2 (AUC = 0.915) with high confidence in predicting AAK were identified. Gene set enrichment analysis (GSEA) analysis of hub genes enriched in the IL-17 signaling pathway.</p

A phylogenic tree of <i>EcR-A and EcR-B</i> genes in insects and other organisms.

<p>A phylogenic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.1 with the sequences obtained from GenBank. The robustness of each cluster was verified in 50,000 replicates. The scale on the x-axis represents the estimated branch lengths and numbers indicate the bootstrap values.</p

The survival rates, developmental duration, food-intake and the changes of SeEcR mRNA and protein in <i>S. exigua</i> after <i>SeEcR</i> RNAi.

<p>(A) The accumulated percentage of survival rates, (B) Developmental duration, (C) Food intake, (D) The relative expression level of <i>SeEcR</i> and (E) The SeEcR protein level of <i>S.exigua</i> larvae in different treatments. For (A, B, C), the following three controls were included: an equivalent amount of <i>GFP</i> dsRNA, the same volume of buffer group (DEPC water) and the CK control group (no treatment). Data are presented as mean ± SE from three independent experiments with 30 fifth-instar larvae in each group. The lowercase letter “a” at each developmental stage in Fig. 3A represents no significant difference among ds<i>GFP</i>, buffer and CK groups but the survival rates in all three control groups are significantly different from that in the ds<i>EcRcom</i>-injection group (labeled with a lowercase “b” at the same developmental stage). Different letters at the same developmental stage in Fig. 3B and Fig. 3C among different treatments indicate significant differences of either developmental duration or food-intake (p<0.05, Duncan multiple comparison test, SPSS). For (D), the expression level of <i>SeEcR</i> and <i>Seβ-actin</i> transcripts were detected using qRT-PCR. By definition, the mRNA level of the target gene was normalized to <i>Seβ-actin</i> in each group; and the relative expression level of the target gene was measured as the level in the ds<i>EcRcom</i>-injection group divided by the level in the ds<i>GFP</i>-injection group. Data are shown as mean ± SE from three independent experiments. An asterisk indicates significant differences in the expression level between the treated and control groups measured at the same time (p<0.05, T test). For (E), the protein levels of target genes were detected using Western blot. A total of 80 ug of proteins from each individual treated with ds<i>GFP</i> or ds<i>EcRcom</i> was applied to each lane and separated by 10% SDS-polyacrylamide gel electrophoresis. Lanes 1 and 3: 36 and 12 hours post-ds<i>GFP</i> injection; Lanes 2 and 4: 36 and 12 hours post- ds<i>EcRcom</i> injection, respectively. The results are representative of four replicates.</p

Lethal phenotypes caused by ds<i>EcRcom</i> injection into fifth-instar larvae.

<p>(A, D, G) Normal prepupa, pupa and adult (B, C) Abnormal prepupae (E, F) Severe misshaped pupae, including typical “half pupation” pupa (E) and “Abnormal eclosion” adults (H, I).</p

Summary of the 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway in <i>S.exigua</i>.

<p>See text for details.</p

The influence of 20E on expression of <i>SeEcR</i> and key genes in the pathway.

<p>The mRNA expression levels of <i>SeEcR</i> and the six chitin biosynthesis pathway genes were detected using qRT-PCR at 4 hr, 12 hr and 36 hr after injection of 20E. Data are represented as mean ± SE from Six independent experiments which were analyzed in the same way as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014058#pone-0014058-g003" target="_blank">Fig. 3</a>. *: significant difference between the ds<i>EcRcom</i> injection group and the ds<i>GFP</i> injection group (p<0.05, T test); NS: no significant difference between the two groups.</p

The effect of injecting <i>EcR</i> dsRNAs on chitin content of different tissues in <i>S. exigua</i> larvae.

<p>Different letters between treatments of ds<i>GFP</i> and ds<i>EcRcom</i> in the same column indicate significant difference in the chitin contents in either the midgut or the epidermis (p<0.05, T test, n = 8).</p

The domain structures and the developmental expression pattern of the two EcR isoforms in <i>S. exigua</i>.

<p>(A) The domain structures of SeEcR-A and SeEcR-B1 and the common fragment used to generate the dsRNAs. The number of amino acids of each domain is indicated. (B) The developmental expression profiles of <i>SeEcR-A</i> and <i>SeEcR-B1</i> mRNA. Total RNAs were prepared from individuals of <i>S. exigua</i> collected at all developmental stages from the first day of eggs to the last day of adults. The levels of the mRNA were detected using qRT-PCR. The mRNA level of β-Acitn was used as a control to normalize for loading. The data represent the mean values ± SE of three replicates. The age of the insects is indicated in: E1, the first day of eggs; I1, the first day of the first instar larvae; pre, prepupal period; P1, the first day of pupae; A1, the first day of adults.</p

Changes in mRNA expression levels for key genes in the pathway following <i>SeEcR</i> RNAi.

<p>The expression levels of six transcripts (<i>SeTre-1, SeTre-2, SeG6PI, SeUAP, SeCHSA</i> and <i>SeCHSB</i>) were detected by qRT-PCR at five detection points after ds<i>EcRcom</i> injection. Data are represented as mean ± SE from three independent experiments, which were analyzed in the same way as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014058#pone-0014058-g003" target="_blank">Fig. 3</a>. *: a significant difference between the ds<i>EcRcom</i> injection group and the ds<i>GFP</i> injection group (p<0.05, T test); NS: no significant difference between the two groups.</p

A brief diagram of insect chitin biosynthesis pathway.

<p>The black italics in parenthesis indicate six genes encoding the enzymes.</p