小分子干扰RNA抑制黏着斑激酶基因对子宫内膜癌细胞生物学特征的影响
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摘要
目的探讨小分子干扰RNA(small interference RNA,si RNA)抑制黏着斑激酶(focal adhesion kinase,FAK)基因对子宫内膜癌细胞生物学特征的影响。方法培养人子宫内膜癌HEC-1A细胞,分为si RNA-FAK组、si RNA-阴性对照组和空白对照组,实时荧光定量PCR检测各组细胞中FAK基因表达,细胞增殖能力、迁移和侵袭能力检测分别采用MTT法和Transwell法,蛋白质印迹法(Western blot)检测各组细胞中FAK、磷酸肌醇3激酶(phosphoinositol 3 kinase,PI3K)、磷酸化-Akt(phosphorylateAkt,p-Akt)、磷酸化-哺乳动物雷帕霉素(phosphorylation-mammalian rapamycin,p-mTOR)蛋白表达。结果 si RNA-FAK组细胞中FAK m RNA和蛋白相对表达量分别为(1. 31±0. 15)、(0. 26±0. 08),均低于si RNA-阴性对照组[(2. 06±0. 18)、(0. 62±0. 11)]和空白对照组[(2. 11±0. 20)、(0. 64±0. 13)],差异有统计学意义(P <0. 05); si RNA-FAK组24 h、48 h、72 h、96 h时细胞吸光度A值均低于si RNA-阴性对照组和空白对照组,均差异有统计学意义(P <0. 05);与si RNA-阴性对照组和空白对照组比较,si RNA-FAK组迁移细胞数和侵袭细胞数均降低,si RNA-FAK组细胞中FAK、PI3K、p-Akt、p-mTOR蛋白表达相对表达量均降低,而Akt和m TOR蛋白相对表达量均升高,均差异有统计学意义(P <0. 05)。结论沉默FAK基因表达可抑制HEC-1A细胞增殖、迁移及侵袭能力,可能与抑制PI3K/Akt/m TOR信号有关。
Objective To investigate the effect of small interfering RNA(si RNA) silencing of focal adhesion kinase(FAK) gene on the biological characteristics of endometrial carcinoma cells. Methods The human endometrial carcinoma HEC-1A cells were cultured,and all cells were divided into si RNA-FAK group,si RNA-negative control group and blank control group. The expression of FAK gene in each group was detected by using real-time quantitative PCR. The cell proliferation,migration and invasion of cells were detected by using MTT assay and Transwell method. The expressions of FAK,phosphoinositol 3 kinase(PI3K),phosphorylate-Akt(p-Akt)and phosphorylation-mammalian rapamycin(p-mTOR) proteins in each group were detected by Western blot. Results The relative expression levels of FAK m RNA and protein in si RNA-FAK group were(1. 31 ± 0. 15) and(0. 26 ± 0. 08),respectively,which were lower than those in the si RNA-negative control group [(2. 06 ± 0. 18) and(0. 62 ± 0. 11) ]and the blank control group [(2. 11 ± 0. 20)and(0. 64 ± 0. 13) ],the differences were statistically significant(P < 0. 05). The absorbance A values at 24 h,48h,72 h and 96 h in si RNA-FAK group were lower than those in the si RNA-negative control group and blank control group,the differences were statistically significant(P < 0. 05). Transwell test results showed that compared with the si RNA-negative control group and blank control group,the number of migrating cells and the number of invasive cells in si RNA-FAK group were decreased,the relative expression levels of FAK,PI3 K,p-Akt and p-mTOR proteins in si RNA-FAK group were decreased,while the relative expression levels of Akt and m TOR proteins were increased,the differences were statistically significant(P < 0. 05). Conclusion Specific silencing of FAK gene could inhibit proliferation,migration and invasion of HEC-1A cells. It might be related to inhibition of PI3K/Akt/m TOR signaling.
