The Expression Of FoxC1 In Nasopharyngeal Carcinoma Tissues And Cells And The Correlation Of FoxC1 With EMT-related Molecular Markers

Objective: To investigate the expression of Fox C1 in human NPC tissues and cell lines, and to evaluate the effects of biological behavior in NPC CNE2 cell, and to discuss the correlation of Fox C1 with EMT-related molecular markers.Methods:(1) Tissue microarrays and IHC was used to detect the expression of Fox C1 in 93 NPC tissue specimens with complete clinical data and 33 chronic inflamed nasopharyngeal tissue specimens.(2) The expression of Fox C1 in Human nasopharyngeal cancer cell lines 5-8F, 6-10 B, CNE1, CNE2 and immortalized nasopharyngeal epithelial cell line NP69 was detected by Western blot assay, and screen the cell line with the highest Fox C1 expression for next experiment.(3) Fox C1 knockdowned by si RNA and the expression of Fox C1 after knockdown was detected by Western blot assay.(4) MTT and DAPI staining assay was used to investigate the effects of proliferation and apoptosis, and migration and invasion assays in vitro was used to evaluate the ability of invasion and migration after Fox C1 knockdowned in CNE2 cells.(5) Immunohistochemistry assay was used to detect the Vimentin, Fibronectin, N-cadherin, E-cadherin and β-Catenin expression in aforementioned 93 NPC tissues. The expression of these biomarkers was investigated by Western blotting assay in CNE2 cells with Fox C1 knockdown.Results:(1) Fox C1 high expression was observed in 40 of 93(43.0%) NPC tissues, while only 4 of 33(12.1%) in chronic inflamed nasopharyngeal tissues(P < 0.01). High expression of Fox C1 was significantly correlated with tumor size, lymph node metastasis, distant metastasis and clinical stage.(2) Western blotting showed that Fox C1 relative expression increased progressively from immortalized nasopharyngeal epithelial cells NP69 to NPC cell lines with low metastatic(6-10B) and well-differentiated(CNE1), and finally to NPC cell lines with high metastatic(6-10B) and low differentiated(CNE1)(P5-8f,NP69<0.01, P6-10 B,NP69<0.01, PCNE2,NP69<0.01, PCNE1,NP69<0.01).(3) The expression of Fox C1 was significantly down-regulated by si RNA in si Fox C1-1657 group CNE2 cells.(4) The MTT showed that the inhibition ratio of cellular proliferation after Fox C1 knockdown by si RNA in CNE2 cells and negative control group had no statistically significant(P>0.05). Transwell assay showed that following Fox C1 knockdown, the number of migration and invasion cells was significantly less than the number in the negative control group cells(Pmigration<0.01, Pinvasion<0.01).(5) Comparing to cases with low Fox C1, mesenchymal markers Vimentin, Fibronectin and N-cadherin(PVimentin<0.01, PFibronectin<0.05, PN-cadherin<0.05) were upregulated in NPC tissues with high Fox C1. While there was no difference between these two groups in the expression of E-cadherin and β-Catenin(PE-cadherin>0.05, Pβ-Catenin>0.05). Among the 40 cases with high Fox C1 expressed, positive expression rate of Vimentin, Fibronectin, N-cadherin were 29/40(72.5%), 25/40(62.5%), 39/40(97.5%), respectively, high expression rate of E-cadherin and β-Catenin were 26/40(65.0%), 24/40(60.0%), respectively. Notably, in the specimens with low Fox C1 expression, positive expression rate of Vimentin, Fibronectin, N-cadherin were 22/53(41.5%), 20/53(37.7%), 42/53(79.3%), respectively, high expression rate of E-cadherin and β-Catenin were 34/53(64.2%), 39/53(73.6%), respectively. After Fox C1 knockdowned by si RNA, mesenchymal markers including Vimentin, Fibronectin and N-cadherin were downregulated in CNE2 cells(PVimentin<0.01, PFibronectin<0.01, PN-cadherin<0.01). However, the expression of E-cadherin and β-Catenin showed no significant difference between the knockdown group and the negative control group(PE-cadherin>0.05, Pβ-Catenin>0.05).Conclusions: Fox C1 may be involved in the development of nasopharyngeal carcinoma. The si RNA of this experiment design can effectively lower the expression of Fox C1 CNE2 cells. Fox C1 may play an important role in NPC metastasis and invasion. There may be a correlation between the expression of Fox C1 and mesenchymal markers, also Fox C1 play an important role in the process of EMT and it is likely to be used as a target of nasopharyngeal carcinoma therapy.