Integrative transcriptomic and proteomic analysis reveals that SERPING1 inhibits neuronal proliferation via the CaMKII-CREB-BDNF pathway in schizophrenia

Abstract

Background: Schizophrenia (SZ), a chronic and widespread brain disorder, presents with complex etiology and pathogenesis that remain inadequately understood. Despite the absence of a universally recognized endophenotype, peripheral blood mononuclear cells (PBMCs) serve as a robust model for investigating intracellular alterations linked to SZ.

Aim: To preliminarily investigate potential pathogenic mechanisms and identify novel biomarkers for SZ.

Methods: PBMCs from SZ patients were subjected to integrative transcriptomic and proteomic analyses to uncover differentially expressed genes (DEGs) and differentially expressed proteins while mapping putative disease-associated signaling pathways. Key findings were validated using western blot (WB) and real-time fluorescence quantitative PCR (RT-qPCR). RNAi-lentivirus was employed to transfect rat hippocampal CA1 neurons in vitro, with subsequent verification of target gene expression via RT-qPCR. The levels of neuronal conduction proteins, including calmodulin-dependent protein kinase II (caMKII), CREB, and BDNF, were assessed through WB. Apoptosis was quantified by flow cytometry, while cell proliferation and viability were evaluated using the Cell Counting Kit-8 assay.

Results: The integration of transcriptomic and proteomic analyses identified 6079 co-expressed genes, among which 25 DEGs were significantly altered between the SZ group and healthy controls. Notably, haptoglobin (HP), lactotransferrin (LTF), and SERPING1 exhibited marked upregulation. KEGG pathway enrichment analysis implicated neuroactive ligand-receptor interaction pathways in disease pathogenesis. Clinical sample validation demonstrated elevated protein and mRNA levels of HP, LTF, and SERPING1 in the SZ group compared to controls. WB analysis of all clinical samples further corroborated the significant upregulation of SERPING1. In hippocampal CA1 neurons transfected with lentivirus, reduced SERPING1 expression was accompanied by increased levels of CaMKII, CREB, and BDNF, enhanced cell viability, and reduced apoptosis.

Conclusion: SERPING1 may suppress neural cell proliferation in SZ patients via modulation of the CaMKII-CREB-BDNF signaling pathway.

Keywords: Pathogenesis; Proteomics; SERPING1; Schizophrenia; Transcriptomics.

Figure 1

Analysis of transcriptomics sequencing results. A: Volcano plot illustrating the distribution of differentially expressed genes between the schizophrenia (SZ) and Healthy controls (HC) groups; B: Cluster heatmap of differentially expressed genes in the SZ and HC groups.

Figure 2

Analysis of quantitative results of data-independent acquisition proteomics. A: Histogram summarizing data-independent acquisition identification statistics for the schizophrenia (SZ) and healthy controls (HC) groups; B: Histogram of quantitative differences in protein expression between the SZ and HC groups; C: Volcano plot depicting quantitative protein differences between the SZ and HC groups; D: Cluster heatmap of differentially expressed proteins between the SZ and HC groups.

Figure 3

Integrated transcriptomics and proteomics analysis. A: Venn diagram showing overlapping transcriptomic and proteomic associations at the quantitative and differential expression levels (DE_Protein numbers represent genes corresponding to proteins); B: Correlation analysis (Spearman) between significantly differentially expressed genes and proteins; C: Cluster heatmap of transcriptomic and proteomic expression patterns for associated molecules; D: KEGG enrichment analysis of differentially expressed genes.

Figure 4

Expression of haptoglobin, lactotransferrin, and SERPING1 (n = 3). A and B: Detected by western blot, Immunoblotting image (A) and statistical bar chart of relative protein expression (B); C: Analyzed by real-time fluorescence quantitative PCR. ^aP < 0.001; HP: Haptoglobin; LTF: Lactotransferrin.

Figure 5

Expression of SERPING1 was detected by western blot (n = 150, including three samples of the results shown in Figure 4A; Here only a subset' western blot images are shown in Figure 5). A: Immunoblot image [schizophrenia (SZ) group samples numbered 1-150, healthy controls (HC) group samples numbered 301-450; representative western blot images are displayed]; B: Statistical bar chart of relative protein expression comparing SZ and HC groups; C: Statistical bar chart of relative protein expression across SZ subtypes. ^aP < 0.001, NS: Not significant.

Figure 6

Analysis of SERPING1 expression in si-SERPING1 Lentiviral infection status by real-time fluorescence quantitative PCR (n = 3). ^aP < 0.01; ^bP < 0.001; NC: Negative control.

Figure 7

Analysis of calmodulin-dependent protein kinase II, cAMP response element-binding protein, and brain-derived neurotrophic factor expression in lentiviral infection status by western blot (n = 3). A: Immunoblotting image; B: Statistical bar chart of relative protein expression. ^aP < 0.001; NC: Negative control.

Figure 8

Apoptosis of rat neuronal cells which were treated with si-SERPING1 Lentiviral was detected by flow cytometry (n = 3). A: Flow cytometry scatter plot; B: Statistical bar chart of apoptosis rate. ^aP < 0.001; NC: Negative control.

Figure 9

The cell viability was analyzed by Cell Counting Kit-8 in rat neuronal cells treated with si-SERPING1 lentiviral (n = 3). ^aP < 0.001. NC: Negative control.