KISS1基因激活ERK1/2磷酸化通路抑制鼻咽癌细胞迁移和侵袭能力的研究
Study on KISS1 gene inhibiting the migration and invasion of nasopharyngeal carcinoma cells via activating the ERK1/2 pathway
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摘要: 目的：探讨KISS1及其受体基因KISS1-R在ERK1/2磷酸化通路中调节鼻咽癌细胞迁移和侵袭能力的分子机制。方法：采用实时荧光定量PCR以及蛋白免疫印迹技术，检测KISS1、KISS1-R基因、黏着斑激酶（FAK）的表达；验证ERK1/2信号通路的磷酸化激活，EZR蛋白的表达与鼻咽癌细胞迁移和侵袭能力的关系。构建过表达KISS1及KISS1-R基因载体，通过划痕实验及Transwell实验分析过表达KISS1（SUNE-KISS1）及KISS1-R（SUNE-1R）基因对SUNE-1-5-8F鼻咽癌细胞系的迁移能力的影响。通过蛋白免疫印迹技术，检测在过表达KISS1及KISS1-R基因后，磷酸化FAK（p-FAK）和磷酸化ERK（p-ERK）以及EZR蛋白的表达水平。通过阻断ERK1/2通路的磷酸化激活，验证KISS1及KISS1-R基因对鼻咽癌细胞迁移能力的影响，以及对细胞转移相关蛋白p-FAK、EZR表达量的影响。最后，通过siRNA抑制KISS1基因表达，进一步验证KISS1基因对鼻咽癌细胞迁移和侵袭的调控作用。结果：实时荧光定量PCR实验发现，KISS1基因在SUNE-1-6-10B细胞中表达量相对于SUNE-1-5-8F细胞中较高。蛋白免疫印迹分析发现，KISS1及KISS1-R的蛋白表达量，以及p-FAK的表达量与鼻咽癌细胞转移能力呈负相关；EZR与鼻咽癌细胞转移能力呈正相关关系。划痕及Transwell实验分析发现，过表达KISS1及KISS1-R基因均能够有效抑制SUNE-1-5-8F细胞的迁移和侵袭能力。蛋白免疫印迹分析发现，过表达KISS1及KISS1-R基因能增加p-FAK及p-ERK1/2的蛋白表达量，抑制EZR蛋白的表达。加入ERK1/2通路特异性的阻断剂PD98059后，由过表达KISS1及KISS1-R基因引起的细胞迁移和侵袭的抑制状态被阻断。同时，由过表达KISS1及KISS1-R引起的p-FAK与p-ERK1/2的上调，EZR的下调均被恢复。通过siRNA的干扰，抑制了KISS1基因表达，从而引起SUNE-1-5-8F细胞的迁移和侵袭能力的增加。结论：过表达KISS1基因可以显著抑制鼻咽癌细胞的迁移和侵袭能力；同时，通过siRNA干扰KISS1基因的表达，能够增加鼻咽癌细胞的迁移能力。KISS1及KISS1-R基因通过磷酸化激活ERK1/2通路，进一步激活p-FAK并抑制EZR表达，从而抑制鼻咽癌细胞的迁移和侵袭能力。
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关键词:
- 鼻咽肿瘤 /
- KISS1基因 /
- 蛋白免疫印迹 /
- 实时荧光定量PCR /
- Transwell实验
Abstract: Objective:To explore the molecular mechanism of KISS1 and its receptor gene KISS1-R in adjusting the migration and invasion of nasopharyngeal carcinoma(NPC) cells via activating the ERK1/2 pathway.Methods:The expression levels of KISS1,KISS1-R and FAK were detected using RT-PCR and Western blot,the phosphorylation activation of ERK1/2 signaling pathway was identified,and the relationship between the expression of EZR protein,and migration and invasion of NPC cells was analyzed.The overexpression vector containing KISS1 and KISS1-R gene was constructed,the effects of overexpressing KISS1 and KISS1-R gene on the migration of SUNE-1-5-8F cells were detected using the wound healing assay and Transwell assay.After KISS1 and KISS1-R gene expressing,the levels of FAK,p-FAK,p-ERK1/2 and EZR protein were measured by Western blot.After the phosphorylation activation of ERK1/2 pathway was blocked,the effects of KISS1 and KISS1-R gene on the migration and invasion of NPC cells,and expression levels of p-FAK and EZR protein were identified.The expression of KISS1 was interfered with siRNA,and the effects of KISS1 gene on migration and invasion of NPC cells were assayed.Results:The results of RT-PCR showed that the expression of KISS1 gene was high in SUNE-1-6-10B cells compared with SUNE-1-5-8F cells.The results of Western blot showed that the expression levels of KISS1,KISS1-R,FAK and p-FAK were negatively related to metastasis of NPC cells,and the expression of EZR was positively correlated with the metastasis of NPC cells.The wound healing and Transwell assay showed that the overexpression of KISS1 and KISS1-R could effectively inhibit the migration and invasion of SUNE-1-5-8F cells.The results of Western blot showed that the overexpression of KISS1 and KISS1-R could improve the protein expression of p-FAK and p-ERK1/2,and inhibit the level of EZR expression.The migration and invasion suppression induced by overexpression of KISS1 and KISS1-R was reversed after the inhibitor PD98059 of ERK1/2 pathway was used.The overexpression of KISS1 and KISS1-R could lead to the recovery of the p-FAK and p-ERK1/2 upregulating and EZR downregulating.Inhibiting the KISS1 gene expression caused by siRNA interference could increase the migration and invasion of SUNE-1-5-8F cells.Conclusions:The KISS1 gene overexpression can significantly inhibit the migration and invasion of NPC cells.Inhibiting the KISS1 gene expression caused by siRNA interference can increase the migration and invasion of NPC cells.The KISS1 and KISS1-R gene can activate the ERK1/2 pathway,p-FAK,and inhibit the EZR expression through phosphorylation,which can inhibit the migration and invasion of NPC cells.-
Key words:
- nasopharynx neoplasms /
- KISS1 gene /
- Western blot /
- RT-PCR /
- Transwell assay
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