lncRNA HOTAIR靶向调控血管舒张剂激活磷蛋白对宫颈癌细胞迁移与侵袭的影响

Effect of targeted regulation of vasodilator activated phosphoprotein by lncRNA HOTAIR on cell migration and invasion of cervical cells

目的 探讨长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)和血管舒张剂激活磷蛋白(VASP)在宫颈癌细胞迁移和侵袭过程中的分子调控机制。方法 敲降HOTAIR实验中,siHOTAIR-Ⅰ和siHOTAIR-Ⅱ作为HOTAIR敲降组。敲降VASP实验中,siVASP作为VASP敲降组。2组实验均以siNC作为对照。过表达VASP实验中,通过脂质体转染pCDNA3.1-VASP使VASP在HeLa细胞中高表达,以空载pCDNA3.1作为对照。实时荧光定量PCR(qRT-PCR)检测敲降HOTAIR后HOTAIR表达变化情况。蛋白质印迹法检测敲降HOTAIR后VASP蛋白表达水平的变化,划痕实验和Transwell实验用于检测敲降HOTAIR或过表达/敲降VASP对HeLa细胞迁移和侵袭的影响。生物信息学分析用于分析VASP与miR-206之间的调控关系。过表达miR-206实验中,转染miR-206 mimics用于HeLa细胞中过表达miR-206,NC(mimics)作为对照组。敲降miR-206实验中,转染miR-206 inhibitor用于抑制miR-206表达,NC(inhibitor)作为对照组。蛋白质印迹法检测过表达或抑制miR-206对VASP蛋白水平的影响。将miR-206 mimics与VASP-UTR进行共转染,通过双荧光素酶报告系统检测miR-206对VASP的调控作用,共转染miR-206 mimics与VASP-UTR-wt作为对照。划痕实验和Transwell实验用于检测NC(mimics)、miR-206 mimics以及共转染miR-206 mimics与siVASP 3种分组对HeLa细胞迁移和侵袭的影响。采用独立样本t检验、单因素方差分析以及Dunnett-t等方法对实验数据进行统计学分析。结果 通过siRNA沉默HOTAIR表达,与siNC组(1.00±0.02)相比,siHOTAIR-Ⅰ和siHOTAIR-Ⅱ组中HOTAIR相对表达量分别为0.36±0.05和0.42±0.02,差异有统计学意义,F=387.724,P<0.001。蛋白质印迹法结果显示,抑制HOTAIR可下调VASP蛋白表达,F=93.722,P<0.001。划痕实验结果显示,siNC组及敲降HOTAIR实验组划痕的相对宽度分别为0.18±0.02、0.41±0.02和0.39±0.03,差异有统计学意义,F=85.941,P<0.001。Transwell实验结果显示,siNC组及敲降HOTAIR实验组中侵袭转移的细胞相对数目分别为1.00±0.07、0.34±0.03和0.28±0.09,差异有统计学意义,F=116.153,P<0.001。生物信息学分析结果表明,VASP可能是miR-206的靶基因。同时,转染miR-206 mimics后,相比于NC(mimics)的0.99±0.05,VASP相对表达量为0.73±0.04,差异有统计学意义,F=35.788,P=0.004。转染miR-206 inhibitor后,VASP的相对表达量为1.65±0.07,相比于NC(inhibitor)中VASP的相对表达量0.96±0.03,差异有统计学意义,F=227.705,P<0.001。双荧光素酶报告基因检测结果显示,共转染miR-206 mimics和VASP UTR-mut荧光素酶活性(6.44±0.42)高于共转染miR-206 mimics和VASP UTR-wt(1.00±0.18),差异有统计学意义,F=425.195,P<0.001。同时,划痕实验结果显示,转染miR-206 mimics在24和48 h划痕的相对宽度分别为0.62±0.03和0.37±0.02,相比于NC(mimics)的0.44±0.02和0.28±0.02,差异有统计学意义(F_(24 h)=74.769,P_(24 h)<0.001;F_(48 h)=30.375,P_(48 h)=0.005)。Transwell实验结果显示,转染miR-206 mimics后侵袭转移的细胞相对数目为0.38±0.06,相比于NC(mimics)的1.00±0.04,差异有统计学意义,F=221.769,P<0.001。而且,沉默HOTAIR表达后,miR-206相对RNA表达水平分别为17.32±3.12和18.52±4.24,高于siNC组(1.00±0.03),差异有统计学意义,F=31.549,P<0.001。结论 HOTAIR调控宫颈癌细胞的迁移和侵袭可能是通过miR-206调控VASP表达来实现的。

