Knockdown of PPFIA4 inhibits hepatocellular carcinoma growth and glycolysis via PFKFB3

Background: Protein tyrosine phosphatase receptor type F polypeptide interacting protein alpha 4 (PPFIA4) has been implicated in the growth and dissemination of tumor cells and is known to be elevated in several malignancies. However, the biological function and regulatory mechanisms of PPFIA4 in hepatocellular carcinoma (HCC) remain poorly understood. Methods: The Ualcan database was used to analyze PPFIA4 expression levels and survival outcomes in patients with HCC. HCC cell lines were transfected with siRNAs targeting PPFIA4 or with PPFIA4 overexpression plasmids. Cell viability at multiple time points was assessed using the Cell Counting Kit-8 (CCK8) assay. A colony formation assay was performed to quantify the number of crystal violet-stained colonies. Commercial kits were used to measure lactate production, glucose uptake, and Adenosine Triphosphate (ATP) content. The protein expression of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) was evaluated by western blotting. To further investigate the mechanistic relationship, HCC cells were co-transfected with si-PPFIA4 and PFKFB3 plasmids, followed by assessment of lactate production, glucose uptake, ATP levels, and cell viability. All experiments were conducted in triplicate. Results: Analysis of the Ualcan database revealed that patients with HCC exhibited significantly elevated PPFIA4 expression (p < 0.05), which was associated with poorer survival outcomes. Functional assays demonstrated that PPFIA4 knockdown reduced HCC cell viability and colony formation, and significantly decreased ATP levels, glucose uptake, lactate production, and PFKFB3 protein expression (p < 0.05). Importantly, overexpression of PFKFB3 partially reversed the inhibitory effects of si-PPFIA4 on HCC cell proliferation and glycolysis (p < 0.05). Conclusions: This study demonstrates that PPFIA4 is highly expressed in HCC and promotes tumor progression by positively regulating PFKFB3 expression, thereby enhancing glycolytic activity and cell proliferation. These findings highlight the PPFIA4/PFKFB3 axis as a potential regulatory pathway in HCC metabolism and suggest that PPFIA4 may represent a promising therapeutic target.

Hepatocellular cancer; PPFIA4; Proliferation; Glycolysis; PFKFB3

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