沉默环指蛋白187对人高转移性肝癌细胞裸鼠移植瘤生长机制的研究

The effects of silencing RNF187 gene on subcutaneous tumor of highly metastatic human hepatocellular carcinoma cells transplanted in nude mice and its possible mechanism

目的观察沉默环指蛋白(RNF)187基因的慢病毒载体对人高转移性肝癌细胞裸鼠中成瘤效应的影响。方法将靶向RNF187基因的短发夹RNA(shRNA)慢病毒(LV-shRNA-RNF187)和阴性对照慢病毒(LV-shRNA-NC)转染至高转移性肝癌细胞(MHCC97-H),并分别作为vshRNF187组和vshNC组,未转染MHCC97-H细胞作为空白对照(Control)组。将3组细胞系接种于裸鼠后观察裸鼠移植成瘤情况及移植瘤生长情况。4周后测量肿瘤的体积和重量,绘制移植瘤生长曲线。实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质印迹法(Western blot)检测裸鼠移植瘤中RNF187的表达。计量资料进行t检验分析,计数资料组间分析采用χ^(2)检验。结果3组裸鼠接种癌细胞后均有肿瘤形成。vshRNF187组裸鼠的肿瘤体积[(746±154)mm],明显低于vshNC组和Control组[(1502±315)、(1590±418)mm],差异有统计学意义(t=5.885、5.083,P<0.01)。vshRNF187组裸鼠瘤体重量[(0.79±0.13)g],低于vshNC组和Control组[(1.57±0.32)、(1.63±0.41)g],差异有统计学意义(t=5.618、4.926,P<0.01)。vshRNF187组瘤组织中RNF187 mRNA的表达量(0.39±0.06)明显低于vshNC组和Control组(1.03±0.06、1.02±0.07,t=21.192、1.623,P<0.01),vshRNF187组瘤组织中RNF187蛋白的表达量(0.365±0.049)明显低于vshNC组和Control组(1.018±0.123、1.168±0.137,t=13.663、14.798,P<0.01),差异均有统计学意义。结论沉默RNF187基因能有效抑制人肝癌裸鼠移植瘤的生长。

Objective To study the effect of lentiviral vector silencing ring finger protein 187(RNF187)gene on tumorigenesis of highly metastatic human hepatocellular carcinoma cells in nude mice.Methods Short hairpin RNA(shRNA)lentivirus(LV-shRNA-RNF187)and negative Control lentivirus(LV-shRNA-NC)targeting RNF187 gene were transfected into highly metastatic human hepatocellular carcinoma cells(MHCC97-H),and used as vsRNF187 group and vshNC group,respectively.The untransfected MHCC97-H cells served as a blank Control(Control)group.After 3 groups of cell lines were inoculated into nude mice,the tumor formation and growth of transplanted tumors were observed.After 4 weeks,the volume and weight of the tumor were measured,and the growth curve of the transplanted tumor was drawn.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of RNF187 in transplanted tumors in nude mice.The measurement data were analyzed by t test,and the count data between groups were analyzed byχ^(2) test.Results Three groups of nude mice all formed tumors after inoculation with cancer cells.The tumor volume of nude mice in the vshRNF187 group was(746±154)mm,which was significantly smaller than that in the vshNC group and control group[(1502±315),(1590±418)mm].The difference was statistically significant(t=5.885,5.083,P<0.01).The tumor weight of vshRNF187 group was(0.79±0.13)g,which was lower than that of vshNC group and control group[(1.57±0.32),(1.63±0.41)g,t=5.618,4.926,P<0.01].The expression of RNF187 mRNA in tumor tissue of vshRNF187 group(0.39±0.06)was significantly lower than that of vshNC group and control group(1.03±0.06,1.02±0.07,t=21.192,1.623,P<0.01).The expression of RNF187 protein in tumor tissue of vshRNF187 group(0.365±0.049)was significantly lower than that of vshNC group and control group(1.018±0.123,1.168±0.137,t=13.663,14.798,P<0.01).Conclusion The silencing of RNF187 gene may effectively inhibit the growth of human hepatocellular carcinoma transplanted tumor in nude mice.