Response gene to complement 32 promotes tumorigenesis by mediating DNA damage repair and inhibits CD8+ T cells infiltration in diffuse large B-cell lymphoma

Background: Response gene to complement 32 (RGC32), a complement activation-inducible factor broadly expressed in normal human tissues, has been implicated in tumorigenesis through its dysregulated expression in various malignancies and its involvement in critical oncogenic processes. Despite its established roles in cancer biology, RGC32 remains uncharacterized in diffuse large B-cell lymphoma (DLBCL). This study provides the first comprehensive investigation of RGC32 expression patterns and functional contributions to DLBCL pathogenesis, elucidating its potential as a novel therapeutic target or prognostic biomarker in this disease. Methods: Immunohistochemical (IHC) staining of RGC32 was performed on specimens from 32 Reactive hyperplasia lymphoid (RHL) patients and 80 DLBCL patients. To evaluate the role of RGC32 in DLBCL, lentivirus vectors either encoding shRGC32 or shControl were transfected into DLBCL cell lines. RNA-sequencing (RNA-seq) analysis was performed between shRGC32 and shControl stably transfected OCI-LY1 cells and functional enrichment analyses used gene ontology (GO) and kyoto encylopaedia of genes and genomes (KEGG). In order to explored its functions in vivo, xenograft models were established by subcutaneously injecting shRGC32 and shControl transfected DLBCL cells into SCID beige mice. Results: Immunohistochemical analysis revealed RGC32 overexpression in DLBCL tissues contrast with RHL, and was associated advanced Ann Arbor stage (p = 0.043), B symptoms (p = 0.020), and poor progression-free survival (p = 0.015) and overall survival (p = 0.035). Functional studies demonstrated that RGC32 knockdown via shRNA significantly suppressed DLBCL cell proliferation in vitro and in vivo, with xenograft models showing reduced tumor growth and Ki-67 expression. RNA-seq analysis linked RGC32 depletion to downregulation of cell proliferation and impaired DNA damage repair (DDR) mechanisms. Western blot showed RGC32 knockdown could suppress ATM/ATR/CHK1 pathway and increase the tumor mutational burden (TMB). Furthermore, after inhibition of RGC32, infiltration of CD8+ T cells was increased in DLBCL tumor microenvironment (TME). Conclusions: This study highlights that RGC32 is a novel molecule in DLBCL progression and might be a potential therapeutic target for DLBCL therapy. This study employed a multifaceted approach to investigate the role of RGC32 in diffuse large B-cell lymphoma (DLBCL). The core design included: Human Samples: Retrospective analysis of 80 FFPE tumor specimens from DLBCL patients (treated with R-CHOP, 2011-2022) compared to 32 reactive hyperplasia control tissues. Histological diagnosis adhered to WHO Classification (5th edition). Ethical approval and informed consent were obtained (Shandong Provincial Hospital Medical Ethics Committee). Peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers served as additional controls. Pathological & Molecular Characterization: H&E Staining & Immunohistochemistry (IHC): Performed on FFPE sections using a validated scoring system (intensity x % positive area) with key antibodies including anti-RGC32. Specimens were stratified into low/high RGC32 expression groups. Western Blot: Analyzed RGC32 and associated signaling pathway proteins (e.g., c-myc, p-ATM, p-ATR, PD-L1) in DLBCL cell line lysates. RGC32 Poly(A) Tail (PAT) Length Assay: Quantified mRNA stability/post-transcriptional regulation using a specific kit and RGC32-targeted primers. Functional Analysis (In Vitro): Genetic Manipulation: DLBCL cell lines (OCI-LY1, OCI-LY10) were transfected with RGC32-specific siRNA or control plasmids (validated by Western blot). Proliferation Assay: Monitored cell growth kinetics post-transfection using CCK-8. Cell Cycle Analysis: Performed via propidium iodide staining and flow cytometry (Beckman Coulter Navios). In Vivo Validation: Xenograft Models: shRGC32-transfected or control OCI-LY1 cells were subcutaneously injected into beige SCID mice (n per group). Tumor growth was monitored by caliper measurement for 3-4 weeks. Ethical approval was obtained (Shandong Provincial Hospital Animal Committee). Tumor-Infiltrating Lymphocytes (TILs) Analysis: TILs were isolated from digested tumors, stained for CD8a and CD3, and analyzed by flow cytometry. Transcriptomic Profiling: RNA-Sequencing: Compared global gene expression profiles of shRGC32 vs. shControl OCI-LY1 cells (biological triplicates). Differential expression was analyzed using DESeq2, followed by GO and KEGG pathway enrichment. Target Validation: Quantitative Real-time PCR (qRT-PCR): Quantified gene expression changes (validating RNA-Seq targets/nominated genes) using SYBR Green chemistry and normalized to GAPDH. Statistical Analysis: Data are presented as mean ± SD. Analyses employed Student's t-test, one-way ANOVA, Kaplan-Meier survival curves (log-rank test), using SPSS 24.0 and GraphPad Prism 5.0 (significance: *p<0.05, **p<0.01, ***p<0.001).