Overexpression of hsa-miR-125a-5p enhances proliferation, migration and invasion of head and neck squamous cell carcinoma cell lines by upregulating C-C chemokine receptor type 7

Abstract

Head and neck squamous cell carcinoma (HNSCC) is usually diagnosed accompanied by lymph node metastasis. C-C chemokine receptor type 7 (CCR7) is associated with the invasion and metastasis of tumors in HNSCC through various signaling pathways. The role of hsa-miR-125a-5p in HNSCC remains unclear. The present study was performed to investigate the association between hsa-miR-125a-5p and CCR7 in HNSCC. Reverse transcription-quantitative polymerase chain reaction was applied to analyze the expression of hsa-miR-125a-5p in clinical samples. Cell Counting Kit-8, Transwell and wound healing assays were used to detect cell proliferation, invasion, and metastasis, respectively, following overexpression of hsa-miR-125a-5p. Changes in protein expression of CCR7 were observed using western blotting. In the survival analysis, Student's t-tests and log rank tests were performed to analyze the association between the expression of hsa-miR-125a-5p, and HNSCC according to the Cancer Genome Atlas database. The expression of hsa-miR-125a-5p was identified to be significantly lower in cancer tissue compared with the corresponding adjacent normal tissues in clinical samples (P=0.038). The results of western blotting indicated that there was a positive regulatory association between hsa-miR-125a-5p and CCR7. Furthermore, overexpression of hsa-miR-125a-5p significantly enhanced the ability of cell proliferation, migration and invasion in HNSCC, with upregulation of CCR7. The results of survival analysis revealed that patients in the low expression group of hsa-miR-125a-5p tended to have longer survival times compared with the high expression group (P=0.045). Altogether, the data raised the possibility that hsa-miR-125a-5p has a significant role in promoting cancer in HNSCC, which may provide a basis for the treatment of HNSCC in molecular targeted therapy. Further studies are required to ascertain the role of hsa-miR-125a-5p in other HNSCC cell lines and in vivo.

Keywords: chemokine receptor type 7; head and neck squamous cell carcinoma; invasion; migration; proliferation.

Figure 1.

Results of transfection and CCR7 protein expression in PCI-37B cells. (A) hsa-miR-125a-5p relative expression of following transfection with hsa-miR-125a-5p overexpression plasmid. Hsa-miR-125a-5p+ vs. 37B, **P<0.01. (B) Results of western blotting for CCR7 protein expression and corresponding gray analysis. Hsa-miR-125a-5p+ vs. 37B, **P<0.01. Western blot analysis of β-actin was performed to confirm equal protein loading. Relative expression of CCR7 was measured by the ratio of absorbance between CCR7 and β-actin. 37B, blank control group; miR-125a-5p+, positive transfection; miR-125a-5p-, negative transfection group; CCR7, C-C chemokine receptor type 7.

Figure 2.

Results of CCK-8 and scratch assays for testing cell proliferation and migration. (A) OD (450 nm) values (mean of five wells) of three groups were detected at five time points (0, 24, 48, 72 and 96 h) by CCK-8 assay. Cell migration was measured by cell scratch assay for the three groups at four time points (0, 6, 12 and 24 h). miR-125a-5p+ vs. PCI-37B, *P<0.05. The (B) cell migration rate was calculated from (C) the mean width of the scratch in the cell scratch image at each time point. miR-125a-5p+ vs. PCI-37B at 6, 12 and 24 h, **P<0.01 separately. Light microscopy with original magnification, ×100. 37B, blank control group; miR-125a-5p+, positive transfection; miR-125a-5p-, negative transfection group; CCK-8, Cell Counting Kit-8; OD, optical density; miR, microRNA.

Figure 3.

Results of Transwell assays for testing invasive ability of the three groups of cells (37B, miR-125a-5p+, miR-125a-5p-). (A) Light microscopy of a frozen section of a lesion stained with crystal violet dyes. (B) The number of invasive cells was obtained by calculating the average number of five different fields under the microscope. miR-125a-5p+ vs. PCI-37B, **P<0.01. Original magnification, ×200. miR, microRNA; 37B, blank control group; miR-125a-5p+, positive transfection; miR-125a-5p-, negative transfection group. **P<0.01.

Figure 4.

(A) Expression of hsa-miR-125a-5p in 383 patients with head and neck squamous cell carcinoma with different clinical stages. I and II, patients with lower clinical stages; III and IV, patient with higher clinical stages. I and II vs. III and IV, *P<0.05. (B) Kaplan-Meier overall survival curves for patients with different expression of has-miR-125a-5p (high or low, *P<0.05). Patients were defined as having high hsa-miR-125a-5p expression when the expression of hsa-miR-125a-5p was greater than or equal to a certain value (659), while patients were defined as having low has-miR-125a-5p expression when the expression was lower than this value. miR, microRNA. *P<0.05.