长链非编码RNA SNHG16对人宫颈癌细胞SiHa迁移侵袭的影响

Effect of long non-coding RNA SNHG16 on migration and invasion of human cervical cancer cell SiHa

目的探讨长链非编码RNA(lncRNA)SNHG16通过调控微RNA(miR)-216-5p/锌指E盒结合同源框1(ZEB1)轴对人宫颈癌细胞SiHa迁移、侵袭的影响及机制。方法将对数生长期人宫颈癌细胞SiHa随机分为干扰小RNA(siRNA)组、阴性对照组和空白组。siRNA组、阴性对照组SiHa细胞应用脂质体转染法分别进行lncRNA SNHG16-siRNA、阴性对照-siRNA质粒转染,空白组细胞不作处理。转染后48 h,采用倒置荧光显微镜观察转染效率,实时荧光定量聚合酶链反应(qRT-PCR)法测定各组SiHa细胞lncRNA SNHG16 mRNA相对表达量,生物信息学软件靶向预测和双荧光素酶实验验证lncRNA SNHG16与miR-216-5p的靶向关系,划痕实验测定各组细胞迁移能力,Transwell小室实验测定各组细胞侵袭能力,qRT-PCR测定各组细胞miR-216-5p、ZEB1、E-钙黏蛋白(E-cadherin)mRNA相对表达量,Western blot法测定各组细胞ZEB1、E-cadherin相对表达量。结果转染48 h后,各组细胞转染效率均>80%;siRNA组SiHa细胞lncRNA SNHG16 mRNA相对表达量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lncRNA SNHG16 mRNA相对表达量比较差异无统计学意义(P>0.05)。LncRNA SNHG16与miR-216-5p基因3′UTR存在互补结合位点;siRNA组野生型荧光素酶报告质粒相对活性值显著高于空白组和阴性对照组(P<0.05),空白组与阴性对照组野生型荧光素酶报告质粒相对活性值比较差异无统计学意义(P>0.05)。siRNA组SiHa细胞迁移率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细胞迁移率比较差异无统计学意义(P>0.05);siRNA组每个视野平均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每个视野平均穿膜细胞数比较差异无统计学意义(P>0.05)。siRNA组SiHa细胞miR-216-5p mRNA相对表达量、E-cadherin mRNA及蛋白相对表达量均高于空白组和阴性对照组(P<0.05),空白组与阴性对照组miR-216-5p mRNA相对表达量、E-cadherin mRNA及蛋白相对表达量比较差异无统计学意义(P>0.05);siRNA组SiHa细胞ZEB1 mRNA及蛋白相对表达量均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ZEB1 mRNA及蛋白相对表达量比较差异均无统计学意义(P>0.05)。结论沉默lncRNA SNHG16可抑制人宫颈癌细胞SiHa的迁移与侵袭能力,其作用机制可能与调控miR-216-5p/ZEB1轴相关基因和蛋白表达有关。

Objective To explore the effect and mechanism of long non-coding(lnc)RNA SNHG16 on the migration and invasion of human cervical cancer cell SiHa by regulating the microRNA(miR)-216-5p/zinc finger E box binding homeobox 1(ZEB1)axis.Methods The human cervical cancer cells SiHa in the logarithmic growth stage were randomly divi-ded into the small interfering RNA(siRNA)group,negative control group and blank group.LncRNA SNHG16-siRNA and negative control plasmids were respectively transfected into SiHa cells in the siRNA group and negative control group by liposome transfection method.The SiHa cells in the blank group were not treated.At 48 h after transfection,the transfection efficiency was observed by inverted fluorescence microscope,the relative expression of lncRNA SNHG16 mRNA in SiHa cell in each group was measured by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR),the targeting relationship between lncRNA SNHG16 and miR-216-5p were verified by bioinformatics software and dual luciferase experiments,the migration ability of SiHa cell in each group was determined by scratch test,the invasion ability of SiHa cell in each group was detected by Transwell chamber test,the relative expression of miR-216-5p,ZEB1 and E-cadherin mRNA of SiHa cell in each group was detected by qRT-PCR,the relative expression of miR-216-5p,ZEB1 and E-cadherin protein of SiHa cell in each group was detected by Western blot.Results At 48 h after transfection,the transfection efficiency of each group were higher than 80%.The relative expression of lncRNA SNHG16 mRNA in SiHa cell in the siRNA group was lower than that in the blank group and negative control group(P<0.05),there was no difference in the relative expression of lncRNA SNHG16 mRNA in SiHa cell between the blank group and negative control group(P>0.05).There was the complementary binding sites between lncRNA SNHG16 and 3′UTR of miR-216-5p.The relative activity value of the wild-type luciferase reporter plasmid in the siRNA group was higher than that in the blank group and negative control group(P<0.05),there was no difference in the relative activity value of the wild-type luciferase reporter plasmid between the blank group and negative control-siRNA group(P>0.05).The cell migration rate of SiHa cell,in the siRNA group was lower than that in the blank group and negative control group(P<0.05),there was no difference in the cell migration rate of SiHa cell between the blank group and the negative control group(P>0.05).The average number of perforating cells per field in the siRNA group was less than that in the blank group and negative control group(P<0.05),there was no difference in the average number of perforating cells per field between the blank group and negative control group(P>0.05).The relative expression levels of miR-216-5p mRNA,E-cadherin mRNA and protein of SiHa cell in the siRNA group were higher than those in the blank group and negative control group(P<0.05),there was no difference in the relative expression levels of miR-216-5p mRNA,E-cadherin mRNA and protein between the blank group and negative control group(P>0.05);the relative expression levels of ZEB1 mRNA and protein were lower than those in the blank group and negative control group(P<0.05),there was no difference in the ZEB1 mRNA and protein between the blank group and negative control group(P>0.05).Conclusion Knockdown of lncRNA SNHG16 can inhibit the migration and invasion of human cervical cancer cells SiHa,the mechanism may be related to regulating the expression of genes and proteins related to miR-216-5p/ZEB1 axis.