Objective To investigate the molecular regulatory mechanism between HOX transcript antisense RNA(HOTAIR) and vasodilator-stimulated phosphoprotein(VASP) in cell migration and invasion of cervical cancer.Methods In HeLa cells, siHOTAIR-Ⅰ and siHOTAIR-Ⅱ were used to knockdown the expression of HOTAIR,and siVASP was used to silence VASP expression, respectively.SiNC was used as a negative control.The plasmid pcDNA3.1-VASP was transfected into HeLa cells to express VASP,and the empty plasmid pCDNA3.1 was used as a control.Quantitative real-time PCR(qRT-PCR) was used to measure the expression of HOTAIR after transfecting siHOTAIR-Ⅰ or siHOTAIR-Ⅱ.Western blotting was employed to measure VASP expression after HOTAIR inhibition.Wound healing and transwell assays were used to determine the capability of migration and invasion of HeLa cells after HOTAIR inhibition or overexpression/knockdown VASP.Bioinformatics analysis and dual luciferase reporter system were used to analyze the regulatory relationship between VASP and miR-206.MiR-206 mimics and miR-206 inhibitor, respectively, were used to overexpress or knockdown miR-206 expression in HeLa cells, comparing with NC(mimics) or NC(inhibitor).Western blotting was used to determine the expression of VASP after overexpression or inhibition miR-206 in HeLa cells.The luciferase activity was measured after co-transfecting with miR-206 mimics and VASP-UTR-wt, compared with co-transfecting with miR-206 mimics and VASP-UTR.Wound healing and Transwell assays were used to assess the capability of migration and invasion of HeLa cells after transfecting with NC(mimics),miR-206 mimics or co-transfected miR-206 mimics.Independent sample t-test, one-way analysis of variance and Dunnett-t were used for statistical analysis.Results After inhibition of HOTAIR by siRNA,compared with the siNC group(1.00±0.02),the relative expression levels of HOTAIR in the siHOTAIR-Ⅰ and siHOTAIR-Ⅱ groups were 0.36±0.05 and 0.42±0.02,respectively, and the difference was statistically significant(F=387.724,P<0.001).The result of Western blotting indicated that inhibition of HOTAIR could down-regulate VASP(F=93.722,P<0.001).The results of wound healing showed that the relative width of wound scratch after HOTAIR inhibition were 0.41±0.02 and 0.39±0.03,respectively, comparing with siNC group(0.18±0.02),indicating a significant difference(F=85.941,P<0.001).Transwell assay showed that the relative number of invading cells in siNC group and the HOTAIR-knockdown group were 1.00±0.07,0.34±0.03 and 0.28±0.09,respectively, with significant difference(F=116.153,P<0.001).Bioinformatics analysis showed that VASP may be a target gene of miR-206.Meanwhile, after overexpression of miR-206,the relative protein expression VASP(0.73±0.04) was significantly decreased compared with the NC(mimics) group(0.99±0.05),F=35.788,P=0.004.However, compared with the NC(inhibitor) group(0.96±0.03),the relative protein expression VASP(1.65±0.07) was significantly increased after transfecting with miR-206 inhibitor in HeLa cells.Dual luciferase assay showed that the luciferase activity of co-transfected miR-206 mimics and VASP UTR-mut(6.44±0.42) was higher than that of co-transfected miR-206 mimics and VASP UTR-wt(1.00±0.18),with a statistically significant difference(F=425.195,P<0.001).Furthermore, wound healing showed that the relative width of scratch after transfecting with miR-206 mimics at 24 and 48 hours were 0.62±0.03 and 0.37±0.02,respectively, compared with the NC(mimics) group(0.44±0.02 and 0.28±0.02),with a statistically significant difference(F_(24 h)=74.769,P_(24 h)0.001;F_(48 h)=30.375,P_(48 h)=0.005).Transwell assay showed that the relative number of invading cells after overexpression of miR-206 was(0.38±0.06),comparing with the NC(mimics) group(1.00±0.04),with significant differences(F=221.769,P<0.001).Moreover, after inhibition of HOTAIR,the relative RNA expression levels of miR-206(17.32±3.12 and 18.52±4.24),were higher than siNC group(1.00±0.03),and the difference was statistically significant(F=31.549,P<0.001).Conclusion HOTAIR may exert its function on the migration and invasion of HeLa cells through the regulation of VASP expression via miR-206.