RNAi介导SALL4基因沉默对SKOV-3细胞裸鼠皮下移植瘤生长的影响机制  

Mechanism of RNAi-mediated SALL4 gene silencing on the growth of SKOV-3 cells subcutaneously xenografted tumor in nude mice

作　　者：胡振宇[1] 刘迎春 何宜静 李理 阳哲 HU Zhen-yu;LIU Ying-chun;HE Yi-jing;LI Li;YANG Zhe(Department of Gynecology and Obstetrics,Nanhua Hospital Affiliated to Nanhua University,Hengyang 421002,China;Department of Obstetrics,Second Affiliated Hospital of Nanhua University,Hengyang 421001,China;Department of Gynecology and Obstetrics,Hengyang Maternal and Child Health Hospital,Hengyang 421009,China)

机构地区：[1]南华大学附属南华医院妇产科,湖南衡阳421002 [2]南华大学附属第二医院产科,湖南衡阳421001 [3]衡阳市妇幼保健院妇产科,湖南衡阳421009

出　　处：《中华肿瘤防治杂志》2022年第2期122-128,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：湖南省卫生健康委2019年度科研计划科研项目(B2019116)

摘　　要：目的探讨婆罗双树样基因4(SALL4)对人卵巢癌SKOV-3细胞裸鼠皮下移植瘤生长的影响及可能机制。方法通过SALL4短发夹RNA(shRNA)慢病毒感染构建稳定低表达SALL4基因的SKOV-3细胞株,分为对照组(blank)、载体组(NC shRNA)和干扰组(SALL4 shRNA);采用qRT-PCR和蛋白质印迹法检测各组细胞SALL4 mRNA和蛋白的表达水平。15只雄性BALB/c裸鼠按随机数字法分成3组,每组5只。将各组细胞经右前肢皮下注射建立裸鼠皮下移植瘤模型,根据注射的细胞类别将裸鼠分为对照组(blank,注射SKOV-3细胞)、载体组(NC shRNA,注射NC shRNA干预的SKOV-3细胞)和干扰组(SALL4 shRNA,注射SALL4 shRNA干预的SKOV-3细胞);监测肿瘤生长情况,并绘制肿瘤生长曲线;采用qRT-PCR法检测各组裸鼠移植瘤组织中SALL4 mRNA的表达水平;TUNEL染色观察各组裸鼠移植瘤组织中细胞凋亡情况;免疫组化法观察移植瘤组织中Ki-67的表达水平;蛋白质印迹法检测各组移植瘤组织中SALL4蛋白、凋亡蛋白cleaved caspase-3以及Wnt/β-catenin通路相关蛋白β-catenin、c-Myc和cyclin D1的表达水平。结果SALL4 shRNA慢病毒感染后,SKOV-3细胞中SALL4 mRNA和蛋白表达水平降低,差异有统计学意义,F_(mRNA)=370.032,F_(蛋白)=81.414,均P<0.001。与对照组[(2.13±0.17)g、(1162.00±62.42)mm^(3)]和载体组[(1.98±0.25)g、(1020.00±69.28)mm^(3)]比较,SALL4干扰组[(0.77±0.11)g、(560.40±70.77)mm^(3)]移植瘤质量和体积均降低,差异有统计学意义,F_(瘤质量)=80.584,F_(体积)=33.271,均P<0.001;同时,与对照组[(11.58±1.73)%、0.08±0.04]和载体组[(14.62±1.36)%、0.07±0.08]比较,SALL4干扰组[(56.84±4.48)%、0.87±0.12]移植瘤组织细胞凋亡率以及cleaved caspase-3蛋白表达水平增加,差异有统计学意义(F值分别为254.180、759.081,均P<0.001),而与对照组[(78.66±2.21)%、0.89±0.06、0.20±0.05、0.24±0.04]和载体组[(74.82±3.07)%、0.82±0.08、0.19±0.02、0.22±0.05]比较,SALL4干扰组[(17.15±0.82)%Objective To investigate the effect of Spalt-like transcription factor 4(SALL4)on the growth of human ovarian cancer SKOV-3 cells subcutaneously transplanted in nude mice and its possible mechanism.Methods SALL4 shRNA lentiviral infection was used to construct SKOV-3 cell lines with stable and low expression of SALL4 gene.The cells were divided into control group(blank),carrier group(NC shRNA)and interference group(SALL4 shRNA).The expression of SALL4 mRNA and protein in each group cells were detected by qRT-PCR and Western bloting.Fifteen male BALB/c nude mice were randomly divided into 3 groups,with 5 in each group.The each group cells were injected subcutaneously through the right forelimb to establish a model of subcutaneous xenograft tumor in nude mice.According to the type of cells injected,nude mice were divided into control group(blank,injected with SKOV-3 cells),carrier group(NC shRNA,injected with SKOV-3 cells intervened by NC shRNA),and interference group(SALL4 shRNA,injected with SALL4 shRNA intervened by SKOV-3 cells);the tumor growth was monitored,and the tumor growth curve was drawn;qRT-PCR was used to detect the expression of SALL4 mRNA in transplanted tumor tissues;TUNEL staining was used to observe the apoptosis of transplanted tumor tissue of each group;Immunohistochemical method was used to observe the expression of Ki-67 in transplanted tumor tissue;Western bloting was used to detect the expression of SALL4 protein,apoptotic protein cleaved caspase-3,Wnt/β-catenin pathway-related proteinsβ-catenin,c-Myc and cyclin D1 in the transplanted tumor tissues.Results After SALL4 shRNA lenticirus infection,the mRNA and protein expression levels of SALL4 in SKOV-3 cells were decreased,the difference was statistically significant(F_(mRNA)=370.032,F_(protein)=81.414,P<0.001).Compared with the blank group[(2.13±0.17)g,(1162.00±62.42)mm^(3)]and the NC shRNA group[(1.98±0.25)g,(1020.00±69.28)mm^(3)],the tumor weight and volume of the transplanted tumor in the SALL4 shRNA group[(0.77±0.11)g,(560.40±70

关 键 词：婆罗双树样基因4 人卵巢癌 移植瘤 WNT/Β-CATENIN信号通路 RNA干扰 

分 类 号：R737.31[医药卫生—肿瘤]