RNA结合蛋白6对喉癌发生发展的调节作用及机制研究

Regulatory effect and mechanism of RNA-binding protein RBM6 on the development of laryngeal carcinoma

目的探讨RNA结合蛋白6(RBM6)对喉癌发生发展的调节作用及机制。方法收集15例原位喉癌患者在术中的喉癌组织及癌旁组织,采用实时荧光定量PCR(RT-qPCR)和Western Blot检测RBM6、表皮生长因子受体(EGFR)、细胞外信号调节激酶(ERK)和磷酸化ERK(p-ERK)的表达。将携带RBM6基因(Lv-RBM6,RBM6组)或空质粒(Lv-Ctrl,对照组)的重组慢病毒载体转染入人喉癌细胞系LCC构建RBM6过表达喉癌细胞及对照细胞,采用CCK-8法和Transwell法分别检测细胞增殖、迁移和侵袭,并检测MMP-2、MMP-9、EGFR、ERK和p-ERK的表达。将稳定表达Lv-RBM6或Lv-Ctrl的LCC细胞注射于裸小鼠右侧颈背部皮下组织,建立Lv-RBM6过表达人喉癌裸小鼠移植瘤模型,每2天计算移植瘤体积。结果与癌旁组织对比,RBM6在喉癌组织中的表达显著降低(P<0.05),EGFR和p-ERK的表达显著增加(P<0.05),ERK无显著变化。RBM6在3/4期患者喉癌组织中的表达显著低于其在1/2期患者中的表达(P<0.05)。体外实验显示,与对照组相比,RBM6组细胞在12 h、24 h、48 h、72 h的细胞增殖活性均显著降低(P<0.05),迁移细胞数及侵袭细胞数均显著降低(P<0.05),MMP-2、MMP-9、EGFR和p-ERK的表达显著降低(P<0.05),ERK无显著变化。异种移植瘤实验显示,与Lv-Ctrl组相比,Lv-RBM6组裸小鼠接种后12 d、14d、16 d、20 d、24 d、28 d肿瘤体积显著降低(P<0.05),EGFR和p-ERK的表达显著降低(P<0.05),ERK无显著变化。结论RBM6可抑制喉癌的生长和进展,作为肿瘤抑制因子抑制喉癌细胞的增殖、迁移和侵袭并诱导其凋亡,其机制可能与抑制EGFR相关信号传导途径的活性有关。

Objective To explore the regulatory effect and mechanism of RBM6 on the occurrence and the development of laryngeal carcinoma.Methods The laryngeal carcinoma tissues and adjacent tissues of 15 patients with orthotopic laryngeal carcinoma were collected.Real-time quantitative PCR(RT-qPCR)and Western Blot were used to detect the expression of RBM6,EGFR,ERK and p-ERK.The recombinant lentiviral vectors carrying RBM6 gene(Lv-RBM6,RBM6 group)or empty plasmid(Lv-Ctrl,control group)were transfected into human laryngeal carcinoma cell line LCC to construct RBM6 overexpressing laryngeal carcinoma cells and control cells.Cell proliferation,migration and invasion were detected by CCK-8 method or Transwell method,and expression of MMP-2,MMP-9,EGFR,ERK and p-ERK was detected.LCC cells stably expressing Lv-RBM6 or Lv-Ctrl were injected into the subcutaneous tissue of the right neck and back of nude mice to establish a nude mouse xenograft model with Lv-RBM6 overexpressing human laryngeal carcinoma,and the tumor volume was calculated every 2 days.Results Compared with adjacent tissues,the expression of RBM6 in laryngeal carcinoma was significantly decreased(P<0.05),the expressions of EGFR and p-ERK was significantly increased(P<0.05),and expression of ERK was not significantly changed.The expression of RBM6 in laryngeal carcinoma tissues of stage 3/4 patients was significantly lower than that in patients with stage 1/2(P<0.05).The experiments in vitro showed that,compared with the control group,the cell proliferation activity of RBM6 group was significantly decreased at 12 h,24 h,48 h,72 h(P<0.05),and the numbers of migrated cells and invasive cells were significantly decreased(P<0.05).The expressions of MMP-2,MMP-9,EGFR and p-ERK were significantly decreased(P<0.05),and expression of ERK was not significantly changed.Xenograft tumor experiments showed that,compared with the Lv-Ctrl group,the tumor volume of the Lv-RBM6 group was significantly decreased on the 12th,14th,16th,20th,24th,and 28th day after inoculation(P<0.05),the expressions of EGFR and p-ERK were significantly reduced(P<0.05),and expression of ERK was not significantly changed.Conclusion RBM6 can inhibit the growth and progression of laryngeal carcinoma.As a tumor suppressor,it inhibits the proliferation,migration and invasion of laryngeal carcinoma cells and induces apoptosis.The mechanism may be related to the inhibition of the activity of EGFR-related signaling pathways.