ZC3H12D gene expression exhibits dual effects on the development and progression of lung adenocarcinoma

Abstract

While precision oncology requires robust biomarkers, current predictors for lung adenocarcinoma (LUAD) often show limited clinical utility. This study investigates the multifaceted roles of ZC3H12D, a novel immunomodulatory molecule, in LUAD progression and tumor microenvironment regulation. Multi-omics analyses integrated ZC3H12D transcriptomic (511 tumors vs 59 normals), proteomic (74 tumors vs 69 normals), and single-cell RNA-seq data (15 tumors vs 11 normals). Immunohistochemistry validated ZC3H12D expression in 51 matched pairs. Computational biology approaches assessed immune infiltration, genomic instability (TMB/MSI/HRD), and pathway enrichment. Functional validation employed ZC3H12D knockdown in PC9 cells with colony formation and transwell assays. Multi-omics verification confirmed ZC3H12D upregulation in LUAD at both mRNA and protein levels (p < 0.001), with single-cell resolution revealing predominant localization in tumor-infiltrating immune cells. Moreover, ZC3H12D expression positively correlated with immune regulatory genes while inversely associating with genes involved in cellular respiration. Its expression was also linked to clinical markers such as TMB, MSI, HRD, tumor purity, and ploidy. Notably, high ZC3H12D expression revealed Immune-infiltrated microenvironment and favorable prognosis, despite silencing ZC3H12D resulted in significant inhibition of tumor cell proliferation and invasion in vitro (p < 0.001). Our findings demonstrate that high ZC3H12D expression in immune cells appears to enhance antitumor immune activity, whereas lower expression in malignant cells contributes to reduced cellular proliferation and migration. This spatial duality challenges conventional biomarker paradigms and provides mechanistic insights for developing cell type-targeted therapies.

Keywords: ZC3H12D; Cell migration; Cell proliferation; Dual effects; Lung cancer.

Fig. 1

ZC3H12D expression levels in lung tumor tissues and paired normal tissues. A), Bodymap illustrating ZC3H12D expression enrichment. Red denotes cancer patients, green denotes normal subjects, and the shading indicates expression levels; B), Differences in ZC3H12D expression between lung tumor and normal tissues. mRNA level differences were derived from LUAD RNAseq data in the TCGA database; C), protein level differences were assessed through quantitative immunohistochemistry (IHC) analysis of 51 matched lung adenocarcinoma tumor and adjacent normal tissues; D), Representative pathological staining and IHC results are presented; E), Proteomics differences of ZC3H12D between lung tumor and normal tissues, derived from CPTAC database; F), t‑SNE plot of LUAD single cell transcriptomic data, illustrating distinct cellular clusters and the differential expression of ZC3H12D across various cell types. **** indicates p < 0.00005.

Fig. 2

Gene sets highly correlated with ZC3H12D expression and their functional analysis. A), Heatmap of the top 10 positively and negatively correlated genes with ZC3H12D expression; B), GO functional enrichment analysis of the top 100 genes most correlated with ZC3H12D expression; C), KEGG pathway enrichment analysis of the top 100 genes most correlated with ZC3H12D expression.

Fig. 3

Correlation analysis of ZC3H12D expression and immune function. A), Heatmap of immune regulatory genes significantly correlated with ZC3H12D expression; B), Heatmap of immune checkpoint genes significantly correlated with ZC3H12D expression; C), Correlation between ZC3H12D expression levels and immune infiltration scores in LUAD patients, including Stromal score, Immune score, ESTIMATE score, and Microenvironment score.

Fig. 4

Correlation analysis between ZC3H12D expression and the genomic mutation landscape. A), Mutation landscape of 24 genes with mutation frequencies greater than 10%, stratified by ZC3H12D expression levels. The numbers in parentheses next to gene names indicate the p-values for group differences; B), GO functional enrichment analysis of the 24 high-frequency mutated genes; C), KEGG pathway enrichment analysis of the 24 high-frequency mutated genes; D), Functional network diagram of the 24 high-frequency mutated genes. The size of each circular node is proportional to the number of genes, and the color indicates cluster identity. Similarity terms with a kappa score > 0.3 are linked by edges, with the thickness reflecting the score. Images were generated using the Metascape (https://metascape.org) online tool.

Fig. 5

Correlation analysis between ZC3H12D expression, genomic heterogeneity, and patient prognosis. A), Correlation between ZC3H12D expression levels and genomic heterogeneity metrics, including TMB (tumor mutation burden), MSI (microsatellite instability), HRD (homologous recombination deficiency), MATH (mutant-allele tumor heterogeneity), and LOH (loss of heterozygosity). * indicates p < 0.05; **** indicates p < 0.00005; B), Evaluation of the correlation between ZC3H12D expression levels and patient survival outcomes using four survival metrics: Overall survival (OS), Progression-free interval (PFI), Disease-free interval (DFI), and Disease-specific survival (DSS).

Fig. 6

Reduced ZC3H12D expression inhibits lung tumor cell proliferation and invasion. A), Western blot comparison between the siRNA group and the negative control (NC) group. ZC3H12D and β-ACTIN were detected on separate gels. The siRNA knockdown group was performed in triplicate with similar results across the three repeats. The band closest to the NC group was cropped and shown as a representative result. Full-length gels for both ZC3H12D and β- ACTIN are provided in Fig. S5; B), Bar plot displaying the quantitative analysis of western blot data; C), Clonogenic assay results comparing the siRNA group with the NC group; D), Bar plot illustrating the quantification of cell colonies; E), Transwell assay results comparing the siRNA group with the NC group, scale bar indicates 100 μm (200 ×); F), Bar plot showing the quantification of invasive cells. *** indicates p < 0.0005; **** indicates p < 0.00005.