The Role Of Transient Receptor Potential Vanilloid 4 In The Development Of Nasopharyngeal Carcinoma

Objective:Nasopharyngeal carcinoma（NPC）is a solid tumor derived from human nasopharyngeal epithelium,and the pharyngeal recess is the most common site of nasopharyngeal carcinoma.TRPV4 is a non-selective cationic channel.Many studies have shown that TRPV4 is abnormally expressed in a variety of tumors and participates in the regulation of tumor cell proliferation,metastasis and epithelial-mesenchymal transformation.The purpose of this study is to explore the expression of TRPV4 in NPC cells,and the effect of TRPV4 on the proliferation and migration in NPC cells.This study will provide new ideas for the treatment of NPC.Methods:To observe the expression of TRPV4 in normal nasopharyngeal epithelial cell line NP69 and NPC cell lines HNE1,C666-1,CNE1 and CNE2,and the effects of TRPV4 on the regulation of Ca²⁺influx,cell proliferation,migration and nuclear expression of NFAT1.1.RT-qPCR experiment:the total RNA was extracted from the five cell lines,and then the RT-PCR was carried out.We observed the expression of TRPV4 mRNA in nasopharyngeal normal epithelial cell line NP69 and NPC cell lines HNE1,C666-1,CNE1 and CNE2 by qPCR.2.Calcium imaging experiment:cells were pretreated with HC067047（1μM）,a selective antagonist of TRPV4,and then stimulated by GSK1016790A（100n M）,a selective agonist of TRPV4,to observe the function of TRPV4 in regulating Ca²⁺influx in NPC cells.3.CCK-8 assay:cells were divided into the experimental group and the control group.HC067047（1μM）was added in the experimental group,and 0.1%DMSO was added in the control group.The cell viability was detected by CCK-8 kit.4.Transwell assay:Transwell assay was used to examine the effect of inhibition of TRPV4 activation on cell migration.5.Western blot assay:nuclear protein was extracted and Western blot method was used to detect the effect of inhibition of TRPV4 activation and removal of Ca²⁺on NFAT1 nuclear expression.Results:1.qPCR assay showed that TRPV4 was overexpressed in NPC cells.2.Calcium imaging experiment showed that TRPV4 was a functional calcium channel in NPC cells.3.CCK-8 assay showed that inhibition of TRPV4 activation significantly decreased the growth of NPC cells.4.Transwell migration assay showed that inhibition of TRPV4 activation could reduce the migration ability of NPC cells.5.Western blot experiments showed that inhibition of TRPV4 activation or elimination of Ca²⁺could reduce the nuclear expression of NFAT1.Concliusion:1.TRPV4 mRNA is overexpressed in NPC cells.2.Inhibition of TRPV4 activation reduces the proliferation and migration of NPC cell.3.TRPV4 is a functional calcium channel in NPC cell,which can mediate calcium influx.TRPV4promotes the nuclear expression of NFAT1.