Phosphatidylinositol glycan anchor biosynthesis class T promotes malignant phenotype of colon cancer through the NF-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2026.09.16

Abstract

Abstract:

Objective To explore the role of phosphatidylinositol glycan anchor biosynthesis class T (PIGT) in progression of colorectal cancer (CRC) and its molecular mechanism. Methods The differential expression of PIGT in CRC and adjacent normal tissues was analyzed using GEPIA, UALCAN and TNMplot databases. Quantitative PCR and Western blotting were used to detect PIGT expression levels in CRC cell lines HCT116 and HCT8 and in normal colorectal mucosal NCM460 cells. The correlation between PIGT expression and 10-year recurrence-free survival of CRC patients was evaluated using Kaplan-Meier survival analysis. In CRC cells, the effects of PIGT knockdown on cell proliferation, clonogenic ability, migration, invasion and angiogenesis capacity were assessed. The NF-κB signaling pathway was identified as a key downstream pathway using transcriptome sequencing and pathway enrichment analysis, and the expressions of NF-κB key proteins and epithelial-mesenchymal transition (EMT) markers were detected using Western blotting. LPS rescue experiments were performed to confirm the regulatory relationship between PIGT and the NF-κB pathway. A nude mouse subcutaneous xenograft model was used to verify the oncogenic role of PIGT in vivo. Results PIGT was significantly upregulated in CRC tissues and cell lines. High PIGT expression was closely associated with a shorter recurrence-free survival in patients with stage II to IV CRC. In CRC cells, PIGT knockdown significantly inhibited cell proliferation, migration, invasion and angiogenesis and effectively reversed EMT. Downregulation of PIGT reduced the phosphorylation level of P65 and suppressed NF-κB pathway activation, and these changes were effectively reversed by LPS treatment. In the tumor-bearing mice, PIGT knockdown markedly reduced subcutaneous tumor growth. Conclusion PIGT promotes CRC progression and EMT by activating the NF‑κB signaling pathway, suggesting the potential of PIGT as both a prognostic biomarker and a therapeutic target for CRC.

Key words: colorectal neoplasms, phosphatidylinositol glycan anchor biosynthesis class T, nuclear factor-κB, epithelial-mesenchymal transition

Baichao LI, Bingzhe WANG, Xianghui MEI, Ye TIAN, Liang HAN, Jiuna ZHANG. Phosphatidylinositol glycan anchor biosynthesis class T promotes malignant phenotype of colon cancer through the NF-κB signaling pathway[J]. Journal of Southern Medical University, 2026, 46(9): 2173-2184.

Figures/Tables 12

Fig.1 Elevated expression of PIGT in colorectal cancer tissues. A: Pan cancer analysis based on the TNMplot database show that PIGT mRNA levels are significantly upregulated in multiple malignancies, including colorectal cancer (CRC), compared with corresponding normal tissues. B: Validation using the UALCAN database demonstrates that PIGT mRNA levels in primary tumor tissues are significantly higher than those in adjacent normal tissues in both colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). C: Analysis using the GEPIA database shows that PIGT mRNA expression is significantly increased in COAD and READ samples compared with normal controls. *P<0.05.

Fig.2 High PIGT expression is associated with poor prognosis in mid-to-late stage colorectal cancer patients.

Fig.3 PIGT expression is upregulated in colorectal cancer cell lines. A: qPCR analysis of PIGT mRNA levels in two colorectal cancer cell lines (HCT116 and HCT8) and the normal colorectal mucosal cell line NCM460. B: Western blotting analysis of PIGT protein expression in the same cell lines. All data represent the Mean±SD from 3 independent experiments. *P<0.05 vs NCM460.

Fig.4 PIGT expression is efficiently knocked down in HCT116 and HCT8 cells (n=3). ***P<0.001.

Fig.5 Knockdown of PIGT inhibits proliferation of colorectal cancer cells. A: Knockdown of PIGT significantly reduces the number and size of colonies compared with the sh-NC control group. B: PIGT knockdown markedly decreases the number of colony spheres formed in soft agar. ***P<0.001 (n=3).

Fig.6 Knockdown of PIGT attenuates migration and invasion of CRC cells. A: Wound healing assay in HCT116 and HCT8 cells transfected with sh NC or sh PIGT. Knockdown of PIGT significantly delays wound closure compared with the control group. B: Transwell migration assay. PIGT knockdown markedly reduces the number of cells migrating through the polycarbonate membrane. C: Matrigel invasion assay. PIGT knockdown similarly impairs the ability of cells to penetrate the Matrigel barrier. ***P<0.001 (n=3).

Fig.7 Knockdown of PIGT impairs tube formation in CRC cells (n=3). ***P<0.001.

Fig.8 PIGT knockdown reverses EMT by downregulating its effector molecules in CRC cells. A: Western blotting of N cadherin, E cadherin, Vimentin, MMP9, and VEGF-C protein levels in HCT116 cells after PIGT knockdown. GAPDH was used as a loading control. B: Western blotting and quantification of the same proteins in HCT8 cells. ***P<0.001 (n= 3).

Fig.9 Functional enrichment analysis reveals key pathways and biological processes associated with PIGT. A: Number of differentially expressed genes upon PIGT knockdown. B: GO enrichment analysis of the differentially expressed genes. C: KEGG pathway enrichment analysis of the differentially expressed genes.

Fig.10 Protein level validation of PIGT-mediated regulation of NF‑κB signaling. A: Western blotting of P65 and phosphorylated P65 (P-P65) in HCT116 cells after PIGT knockdown. B: Western blotting analysis of P65 and P-P65 in HCT8 cells after PIGT knockdown. ***P<0.001 (n=3).

Fig.11 LPS reverses PIGT knockdown-induced inhibition of NF-κB pathway and EMT in colorectal cancer cells. A: Western blotting of P P65, E cadherin, N cadherin, vimentin, MMP9, and VEGF-C in HCT116 cells in sh NC, sh PIGT, and sh PIGT+LPS (1 μg/mL LPS, 24 h) groups. B: Western blotting analysis of the same proteins in HCT8 cells. *P<0.05, **P<0.01, ***P<0.001 (n=3).

Fig.12 Knockdown of PIGT inhibits growth of colorectal cancer xenografts in nude mice. A: Representative images of xenograft tumors derived from HCT116 cells transfected with sh NC or sh PIGT. B: Western blotting of PIGT protein expression in tumor tissues, confirming efficient PIGT knockdown in vivo. **P<0.01, ***P<0.001.

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