HCRP-1 alleviates the malignant phenotype and angiogenesis of oral squamous cell carcinoma cells via the downregulation of the EGFR/STAT3 signaling pathway

Abstract

It has previously been reported that human hepatocellular carcinoma-related protein 1 (HCRP-1), which is a tumor suppressor gene, and epidermal growth factor receptor (EGFR) are abnormally expressed in certain solid tumors. Therefore, in the present study, the expression patterns of HCRP-1 in oral squamous cell carcinoma (OSCC) are discussed. Moreover, the present study investigated whether HCRP-1 regulated EGFR expression levels and its downstream effectors to further determine the regulation of tumor cell behavior. Therefore, the expression levels of HCRP-1 in normal oral epithelial and OSCC cells were determined, and the effects of HCRP-1 overexpression on OSCC cell proliferation, migration and invasion were assessed. Moreover, the culture medium from the different groups of OSCC cells was separately supplemented into the human umbilical vein endothelial cell (HUVEC) cultures, and the migration and angiogenesis of the HUVECs were assessed. To determine the roles of EGFR/STAT3 in the regulation of HCRP-1, EGF and colivelin, a STAT3 agonist, were used to treat CAL-27 cells and their effects on the cells were assessed using the aforementioned functional experiments. The results demonstrated that HCRP-1 expression levels were downregulated in OSCC cells and that HCRP-1 overexpression could suppress OSCC cell proliferation, migration and invasion. Moreover, the culture medium from OSCC cells overexpressing HCRP-1 facilitated the migration and angiogenesis of HUVECs. Furthermore, HCRP-1 was demonstrated to function in cells by regulating the EGFR/STAT3 signaling pathway. In summary, the present study indicated that HCRP-1 alleviated the malignant phenotype and angiogenesis of OSCC cells via the downregulation of the EGFR/STAT3 signaling pathway.

Keywords: EGFR; angiogenesis; hepatocellular carcinoma-related protein 1; malignant phenotypes; oral squamous cell carcinoma.

Figure 1.

HCRP-1 overexpression inhibits OSCC cell proliferation. HCRP-1 mRNA and protein expression levels in oral epithelial and OSCC cells were determined using (A) RT-qPCR and (B) western blotting, respectively. CAL-27 cells were transfected to overexpress HCRP-1, and (C) RT-qPCR and (D) western blotting were performed to verify transfection efficiency. ^*P<0.05 and ***P<0.001. Transfected cell proliferation was assessed using the (E) Cell Counting Kit-8 and (F) colony formation assays. ***P<0.001 vs. Ov-NC. HCRP-1, hepatocellular carcinoma-related protein 1; OSCC, oral squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; Ov-, overexpression; NC, negative control; HIOEC, human immortalized oral epithelial cell.

Figure 2.

HCRP-1 overexpression inhibits oral squamous cell carcinoma cell migration and invasion. Effects of HCRP-1 overexpression on cell migration and invasion were assessed using (A) wound healing (scale bar, 100 µm) and (B) Transwell (scale bar, 50 µm) assays. (C) Protein expression levels of MMP9 and MMP14 were determined via western blotting. ^*P<0.05, **P<0.01 and ***P<0.001. HCRP-1, hepatocellular carcinoma-related protein 1; Ov-, overexpression; NC, negative control.

Figure 3.

HCRP-1 overexpression inhibits HUVEC migration and angiogenesis. (A) CM from the control, Ov-NC and Ov-HCRP-1 groups of CAL-27 cells were separately supplemented into the HUVEC CM, and the migration of HUVECs was assessed using the Transwell assay (scale bar, 50 µm). (B) Angiogenesis capacity in each group of HUVECs was assessed using the angiogenesis assay (scale bar, 250 µm). **P<0.01 and ***P<0.001. HCRP-1, hepatocellular carcinoma-related protein 1; Ov, overexpression; NC, negative control; CM, culture medium.

Figure 4.

HCRP-1 overexpression inhibits the EGFR/STAT3 signaling pathway in oral squamous cell carcinoma cells. (A) Western blotting was used to determine the protein expression levels of proteins associated with the EGFR/STAT3 signaling pathway. ^*P<0.05 and ***P<0.001. Cell proliferation was determined using the (B) Cell Counting Kit-8 (***P<0.001 vs. control; ^#P<0.05 and ^###P<0.001 vs. Ov-HCRP-1) and (C) colony formation assays. HCRP-1, hepatocellular carcinoma-related protein 1; Ov, overexpression; EGFR, epidermal growth factor receptor; p/t-, phosphorylated/total.

Figure 5.

HCRP-1 in CAL-27 cells functions via the regulation of the EGFR/STAT3 signaling pathway. Effects of EGF and colivelin on the migration and invasion of CAL-27 cells were assessed using the (A) wound healing (scale bar, 100 µm) and (B) Transwell assays (scale bar, 50 µm). (C) Western blotting was performed to determine the protein expression levels of MMP9 and MMP14. ^*P<0.05, **P<0.01 and ***P<0.001. HCRP-1, hepatocellular carcinoma-related protein 1; EGF, epidermal growth factor; Ov-, overexpression.

Figure 6.

HCRP-1 in HUVECs functions via the regulation of the EGFR/STAT3 signaling pathway. (A) HUVEC migration was assessed using the Transwell assay (scale bar, 50 µm). (B) Angiogenesis in each group of HUVECs was assessed using the angiogenesis assay (scale bar, 250 µm). **P<0.01 and ***P<0.001. HCRP-1, hepatocellular carcinoma-related protein 1; EGF, epidermal growth factor; CM, culture medium; Ov-, overexpression; HUVEC, human umbilical vein endothelial cell.