Title:H19/miR-675-5p Targeting SFN Enhances the Invasion and Metastasis of Nasalpharyngeal Cancer Cells
Volume: 12
Author(s): Ting Zhang, Fanghong Lei, Tao Jiang, Lisha Xie, Pin Huang, Pei Li, Yun Huang, Xia Tang, Jie Gong, Yunpeng Lin, Ailan Cheng*Weiguo Huang*
Affiliation:
- Cancer Research Institute, Hengyang Medical College of University of South China, Hengyang 421001, Hunan Province,China
- Cancer Research Institute, Hengyang Medical College of University of South China, Hengyang 421001, Hunan Province,China
Keywords:
NPC, miR-675-5p, 14-3-3σ, invasion, metastasis, nasalpharyngeal, cancer cells.
Abstract:
Aims: The aim is to study the role of miR-675-5p coded by long non-coding RNA H19 in the
development of Nasopharyngeal Cancer (NPC) and whether miR-675-5p regulates the invasion and metastasis
of NPC through targeting SFN (14-3-3σ). The study further validated the relationship between H19,
miR-675-5p and SFN in NPC and their relationship with the invasion and metastasis of NPC.
Methods: Western blot was used to detect the expression of 14-3-3σ protein in immortalized normal nasopharyngeal
epithelial cells NP69 and different metastatic potential NPC cells, 6-10B and 5-8F. At the same
time, to find out the relationship between 14-3-3σ protein and the expression of H19 and miR-675-5p, the
expression of H19 and miR-675-5p in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal
carcinoma cells 6-10B and 5-8F were quantified by real-time PCR. MiR-675-5p mimic and inhibitor
were transfected into NPC 6-10B to over-express and down-express miR-675-5p; miR-675-5p mimic
negative control and inhibitor negative control were transfected into NPC 6-10B as control groups. The
effect of over-expression and down-expression by miR-675-5p on the expression of 14-3-3σ protein was
detected by Western blotting. The 3’-UTR segments of SFN, containing miR-675-5p binding sites were
amplified by PCR and the luciferase activity in the transfected cells was assayed to detect whether SFN is
the direct target of miR-675-5p. Transwell and scratch assays were used to verify the changes in NPC
invasion and metastasis ability of mimics and inhibitors transfected with miR-675-5p.
Results: The expression of 14-3-3σ protein in normal nasopharynx epithelial cells NP69 is significantly
higher than in varied nasopharyngeal carcinoma cells, 6-10B and 5-8F (P<0.05), and the 14-3-3σ protein
levels in low-metastatic nasopharyngeal carcinoma cell 6-10B is higher than in high-metastatic nasopharyngeal
carcinoma cell 5-8F. The expression of H19 and miR-675-5p are significantly higher in NPC
cells than in NP69 cell (P<0.05). The expression of H19 and miR-675-5p in high-Metastatic nasopharyngeal
carcinoma cell 5-8F was higher than in low-Metastatic nasopharyngeal carcinoma cell 6-10B. The
expression of 14-3-3σ protein in miR-675-5p mimic cells was significantly lower than in mimic NC (negative
control) group and blank control group. However, compared with the blank control group, mimic NC
showed no significant difference in 14-3-3σ protein between the two groups. The miR-675-5p inhibitor
group was significantly higher than the inhibitor NC group and the blank control group (p<0.05), but there
was no significant difference in the expression of 14-3-3σ protein in the inhibitor NC group and the blank
control group (p>0.05). Dual-luciferase reporter assay system shows the 3’-UTR segments of SFN containing
miR-675-5p binding sites. SFN was the target gene of miR-675-5p.
Conclusion: 14-3-3σ is downregulated in NPC and is involved in the development of NPC. H19 and miR-
675-5p are upregulated in NPC, which is related to the development of NPC. The over-expression of miR-
675-5p inhibits the expression of 14-3-3σ protein. SFN is the target gene of miR-675-5p. MiR-675-5p
targets SFN, downregulates its protein expression and promotes the invasion and metastasis of NPC.
