GABRD Accelerates Tumour Progression via Regulating CCND1 Signalling Pathway in Gastric Cancer

Abstract

Neurotransmitters and their receptors were reported to be involved in tumour initiation and progression. However, little is known about their roles in gastric cancer (GC). Here, we first identified gamma-aminobutyric acid type A receptor subunit delta (GABRD) as a novel oncogene in GC. GABRD was preferentially upregulated in GC tissues compared with adjacent normal tissues. High GABRD expression was significantly associated with poor survival prognosis. Knockdown of GABRD could markedly induce cell apoptosis and cell cycle arrest while repressing proliferation and migration in vitro, and suppress tumour growth in vivo. The results of transcriptomic analysis and Ingenuity pathway analysis (IPA) highlighted that cyclin D1(CCND1) was a potential downstream target. Immunohistochemistry results also indicated that CCND1 expression was associated with GABRD in GC. Functional experiments also confirmed that the role of GABRD in regulating proliferation, migration, invasion, and apoptosis was dependent on CCND1. Mechanically, further research confirmed that GABRD knockdown could induce p53-dependent apoptosis through CCND1, and GABRD upregulated CCDN1 through inhibiting its ubiquitin-mediated degradation. Overall, these findings uncover a role for the neurotransmitter receptor GABRD in regulating the proliferation and apoptosis of gastric cancer cells. Our present study provides novel insights into the mechanism of tumourigenesis in gastric cancer.

Keywords: cyclin D1; gamma‐aminobutyric acid type A receptor; gastric cancer; neurotransmitter receptors.

FIGURE 1

GABRD expression analysis in gastric cancer tissues and its prognostic implications. (A) GABRD mRNA expression levels in stomach adenocarcinoma tissues versus normal tissues from TCGA database. (B) Comparison of GABRD mRNA expression in 27 pairs of gastric cancer tissues and adjacent normal tissues. (C) Immunohistochemical analysis of GABRD protein expression in gastric adenocarcinoma versus normal gastric tissues. (D) GABRD protein expression in 86 paired samples of gastric adenocarcinoma and adjacent normal tissues. (E) Percentage of high‐level GABRD expression in cancerous versus normal tissues. (F) Correlation between GABRD expression and selected clinicopathological characteristics (T category, N category, and stage grouping) in gastric cancer patients. Subgroups with extremely small sample sizes—T1 (n = 2) and stage IV (n = 1)—were excluded from the statistical analysis. ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001. Complete data are presented in Figure S1. (G) Representative immunohistochemical staining images of GABRD in different stages of gastric cancer and normal tissues. (H) Kaplan–Meier plots showing the association between GABRD protein expression levels and overall survival (OS) in gastric cancer patients. (I) Multivariate Cox regression analysis demonstrating GABRD expression as an independent prognostic factor for patient survival.

FIGURE 2

Impact of GABRD silencing on gastric cancer cell growth and apoptosis. (A) Celigo Cell Counting assay results show the effect of GABRD knockdown on the proliferation of AGS and MGC‐803 cell lines. ***: p <= 0.001, ****: p <= 0.0001. (B) Cell colony formation assay demonstrating the impact of GABRD knockdown on the clonal formation ability of AGS and MGC‐803 cell lines. (C) Alterations in cell cycle distribution induced by GABRD knockdown, as assessed by flow cytometry. *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001. (D) Transwell migration assay results illustrate the effect of GABRD knockdown on cell migration in AGS and MGC‐803 cell lines. (E) Wound‐healing assay showing the impact of GABRD knockdown on cell invasion in AGS and MGC‐803 cell lines. (F) Annexin V‐APC staining and flow cytometry analysis indicating the percentage of apoptotic cells in AGS and MGC‐803 cell lines following GABRD knockdown.

FIGURE 3

Investigation of GABRD's role in enhancing gastric cancer cell tumorigenicity in vivo. (A) In vivo bioluminescence imaging reflecting tumour size in mice injected with MGC‐803 cells treated with either shGABRD or shCtrl. (B) Measurement of tumour volume in mice injected with shGABRD or shCtrl‐treated MGC‐803 cells. *: p <= 0.05, **: p <= 0.01. (C) Comparison of average tumour weight between mice injected with shGABRD or shCtrl‐treated MGC‐803 cells.

