葡萄糖调节蛋白78上调MMP-2和MMP-9促进鼻咽癌细胞CNE1的增殖与侵袭

Glucose regulatory protein 78 up-regulates MMP-2 and MMP-9 to promote the proliferation and invasion of nasopharyngeal carcinoma cell CNE1

目的探讨葡萄糖调节蛋白78(GRP78)对鼻咽癌细胞CNE1的增殖与侵袭的作用机制。方法收集2018年6月—2019年6月手术切除病灶的56例经病理确诊鼻咽癌患者的肿瘤组织作为鼻咽癌组织的研究对象,男40例,女16例;均为初次确诊鼻咽癌,并且尚未经过放/化疗干预。另同期选取进行治疗的50例慢性鼻咽炎组织作为比对,男38例,女12例。用免疫组化分别检测鼻咽癌组织和慢性鼻咽炎组织中GRP78的表达。CNE1细胞实验分为对照组(CNE1鼻咽癌细胞常规培养,不进行外界干预)、NC组(CNE1鼻咽癌细胞转染LvGFP-Puro-GRP78-NC后,常规培养)、Sh-GRP78组(CNE1鼻咽癌细胞转染LvGFP-Puro-GRP78后,常规培养),转染完成48 h后,可在荧光显微镜下观察到各组细胞内的绿色荧光蛋白(GFP),PCR检测各组细胞中GRP78的mRNA的表达。Brdu标记检测各组细胞的增殖能力,Transwell小室检测各组细胞的侵袭能力,小管形成实验检测各组细胞的血管形成能力;双荧光素酶实验分析GRP78和基质金属蛋白酶-2(MMP-2)、MMP-9的作用,Western blot检测各组细胞GRP78、MMP-2和MMP-9表达水平。结果与慢性鼻咽炎组织相比,GRP78在鼻咽癌组织中的表达明显升高,Sh-GRP78组CNE1细胞增殖、侵袭和迁移能力、血管形成能力明显增强,差异均具有统计意义(P<0.05)。双荧光素酶实验结果显示MMP-2和MMP-9是GRP78作用的靶基因。与对照组相比,Sh-GRP78组细胞中GRP78、MMP-2和MMP-9的表达水平明显上升,差异均具有统计意义(P<0.05)。结论GRP78在鼻咽癌组织中存在高表达的现象,并且GRP78靶向调控MMP-2和MMP-9的表达增强CNE1细胞的增殖、侵袭与血管新生的能力。

Objective To investigate the mechanism of glucose-regulated protein 78(GRP78)on the proliferation and invasion of nasopharyngeal carcinoma(NPC)cells CNE1.Methods Specimens were collected from 56 newly diagnosed NPC patients without chemo/radiotherapy(40 males and 16 females)and 50 chronic nasopharyngitis patients(38 males and 12 females)from June 2018 to June 2019.Immunohistochemistry was used to detect the expression of GRP78 in NPC tissue and chronic nasopharyngitis tissue.Cell experiments were divided into control group(CNE1 cells were routinely cultured without external intervention),negative control(NC)group(CNE1 cells were transfected with LvGFP-Puro-GRP78-NC,and then routinely cultured),and Sh-GRP78 group(CNE1 cells were transfected with LvGFP-Puro-GRP78,and then routinely cultured).48h after transfection,intracellular green fluorescent protein(GFP)was observed under fluorescence microscope and the expression of GRP78 mRNA was detected by polymerase chain reaction(PCR).In cells of each group,Brdu labeling was used to detect the proliferation ability,Transwell chamber assay to detect the invasion ability,tubule formation experiment to detect the angiogenesis ability,double luciferase assay to analyze the relationship between GRP78 and matrix metalloproteinase-2(MMP-2),MMP-9,and Western blot to detect the expression levels of GRP78,MMP-2 and MMP-9.Results The expression of GRP78 in NPC specimens was significantly higher than that the in chronic nasopharyngitis ones(P<0.05).Compared with the control groups,the abilities of proliferation,invasion and migration,as well as blood vessel formation in the Sh-GRP78 group were significantly enhanced,and the differences were all statistically significant(all P<0.05).Dual luciferase assay showed that MMP-2 and MMP-9 were the target genes of GRP78.Compared with the control group and the NC group,the expression levels of GRP78,MMP-2 and MMP-9 in the Sh-GRP78 group were significantly increased,and the differences were statistically significant(all P<0.05).Conclusion GRP78 is highly expressed in NPC,and GRP78 targets to regulate the expressions of MMP-2 and MMP-9 to enhance the proliferation,invasion and angiogenesis of CNE1 cells.