摘要
目的探讨小干扰RNA(siRNA)靶向沉默整合素连接激酶(ILK)基因对宫颈鳞状上皮细胞(H8细胞)增殖、迁移和侵袭等生物学行为的影响。方法设计高效的靶向沉默ILK的siRNA,构建ILK-siRNA脂质体和无关对照质粒。将H8细胞随机分为ILK基因沉默组、阴性对照组和空白组,ILK基因沉默组H8细胞转染ILK-siRNA,阴性对照组细胞转染无关对照质粒,空白组细胞不转染任何序列,转染后继续培养48 h。采用实时荧光定量聚合酶链反应法检测H8细胞中ILK mRNA相对表达量,Western blot法检测H8细胞中ILK蛋白相对表达量,四甲基偶氮唑盐法检测H8细胞活力,Transwell实验检测H8细胞侵袭能力,划痕实验检测细胞迁移能力,免疫组织化学法检测H8细胞内Ki-67蛋白表达。结果ILK基因沉默组H8细胞中ILK mRNA和蛋白相对表达量显著低于阴性对照组和空白组(P<0.05),阴性对照组与空白组H8细胞中ILK mRNA和蛋白相对表达量比较差异无统计学意义(P>0.05)。ILK基因沉默组H8细胞活力显著低于空白组和阴性对照组(P<0.05),空白组与阴性对照组H8细胞活力比较差异无统计学意义(P>0.05)。空白组、阴性对照组和ILK基因沉默组H8细胞Ki-67蛋白阳性表达率分别为(20.01±0.31)%、(21.01±0.11)%、(12.01±0.01)%,ILK基因沉默组H8细胞Ki-67蛋白阳性表达率显著低于阴性对照组和空白组(P<0.05),阴性对照组与空白组H8细胞Ki-67蛋白阳性表达率比较差异无统计学意义(P>0.05)。空白组、阴性对照组和ILK基因沉默组穿膜细胞数分别为28.13±2.44、30.24±2.15、18.52±2.25,ILK基因沉默组穿膜细胞数显著少于空白组和阴性对照组(P<0.05),空白组与阴性对照组穿膜细胞数比较差异无统计学意义(P>0.05)。空白组、阴性对照组和ILK基因沉默组H8细胞迁移率分别为(15.51±4.88)%、(15.11±5.21)%、(6.91±5.12)%,ILK基因沉默组H8细胞迁移率显著低于阴性对照组和空白组(P<0.05),阴性对照组与空白组H8细胞迁移率比较差异无统计学意义(P>0.05)。结论ILK参与调控宫颈鳞状上皮细胞的增殖、迁移以及侵袭,可能在宫颈癌的发生、发展中发挥重要作用;沉默ILK基因可以抑制H8细胞的增殖、迁移和侵袭能力,ILK有望成为宫颈癌靶向治疗的新靶点。
Objective To investigate the effect of small interfering RNA(siRNA)targeted silencing integrin-linked kinase(ILK)gene on the proliferation,migration and invasion of cervical squamous epithelial cells(H8 cells).Methods The high efficient siRNA targeted silencing ILK gene was designed,and ILK-siRNA liposomes and the unrelated control plasmids were constructed.The H8 cells were randomly divided into ILK gene silencing group,negative control group and blank group,the H8 cells in the ILK gene silencing group and negative control group and blank group were transfected with ILK-siRNA and unrelated control plasmids,respectively;while the cells in the blank group were not transfected with any sequence.All cells were cultured for 48 hours after transfection.The relative expression of ILK mRNA in H8 cells was detected by real-time fluorescence quantitative polymerase chain reaction,and the relative expression of ILK protein in H8 cells was detected by Western blot method.The activity of H8 cells were detected by methyl thiazolyl tetrazolium method.The invasion ability of H8 cells was detected by Transwell test,and the migration ability of H8 cells was detected by scratch test.The expression of Ki-67 protein in H8 cells was detected by immunohistochemistry.Results The relative expression of ILK mRNA and protein in H8 cells of ILK gene silencing group was significantly lower than that of the negative control group and blank group(P<0.05),and there was no significant difference in the relative expression of ILK mRNA and protein in H8 cells between the negative control group and the blank group(P>0.05).The activity of H8 cells in the ILK gene silencing group were significantly lower than those in the blank group and the negative control group(P<0.05),and there was no significant difference in the activity of H8 cells between the blank group and the negative control group(P>0.05).The positive expression rate of Ki-67 protein in H8 cells of the blank group,negative control group and ILK gene silencing group was(20.01±0.31)%,(21.01±0.11)%and(12.01±0.01)%,respectively.The positive expression rate of Ki-67 protein in H8 cells of the ILK gene silencing group was significantly lower than that of the negative control group and the blank group(P<0.05),and there was no significant difference in the positive expression rate of Ki-67 protein in H8 cells between the negative control group and the blank group(P>0.05).The number of transmembrane cells in the blank group,the negative control group and the ILK gene silencing group was 28.13±2.44,30.24±2.15 and 18.52±2.25,respectively.The number of transmembrane cells in the ILK gene silencing group was significantly less than that in the blank control group and the negative control group(P<0.05),and there was no significant difference in the number of transmembrane cells between the blank group and the negative control group(P>0.05).The migration rate of H8 cells in the blank group,negative control group and ILK gene silencing group was(15.51±4.88)%,(15.11±5.21)%and(6.91±5.12)%,respectively.The migration rate of H8 cells in the ILK gene silencing group was significantly lower than that in the negative control group and the blank group(P<0.05),and there was no significant difference in the migration rate of H8 cells between the negative control group and the blank group(P>0.05).Conclusion ILK is involved in the regulation of proliferation,migration and invasion of cervical squamous epithelial cells,which may play an important role in the occurrence and development of cervical cancer.Silencing ILK gene can inhibit the proliferation,migration and invasion of H8 cells,ILK is expected to be a new target of targeted therapy for cervical cancer.
作者
李晓丹
费晓莺
王敬苗
霍晓燕
于华
LI Xiaodan;FEI Xiaoying;WANG Jingmiao;HUO Xiaoyan;YU Hua(Department of Gynecology,the First Hospital of Handan City,Handan 056002,Hebei Province,China)
出处
《新乡医学院学报》
CAS
2020年第5期425-429,共5页
Journal of Xinxiang Medical University
基金
邯郸市科学技术研究与发展计划项目(编号:1723208066-2)。
关键词
整合素连接激酶
宫颈鳞状上皮细胞
宫颈癌
小干扰RNA
靶向沉默
细胞增殖
细胞侵袭
integrin-linked kinase
cervical squamous epithelial cells
cervical cancer
small interfering RNA
targeted silencing
cell proliferation
cell invasion
