CAJ | 학술논문

目的观察小于扰RNA(siRNA)沉默结直肠癌HT-29细胞TCF21基因对Kiss-1基因表达的影响,以及细胞增殖、侵袭及迁移能力等生物学行为的变化.方法利用脂质体lipofectamineTM 2000将siRNA稳定转染结直肠癌HT-29细胞,实验分为干扰组、阴性对照组、空白对照组；应用实时定量逆转录聚合酶链反应(RT-qPCR)与Western blot技术检测TCF21与Kiss-1基因mRNA与蛋白的表达水平,分别采用细胞计数试剂盒(CCK-8)法和Transwell小室法检测干扰前后细胞增殖、侵袭与迁移能力的变化.结果 siRNA干扰后成功沉默了HT-29细胞TCF21基因的表达,Kiss-1基因mRNA的表达量为0.58 ±0.02,较对照组的1.00±0.00明显降低(P＜0.05),蛋白表达量为0.3491±0.0009,与对照组比较(0.8485 ±0.0016)亦出现明显下降(P＜0.05),同时细胞增殖、侵袭及迁移能力均明显增强(P＜0.01).结论沉默TCF21基因的表达可以下调结直肠癌细胞Kiss-1基因的表达水平,并导致相应生物学行为的变化,因此TC F21基因可能是Kiss-1基因的上游调控因子.
목적 관찰소우우RNA(siRNA)침묵결직장암HT-29세포TCF21기인대Kiss-1기인표체적영향,이급세포증식、침습급천이능력등생물학행위적변화.방법 이용지질체lipofectamineTM 2000장siRNA은정전염결직장암HT-29세포,실험분위간우조、음성대조조、공백대조조；응용실시정량역전록취합매련반응(RT-qPCR)여Western blot기술검측TCF21여Kiss-1기인mRNA여단백적표체수평,분별채용세포계수시제합(CCK-8)법화Transwell소실법검측간우전후세포증식、침습여천이능력적변화.결과 siRNA간우후성공침묵료HT-29세포TCF21기인적표체,Kiss-1기인mRNA적표체량위0.58 ±0.02,교대조조적1.00±0.00명현강저(P＜0.05),단백표체량위0.3491±0.0009,여대조조비교(0.8485 ±0.0016)역출현명현하강(P＜0.05),동시세포증식、침습급천이능력균명현증강(P＜0.01).결론 침묵TCF21기인적표체가이하조결직장암세포Kiss-1기인적표체수평,병도치상응생물학행위적변화,인차TC F21기인가능시Kiss-1기인적상유조공인자.
Objective To investigate the effect of silencing transcription factor 21 (TCF21) gene expression by small interfering RNA (siRNA) on the expression of Kiss-1 gene and the cell biological behaviors of proliferation,invasion and migration in human colorectal cancer cell line HT-29 in vivo.Methods A specific siRNA molecule fragment for TCF21 was designed,and transfected into HT-29 cells by lipofectamineTM 2000.The experimental group,negative control group and blank control group were set up.Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression levels of TCF21 and Kiss-1 mRNA at 48 h after transfection.By using Western blotting,the expression levels of TCF21 and Kiss-1 proteins at 72nd h after transfection was examined.The Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation capacity,and Transwell assay to detect the invasion and migration capacity at 72nd h post-transfection.Results The siRNA fragment could silence TCF21 gene expression successfully,then leading to a substantial drop-off in the expression of Kiss-1 mRNA (0.58 ± 0.02) and protein (0.3491 ±0.0009,P ＜0.05).CCK-8 test and Transwell assay revealed that cells after transfection proliferated more rapidly than before transfection and had an increasing capability of invasion and migration (P ＜ 0.01).Conclusion The silencing of TCF21 expression in colorectal cancer cell line HT-29 cells could decline the expression level of Kiss-1 mRNA and protein,and lead to the corresponding changes of cell biological behaviors,suggesting TCF21 gene might be the upstream regulator of Kiss-1 gene.