FIGURE 4

Exploring GABRD's influence on gene expression and identification of key downstream targets in gastric cancer. (A) Affymetrix Clariom S human assay results showing gene expression alterations in AGS cells following GABRD silencing. (B) Ingenuity Pathway Analysis (IPA) depicting significant alterations in key signalling pathways influenced by GABRD. (C) Schematic representation of the impact of GABRD silencing on cancer‐related pathways, highlighting Ephrin Receptor and IL‐8 signalling. (D) Protein–protein interaction (PPI) network analysis reveals potential regulation of critical oncogenic pathways by GABRD. (E and F) Results of qPCR and Western blot analyses confirming the downregulation of key genes following GABRD knockdown in AGS cells. (G) Functional significance of downregulated genes demonstrated by the Celigo Cell Counting assay. *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001.

FIGURE 5

GABRD regulation of cell proliferation, migration, invasion, and apoptosis through CCND1. ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001. (A) CCND1 expression levels in gastric cancer cell lines and a normal cell line (GES1), highlighting higher expression in AGS and MGC‐803 cells. (B) GABRD's impact on CCND1 expression validated by qRT‐PCR in AGS cells. (C) Western blot confirms GABRD's influence on CCND1 protein levels in AGS cells. (D) Celigo cell counting assays reveal the proliferative effects of CCND1 knockdown and GABRD overexpression in AGS cells. (E) Counteraction of GABRD‐induced proliferation by CCND1 knockdown in AGS cells, as demonstrated by colony formation assays. (F) Wound‐healing assays showing reversal of GABRD‐mediated cell migration promotion by CCND1 silencing in AGS cells. (G) Transwell assays confirming the attenuation of GABRD‐induced cell invasion by CCND1 knockdown in AGS cells. (H) Flow cytometry analysis illustrating the impact of CCND1 knockdown on GABRD‐mediated apoptosis in AGS cells.

FIGURE 6

Correlation between CCND1 expression, prognosis, and association with GABRD in gastric cancer. (A) Differential CCND1 mRNA expression in gastric cancer versus normal tissues, highlighting elevated GABRD levels in tumour tissues. (B) Confirmation of elevated GABRD mRNA expression in tumour tissues compared to adjacent non‐tumour tissues. (C) Immunohistochemistry (IHC) reveals pronounced CCND1 protein overexpression in gastric cancer samples. (D) IHC validation of CCND1 protein overexpression in paired gastric cancer and normal tissues. (E) High CCND1 expression frequency in cancerous versus non‐cancerous tissues. (F) Kaplan–Meier survival curves indicate a trend toward poorer prognosis with higher CCND1 expression. (G) Multivariate Cox regression analysis establishing CCND1 as an independent prognostic indicator. (H) Correlation analysis between GABRD and CCND1 mRNA expression levels in TCGA‐STAD dataset, revealing a significant yet weak correlation. (I) Pearson's correlation confirms a significant association between GABRD and CCND1 protein expressions. (J) Co‐immunoprecipitation (Co‐IP) assay demonstrating physical interaction between CCND1 and GABRD in AGS cells.

FIGURE 7

GABRD knockdown induces p53‐dependent apoptosis through CCND1 and GABRD stabilises CCND1 via inhibiting its ubiquitin‐mediated degradation. ns: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001. (A) Human Apoptosis Antibody Array results showing alterations in apoptosis‐related protein expression in AGS cells following GABRD knockdown. (B) Western blot analysis demonstrating the effect of GABRD knockdown on p53 expression in AGS cells. (C) Western blot analysis showing the impact of CCND1 knockdown on p53 expression in AGS cells. (D) Inhibition of p53 negating the induction of downstream p53 genes by shGABRD or shCCND1. (E) CCK‐8 assay results indicate the impact of pifithrin‐α on the apoptosis‐promoting effect of GABRD knockdown in AGS cells. (F) Annexin V‐APC staining demonstrating the effect of pifithrin‐α on apoptosis induction following GABRD knockdown in AGS cells. (G) Cycloheximide (CHX) treatment reveals accelerated CCND1 degradation in GABRD‐knockdown AGS cells. (H) Proteasome inhibitor MG132 rescues GABRD‐knockdown‐induced CCND1 degradation. (I) In vitro ubiquitination assay indicates increased CCND1 ubiquitination upon GABRD knockdown.