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COPB1 promotes the development and progression of esophageal squamous cell carcinoma by activating the PI3K/AKT pathway and regulating the tumor immune microenvironment
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Abstract:
[Abstract] Objective: To investigate the expression of coatomer protein complex subunit beta 1 (COPB1) in esophageal squamous cell carcinoma (ESCC) and its impact on the malignant biological behavior of ESCC cells, as well as the underlying mechanism of action and its clinical significance. Methods: A total of 82 pairs of ESCC tissues and adjacent non-cancerous were collected from the biobank of the Fourth Hospital of Hebei Medical University during 2014 and 2018. Normal esophageal squamous cells (HEEC) and ESCC cell lines (KYSE-150, KYSE-170, Eca109, TE1, KYSE-30, KYSE-450) were routinely cultured. The pcDNA3.1-vector (empty vector), pcDNA3.1-COPB1 vector, si-NC, and si-COPB1 were transfected into KYSE-150 and TE1 cells using transfection reagents. The resulting groups were designated as the NC, COPB1-OE, si-NC, and si-COPB1 groups, respectively. A comprehensive database analysis was conducted to assess the mRNA expression of COPB1 in pan-cancer tissues and its correlation with immune cell infiltration. Quantitative real-time polymerase chain reaction (qPCR) was utilized to assess the mRNA expression of COPB1, PIK3CB, CD68, CD163, CD206, ARG1, and IL-10 in ESCC tissues and cells. Western blotting (WB) analysis was performed to detect COPB1, PI3K, CD68, CD163, CD206 and p-AKT protein expression in ESCC tissues and various cell groups. Colony formation and MTS assays were conducted to assess cell proliferation, while wound healing assay and Transwell assay were used to determine cell migration and invasion. Immunohistochemistry (IHC) was employed to detect the protein expression of COPB1 and CD206 in ESCC tissues. A macrophage model was established using human monocytic leukemia cells (THP-1). Macrophage polarization was induced with phorbol myristate acetate (PMA), interleukin-3 (IL-3), IL-4, and ESCC cell supernatant. WB and qPCR were performed to detect the protein and mRNA expression of CD68 and CD206, respectively. Results: COPB1 was highly expressed in both pan-cancer and ESCC tissues, and its expression was associated with lymph node metastasis and TNM staging (both P < 0.01). ESCC patients with high COPB1 expression exhibited shorter overall survival (P < 0.05), making COPB1 a potential diagnostic biomarker for ESCC. COPB1 was also highly expressed in KYSE-150 and TE1 cells (both P < 0.05). Overexpression or knockdown of COPB1 significantly inhibited or promoted the proliferation, migration and invasion capabilities of KYSE-150 and TE1 cells (both P < 0.05). Differentially expressed genes induced by COPB1 expression changes were primarily enriched in the PI3K/AKT pathway (both P < 0.001), and COPB1 activated the PI3K/AKT pathway (P < 0.05). Additionally, high COPB1 expression increased M2 macrophage infiltration (P < 0.05) and promoted TAM/M2 polarization (P < 0.05). Conclusion: COPB1 is highly expressed in ESCC tissues and can activate the PI3K/AKT pathway and modulates the tumor immune microenvironment, thereby promoting ESCC occurrence and development. COPB1 holds promise as a diagnostic and prognostic biomarker, as well as a therapeutic target for ESCC.
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