HBB as a Novel Biomarker for the Diagnosis and Monitoring of Lung Cancer Regulates Cell Proliferation via ERK1/2 Pathway

Abstract

Objective: Recent studies have revealed that hemoglobin beta (HBB) plays an important role not only in blood disorders but also in malignancies. The aim of this study is to investigate the clinical significance, diagnostic value, and biological function of HBB in lung cancer. Methods: HBB expression was examined in lung cancer tissues and plasma samples using quantitative real-time polymerase chain reaction, and its relationship with clinical pathological characteristics was analyzed. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic value of HBB in lung cancer. The proliferation of A549 and SPCA1 cells was analyzed using a cell counting kit-8 assay and protein expressions were detected by western blot. Results: The expressions of HBB were found to be down-regulated in both lung cancer tissues and plasma samples. Notably, plasma HBB levels were significantly elevated in postoperative samples when compared to their preoperative counterparts. Across 66 cases of lung cancer tissues, a correlation was observed between HBB levels and both gender and tumor, node, metastasis staging. ROC curve analysis further confirmed the high diagnostic potential of HBB expression in lung cancer. Moreover, the combination of HBB and carcinoembryonic antigen (CEA) had greater significance than HBB or CEA alone in the diagnosis of lung cancer. Knocking out or overexpressing HBB could affect lung cancer cell proliferation through the ERK1/2 signaling pathway. Conclusion: HBB can serve as a novel biomarker for the diagnosis and monitoring of lung cancer, regulating cell proliferation via the ERK1/2 pathway and playing a pivotal role in the oncogenesis and progression of the disease.

Keywords: biomarker; diagnosis; hemoglobin beta; lung cancer; proliferation.

Figure 1.

(A) The expressions of HBB in lung cancer tissues and adjacent tissues, ***P < .001. (B) HBB expression was significantly down-regulated in 87.9% (58 of 66 paired). The results were normalized to adjacent tissues and shown as log2 (2^−△△Ct). (C) Kaplan-Meier survival curve of patients with lung cancer downloaded from Kaplan-Meier plotter database.

Figure 2.

(A) The expressions of plasma HBB in lung cancer patients (n = 80) and healthy controls (n = 80), ***P < .001. (B) ROC curve for prediction of lung cancer based on HBB expression level, the AUC was 0.862 (95% CI: 0.805-0.920). (C) The expressions of plasma CEA in lung cancer patients (n = 80) and healthy controls (n = 80), ***P < 0.001. (D) ROC curve for prediction of lung cancer based on CEA expression level, the AUC was 0.825 (95% CI: 0.762-0.887). (E) ROC curve for prediction of lung cancer based on a combination of HBB and CEA expression levels, the AUC was 0.930 (95% CI: 0.892-0.969). (F) Comparison of plasma HBB levels between pre- and postoperative samples, **P < .01.

Figure 3.

(A) The level of HBB in normal bronchial epithelial cell line (BEAS-2B) and lung cancer cell lines (H1299, A549, SPCA1, H1650, and H1975). (B and C) qRT-PCR and western blot demonstrated that the HBB level was significantly decreased, while the expression of p-ERK1/2 markedly increased after transfection with siRNA-HBB in A549 and SPCA1 cells. (D) CCK8 assay revealed that the proliferation of A549 and SPCA1 cells was promoted after silencing HBB for 72 h. All experiments were repeated 3 times, *P < .05, **P < .01, ***P < .001.

Figure 4.

(A and B) qRT-PCR and western blot demonstrated that the HBB level was significantly increased, while the expression of p-ERK1/2 was markedly decreased after transfection with oeHBB in A549 and SPCA1 cells. (C) Overexpression of HBB inhibited the proliferation of A549 and SPCA1 cells for 72 h as shown by CCK8 assay. All experiments were repeated 3 times, *P < .05, **P < .01, ***P < .001.