Investigating aberrantly expressed microRNAs in peripheral blood mononuclear cells from patients with treatment‑resistant schizophrenia using miRNA sequencing and integrated bioinformatics

Abstract

Treatment‑resistant schizophrenia (TRS) is a common phenotype of schizophrenia that places a considerable burden on patients as well as on society. TRS is known for its tendency to relapse and uncontrollable nature, with a poor response to antipsychotics other than clozapine. Therefore, it is urgent to identify objective biological markers, so as to guide its treatment and associated clinical work. In the present study, the peripheral blood mononuclear cells (PBMCs) of patients with TRS and a healthy control group, which were gender‑, age‑ and ethnicity‑matched, were subjected to microRNA (miRNA/miR) sequencing to screen out the top three miRNAs with the highest fold change values. These were then validated in the TRS (n=34) and healthy control (n=31) groups by reverse transcription‑quantitative PCR. For two of the top three miRNAs, the PCR results were in accordance with the sequencing result (P<0.01), while the third miRNA exhibited the opposite trend (P<0.01). To elucidate the functions of these two miRNAs, Homo sapiens (hsa)‑miR‑218‑5p and hsa‑miR‑1262 and their regulatory network, target gene prediction was first performed using online TargetScan and Diana‑micro T software. Bioinformatics analysis was then performed using functional enrichment analysis to determine the Gene Ontology terms in the category biological process and the Kyoto Encyclopedia of Genes and Genomes pathways. It was revealed that these target genes were markedly associated with the nervous system and brain function, and it was obvious that the differentially expressed miRNAs most likely participated in the pathogenesis of TRS. A receiver operating characteristic curve was generated to confirm the distinct diagnostic value of these two miRNAs. It was concluded that aberrantly expressed miRNAs in PMBCs may be implicated in the pathogenesis of TRS and may serve as specific peripheral blood‑based biomarkers for the early diagnosis of TRS.

Figure 1.

Heat map of 34 differentially expressed miRNAs between 6 cases of TRS and 6 controls. The columns display miRNA species and the rows individual samples. The color labels of each cell represent the expression level of miRNAs. Red represents upregulation and blue downregulation. Con, control; TRS, treatment-resistant schizophrenia; miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 2.

Reverse transcription-quantitative PCR was performed to verify the expression of miRNAs from peripheral blood mononuclear cells in a large sample (TRS, n=34; control, n=31). miRNA levels are normalized to H-actin. GraphPad Prism 7 was used to plot the data and the Mann-Whitney U-test was performed and analyze significant differences. Data are presented as the mean ± SD. TRS, treatment-resistant schizophrenia; miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 3.

Venn diagram of genes from three miRNA target prediction datasets. Target genes of (A) hsa-miR-218-5p and (B) hsa-miR-1262. miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 4.

miRNA-gene co-expression network. Red represents the miRNA and blue represents the targets. miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 5.

GO annotation and KEGG analysis of target genes of hsa-miR-218-5p and hsa-miR-1262. (A) Top 20 GO terms in the categories BB, CC and MF of the target genes of hsa-miR-218-5p (P<0.05). (B) A total of 14 statistically significantly enriched KEGG pathways were identified (P<0.05). (C) Top 20 GO terms (BB, CC and MF) of the target genes of hsa-miR-1262 (P<0.05). (D) A total of four statistically significantly enriched KEGG pathways are presented (P<0.05). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, Biological Process; CC, Cellular Component; MF, Molecular Function; miRNA/miR, microRNA; hsa, Homo sapiens.

Figure 6.

Diagnostic evaluation of 2 miRNAs. ROC curves of (A) hsa-miR-218-5P and (B) hsa-miR-1262, and (C) their combined ROC curve. ROC, receiver operating characteristic; AUC, area under the curve; miRNA/miR, microRNA; hsa, Homo sapiens.