RNAi沉默Arl4c对结肠癌细胞株HCT116增殖能力的影响
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摘要
目的探讨利用RNA干扰技术沉默Arl4c基因对结肠癌HCT116细胞株增殖的影响。方法将Arl4c基因的siRNA稳定转染至HCT116细胞,采用RT-PCR、Western blot鉴定干扰效果,以HCT116细胞沉默Arl4c基因组为实验组,以转染空质粒的HCT116细胞和空白的HCT116细胞为对照组,分别采用四甲基偶氮唑蓝(MTT)法、细胞倍增时间、平板克隆形成实验、流式细胞术检测沉默Arl4c基因后,HCT116细胞生长速度、细胞周期、克隆形成能力的改变。结果沉默Arl4c基因后,实验组细胞的生长速度明显下降,G1期细胞增多、S期细胞减少,空白对照、空质粒对照组和实验组细胞平均倍增时间分别为(4.718±0.212)h,(4.981±0.24)h和(6.0±0.137)h;空白对照、空质粒对照组和实验组平均细胞克隆形成数目分别为121±9.00,117±10.00和80±9.00。实验组与空白对照、空质粒对照组相比较,差异有统计学意义(P<0.05)。结论 Arl4c基因的沉默能够抑制结肠癌细胞HCT116的增殖,可能在结肠癌的发生发展中起重要作用。
Objective To investigate the effects of RNA interference-mediated gene silence of Arl4c on the proliferation of colon cancer cell line HCT116. Methods Colon cancer cell line HCT116 was transfected with Arl4c small interfering RNA( siRNA),and then the expression of Arl4c was determined by RT-PCR and Western blot. HCT116 cells after Arl4c gene knockdown were chosen as experimental group,and HCT116 cells transfected with empty plasmid transfection and HCT116 cells were chosen as control groups. Then the proliferation of HCT116 was examined by 3-( 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide( MTT) assay,the cell doubling time of HCT116 was examined by double time assay,the colony formation ability of HCT116 was examined by colony formation assay,and the cell cycles of HCT116 were examined by flow cytometry assay. Results Compared with the blank control group and empty plasmid control group,the growth ability in experimental group was significantly decreased,G1 phase cells increased,and S phase cells decreased. The doubling time in blank control group,empty plasmid group and experimental group was( 4. 718 ± 0. 212) h,( 4. 981 ± 0. 24) h,( 6. 0 ± 0. 137) h. The colony number was 121 ± 9. 00 in blank control group,117 ± 10. 00 in empty plasmid control group,and 80 ± 9. 00 in experimental group,and there was significant difference between control groups and experimental group( P <0. 05). Conclusion Arl4c gene silencing by RNAi can suppress the proliferation of colon cancer cell line HCT116,and it may be involved in colon cancer progression.
Objective To investigate the effects of RNA interference-mediated gene silence of Arl4c on the proliferation of colon cancer cell line HCT116. Methods Colon cancer cell line HCT116 was transfected with Arl4c small interfering RNA( siRNA),and then the expression of Arl4c was determined by RT-PCR and Western blot. HCT116 cells after Arl4c gene knockdown were chosen as experimental group,and HCT116 cells transfected with empty plasmid transfection and HCT116 cells were chosen as control groups. Then the proliferation of HCT116 was examined by 3-( 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide( MTT) assay,the cell doubling time of HCT116 was examined by double time assay,the colony formation ability of HCT116 was examined by colony formation assay,and the cell cycles of HCT116 were examined by flow cytometry assay. Results Compared with the blank control group and empty plasmid control group,the growth ability in experimental group was significantly decreased,G1 phase cells increased,and S phase cells decreased. The doubling time in blank control group,empty plasmid group and experimental group was( 4. 718 ± 0. 212) h,( 4. 981 ± 0. 24) h,( 6. 0 ± 0. 137) h. The colony number was 121 ± 9. 00 in blank control group,117 ± 10. 00 in empty plasmid control group,and 80 ± 9. 00 in experimental group,and there was significant difference between control groups and experimental group( P <0. 05). Conclusion Arl4c gene silencing by RNAi can suppress the proliferation of colon cancer cell line HCT116,and it may be involved in colon cancer progression.
引文
[1]訾永宏,姬乐,陈红梅,等.Arl4c在结肠癌中的表达及其临床意义[J].山西医科大学学报,2017,48(1):44-46.
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[8]Sun D,Zhang J,Xie J,et al.MiR-26 controls LXR-dependent cholesterol efflux by targeting ABCA1 and ARL7[J].FEBS Lett,2012,586(10):1472-1479.
[9]Fujii S,Shinjo K,Matsumoto S,et al.Epigenetic upregulation of ARL4C,due to DNA hypomethylation in the 3'-untranslated region,promotes tumorigenesis of lung squamous cell carcinoma[J].Oncotarget,2016,7(49):81571-81587.
[10]Arend RC,Londono-Joshi AI,Straughn JM Jr,et al.The Wnt/β-catenin pathway in ovarian cancer:a review[J].Gynecol Oncol,2013,131(3):772-779.
[2]Gillingham AK,Munro S.The small G proteins of the Arffamily and their regulators[J].Annu Rev Cell Dev Biol,2007,23:579-611.
[3]Fujii S,Matsumoto S,Nojima S,et al.Arl4c expression in colorectal and lung cancers promotes tumorigenesis and may represent a novel therapeutic target[J].Oncogene,2015,34(37):4834-4844.
[4]Su D,Katsaros D,Xu S,et al.ADP-ribosylation factor-like 4C(ARL4C),a novel ovarian cancer metastasis suppressor,identified by integrated genomics[J].Am J Transl Res,2015,7(2):242-256.
[5]Burd CG,Strochlic TI,Setty SR.Arf-like GTPases:not so Arflike after all[J].Trends Cell Biol,2004,14(12):687-694.
[6]Wei SM,Xie CG,Abe Y,et al.ADP-ribo-sylation factor like 7(ARL7)interacts with alpha-tubulin and modulates intracellular vesicular transport[J].Biochem Biophys Res Commun,2009,384(3):352-356.
[7]Hofmann I,Thompson A,Sanderson CM,et al.The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane[J].Curr Biol,2007,17(8):711-716.
[8]Sun D,Zhang J,Xie J,et al.MiR-26 controls LXR-dependent cholesterol efflux by targeting ABCA1 and ARL7[J].FEBS Lett,2012,586(10):1472-1479.
[9]Fujii S,Shinjo K,Matsumoto S,et al.Epigenetic upregulation of ARL4C,due to DNA hypomethylation in the 3'-untranslated region,promotes tumorigenesis of lung squamous cell carcinoma[J].Oncotarget,2016,7(49):81571-81587.
[10]Arend RC,Londono-Joshi AI,Straughn JM Jr,et al.The Wnt/β-catenin pathway in ovarian cancer:a review[J].Gynecol Oncol,2013,131(3):772-779.