Objective To investigate the effect of small interfering RNA(si RNA) silencing of focal adhesion kinase(FAK) gene on the biological characteristics of endometrial carcinoma cells. Methods The human endometrial carcinoma HEC-1A cells were cultured,and all cells were divided into si RNA-FAK group,si RNA-negative control group and blank control group. The expression of FAK gene in each group was detected by using real-time quantitative PCR. The cell proliferation,migration and invasion of cells were detected by using MTT assay and Transwell method. The expressions of FAK,phosphoinositol 3 kinase(PI3K),phosphorylate-Akt(p-Akt)and phosphorylation-mammalian rapamycin(p-mTOR) proteins in each group were detected by Western blot. Results The relative expression levels of FAK m RNA and protein in si RNA-FAK group were(1. 31 ± 0. 15) and(0. 26 ± 0. 08),respectively,which were lower than those in the si RNA-negative control group [(2. 06 ± 0. 18) and(0. 62 ± 0. 11) ]and the blank control group [(2. 11 ± 0. 20)and(0. 64 ± 0. 13) ],the differences were statistically significant(P < 0. 05). The absorbance A values at 24 h,48h,72 h and 96 h in si RNA-FAK group were lower than those in the si RNA-negative control group and blank control group,the differences were statistically significant(P < 0. 05). Transwell test results showed that compared with the si RNA-negative control group and blank control group,the number of migrating cells and the number of invasive cells in si RNA-FAK group were decreased,the relative expression levels of FAK,PI3 K,p-Akt and p-mTOR proteins in si RNA-FAK group were decreased,while the relative expression levels of Akt and m TOR proteins were increased,the differences were statistically significant(P < 0. 05). Conclusion Specific silencing of FAK gene could inhibit proliferation,migration and invasion of HEC-1A cells. It might be related to inhibition of PI3K/Akt/m TOR signaling.
引文
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[2]施晓燕,施晓莺,田梅,等.子宫恶性肿瘤根治术中盆腔淋巴结清扫的手术入路探讨[J].安徽医药,2015,19(3):549-551.
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[7]陈家骅,李元明,王磊,等. MBP-1在人食管癌细胞株Ec109细胞增殖、凋亡和侵袭中的作用[J].重庆医学,2018,47(10):1318-1321.
[8]吴磊.胰岛素样生长因子结合蛋白7对结直肠癌生物学特性的作用[J].中国现代普通外科进展,2018,21(3):181-185.
[9]李大刚,李辉宗,康乐. Vav3在小细胞肺癌组织中的表达及对细胞迁移和侵袭能力的影响[J].中国现代医学杂志,2018,28(24):32-37.
[10]李国彬,张占成,王新颜.上调miR-200b对人喉癌Hep-2细胞增殖、迁移和侵袭能力的影响[J].山东大学耳鼻喉眼学报,2018,32(4):53-57.
[11]郑建平(综述),易村犍(审校).子宫内膜癌分子靶向治疗研究进展[J].医学综述,2016,22(21):4211-4214.
[12] PIULATS JM,GUERRA E,GIL-MARTN M,et al. Molecular approaches for classifying endometrial carcinoma[J]. Gynecol Oncol,2017,145(1):200-207.
[13]夏蕾,罗宝华.中晚期宫颈鳞癌放化疗后复发未控的相关因素及疗效分析[J].安徽医药,2015,19(12):2347-2349.
[14]张艳青,周燕飞,龙玲,等. Dicer和Drosha与子宫内膜癌的相关性研究[J].医学临床研究,2015,32(3):514-518.
[15]张思雨,高惠英,李小峰.黏着斑激酶和p53与类风湿关节炎的研究进展[J].中华风湿病学杂志,2016,20(11):790-792.
[16] KANTETI R,BATRA SK,LENNON FE,et al. FAK and paxillin,two potential targets in pancreatic cancer[J]. Oncotarget,2016,7(21):31586-31601.
[17] YOON H,DEHART JP,MURPHY JM,et al. Understanding the roles of FAK in cancer:inhibitors,genetic models,and new insights[J]. J Histochem Cytochem,2015,63(2):114-128.
[18] ZHOU J,ROH JW,BANDYOPADHYAY S,et al. Overexpression of enhancer of zeste homolog 2(EZH2)and focal adhesion kinase(FAK)in high grade endometrial carcinoma[J]. Gynecol Oncol,2013,128(2):344-348.
[19]封菊,苑中甫.邱海峰. RNA干扰FAK表达抑制宫颈癌细胞侵袭、迁移能力的研究[J].癌症进展,2018,16(2):179-182.
