摘要
目的探讨胶原三螺旋重复蛋白-1(CTHRC1)对口腔鳞状细胞癌细胞血管生成拟态(VM)的影响.方法采用实时荧光定量聚合酶链反应(qRT-PCR)法检测口腔鳞状细胞癌细胞系(SCC-9、SCC-15、SCC-25、SAS和Tca8113)和人口腔上皮角质细胞HOK中CTHRC1 mRNA表达水平.选择CTHRC1 mRNA表达量最高的口腔鳞状细胞癌细胞系SCC-9为研究对象,通过慢病毒感染构建CTHRC1基因低表达的SCC-9细胞,将细胞分为空白对照组(Blank)、阴性对照干扰组(NC-shRNA)和CTHRC1干扰组(CTHRC1-shRNA).采用qRT-PCR法和蛋白质印迹法检测慢病毒感染的各组SCC-9细胞中CTHRC1基因mRNA和蛋白表达水平;三维培养观察各组SCC-9细胞VM管腔数量;酶谱凝胶法检测各组SCC-9细胞上清液中分泌型基质金属蛋白酶MMP-2和MMP-9的活性;Transwell法检测各组SCC-9细胞迁移侵袭情况;蛋白质印迹法检测各组SCC-9细胞血管生成相关蛋白VEGF和VE-cadherin的表达水平.结果6种细胞株(HOK、SCC-9、SCC-15、SCC-25、SAS和Tca8113)的CTHRC1 mRNA表达水平差异有统计学意义,F=27.318,P<0.05.两两多重比较显示,其他细胞株较HOK细胞表达均升高,差异均有统计学意义,均P<0.05.Blank组、NC-shR-NA组和CTHRC1-shRNA组SCC-9细胞中CTHRC1 mRNA和蛋白表达水平(F_(mRNA)=6.766;F_(蛋白)=9.757)、VM管腔形成数量(F=48.211)、上清液MMP-2和MMP-9的活性(FMMP-2=45.121;FMMP-9=49.420)、迁移和侵袭细胞数量占比(F_(迁移)=361.176;F_(侵袭)=203.144)、VEGF和VE-cadherin蛋白表达水平(FVEGF=37.402;F_(VE-cadherin)=19.816)差异均有统计学意义,均P<0.05.与Blank组比较,NC-shRNA组SCC-9细胞各指标水平差异无统计学意义,均P>0.05,CTHRC1-shRNA组SSC-9细胞各指标水平均下降,差异均有统计学意义,均P<0.05.结论CTHRC1在人口腔鳞状细胞癌细胞中高表达,沉默CTHRC1能通过下调VEGF和VE-cadherin蛋白表达水平,抑制人口腔鳞状细胞癌细胞SCC-9的VM形成.
Objective To investigate the effect of collagen triple helix repeat containing 1(CTHRC1)on vasculogenic mimicry(VM)of oral squamous cell carcinoma cells.Methods The expression of CTHRC1mRNA in oral squamous cell carcinoma cell lines(SCC-9,SCC-15,SCC-25,SAS,Tca8113)and human oral epithelial keratinocyte HOK were detected by qRT-PCR assay.The oral squamous cell carcinoma cell line SCC-9with the highest CTHRC1mRNA expression was selected as the research object.CTHRC1gene low-expression of SCC-9cells were constructed by lentivirus infection,so the cells were divided into blank control group(Blank),negative control interference group(NC-shRNA)and CTHRC1interference group(CTHRC1-shRNA).qRT-PCR and Western blot methods were used to measure the expression of CTHRC1 mRNA and protein;Three-dimensional culture was used to observe the number of VM tube of SCC-9cells in each group;Zymogram gel assay was used to detect the activity of matrix metalloproteinases MMP-2and MMP-9in the supernatant of SCC-9cells in each group;The migration and invasion of SCC-9cells were measured by Transwell assay in each group;The expression of angiogenesis-related proteins VEGF and VE-cadherin were detected by Western blot assay in SCC-9 cells of each group.Results The difference in CTHRC1mRNA expression level of 6cell lines(HOK,SCC-9,SCC-15,SCC-25,SAS,Tca8113)was statistically significant(F=27.318,P<0.05).Pairwise multiple comparisons showed that compared with HOK cells,the expression of CTHRC1mRNA in other cell lines increased,and the difference was statistically significant,all P<0.05.The difference in CTHRC1mRNA and protein expression levels(FmRNA=6.766;Fprotein=9.757),the number of VM lumens(F=48.211),the activity of MMP-2and MMP-9in the supernatant(FMMP-2=45.121;FMMP-9=49.420),the proportion of migration and invasion cells(F_(migration)=361.176;F_(invasion)=203.144),VEGF and VE-cadherin protein expression levels(F_(VEGF)=37.402;F_(VE-cadherin)=19.816)were statistically significant in SCC-9cells in the Blank group,NC-shRNA group and CTHRC1-s
作者
许玉萍
王金龄
邓金勇
李欢
XU Yu-ping;WANG Jin-ling;DEGN Jin-yong;LI Huan(Sanya People's Hospital,Sanya572000,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2021年第23期1785-1791,共7页
Chinese Journal of Cancer Prevention and Treatment
关键词
胶原三螺旋重复蛋白-1
口腔鳞状细胞癌
血管生成拟态
迁移
侵袭
collagen triple helix repeat containing-1
oral squamous cell carcinoma cell
vasculogenic mimicry
migration
invasion