[20] XIA P,XU XY. PI3K/Akt/mTOR signaling pathway in cancer stem cells:from basic research to clinical application[J]. Am J Cancer Res,2015,5(5):1602-1609.
[21]李小静,李志锋,李显平,等.丹参酮ⅡA体外促进黑素瘤A375细胞自噬及信号通路的实验研究[J].中华皮肤科杂志,2017,50(1):29-32.
[2]施晓燕,施晓莺,田梅,等.子宫恶性肿瘤根治术中盆腔淋巴结清扫的手术入路探讨[J].安徽医药,2015,19(3):549-551.
[3] SUN Y,CHEN Y,ZHAN L,et al. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors[J]. Rev Neurosci,2016,27(3):283-289.
[4]余俊,刘彤鸥,李晓兰.小分子干扰RNA沉默黏着斑激酶基因对人宫颈癌Hela细胞生物学特征的影响[J].新乡医学院学报,2018,35(4):266-271.
[5]李玲妹(综述),曹文枫(审校). FAK与肿瘤关系的研究进展[J].中国肿瘤临床,2015,42(2):116-119.
[6]司晓辉,孙攀兴,杜增利.沉默WISP3基因抑制子宫内膜癌细胞增殖及侵袭力[J].齐鲁医学杂志,2017,32(6):631-635.
[7]陈家骅,李元明,王磊,等. MBP-1在人食管癌细胞株Ec109细胞增殖、凋亡和侵袭中的作用[J].重庆医学,2018,47(10):1318-1321.
[8]吴磊.胰岛素样生长因子结合蛋白7对结直肠癌生物学特性的作用[J].中国现代普通外科进展,2018,21(3):181-185.
[9]李大刚,李辉宗,康乐. Vav3在小细胞肺癌组织中的表达及对细胞迁移和侵袭能力的影响[J].中国现代医学杂志,2018,28(24):32-37.
[10]李国彬,张占成,王新颜.上调miR-200b对人喉癌Hep-2细胞增殖、迁移和侵袭能力的影响[J].山东大学耳鼻喉眼学报,2018,32(4):53-57.
[11]郑建平(综述),易村犍(审校).子宫内膜癌分子靶向治疗研究进展[J].医学综述,2016,22(21):4211-4214.
[12] PIULATS JM,GUERRA E,GIL-MARTN M,et al. Molecular approaches for classifying endometrial carcinoma[J]. Gynecol Oncol,2017,145(1):200-207.
[13]夏蕾,罗宝华.中晚期宫颈鳞癌放化疗后复发未控的相关因素及疗效分析[J].安徽医药,2015,19(12):2347-2349.
[14]张艳青,周燕飞,龙玲,等. Dicer和Drosha与子宫内膜癌的相关性研究[J].医学临床研究,2015,32(3):514-518.
[15]张思雨,高惠英,李小峰.黏着斑激酶和p53与类风湿关节炎的研究进展[J].中华风湿病学杂志,2016,20(11):790-792.
[16] KANTETI R,BATRA SK,LENNON FE,et al. FAK and paxillin,two potential targets in pancreatic cancer[J]. Oncotarget,2016,7(21):31586-31601.
[17] YOON H,DEHART JP,MURPHY JM,et al. Understanding the roles of FAK in cancer:inhibitors,genetic models,and new insights[J]. J Histochem Cytochem,2015,63(2):114-128.
[18] ZHOU J,ROH JW,BANDYOPADHYAY S,et al. Overexpression of enhancer of zeste homolog 2(EZH2)and focal adhesion kinase(FAK)in high grade endometrial carcinoma[J]. Gynecol Oncol,2013,128(2):344-348.
[19]封菊,苑中甫.邱海峰. RNA干扰FAK表达抑制宫颈癌细胞侵袭、迁移能力的研究[J].癌症进展,2018,16(2):179-182.
[20] XIA P,XU XY. PI3K/Akt/mTOR signaling pathway in cancer stem cells:from basic research to clinical application[J]. Am J Cancer Res,2015,5(5):1602-1609.
[21]李小静,李志锋,李显平,等.丹参酮ⅡA体外促进黑素瘤A375细胞自噬及信号通路的实验研究[J].中华皮肤科杂志,2017,50(1):29-32.