RNA干扰FLOT2基因表达下调NF-κB信号对胃癌细胞凋亡诱导作用研究
|
摘要
目的 探讨RNA干扰FLOT2基因表达对胃癌细胞凋亡及NF-κB信号的影响。方法 Western blotting检测人胃癌BGC823、SGC7901和MKN28细胞中FLOT2蛋白表达。BGC823细胞分为空白组、阴性组和si-FLOT2组,参照脂质体Lipofectamine~(TM)2000将siRNA转染细胞,细胞转染48 h,Western blotting检测FLOT2、NF-κB p65、IKK-β、p-IKK-β和Bax的蛋白表达。流式细胞术检测细胞凋亡率。结果 BGC823、SGC7901和MKN28胃癌细胞中FLOT2蛋白表达均显著高于人正常胃黏膜上皮细胞GES1(P<0.05)。转染si-FLOT2的BGC823细胞FLOT2蛋白表达显著低于空白组(P<0.05)。与空白组比较,si-FLOT2组细胞凋亡率显著升高,NF-κB p65、IKK-β和p-IKK-β蛋白表达显著降低,Bax蛋白表达显著升高(P<0.05)。结论 RNA干扰FLOT2基因表达可诱导胃癌细胞凋亡,机制可能与下调NF-κB信号通路有关。
Objective To investigate the effect of FLOT2 gene expression with RNA interference on apoptosis and NF-κB signaling in gastric cancer cells.Methods Western blotting was used to detect the expression of FLOT2 protein in human gastric cancer BGC823, SGC7901 and MKN28 cells. BGC823 cells were divided into blank group, negative group and si-FLOT2 group. siRNA was transfected into cells according to the liposome Lipofectamine~(TM)2000. The cells were transfected for 48 hours, expressions of FLOT2, NF-κB p65, IKK-β, p-IKK-β and Bax proteins were detected by Western blotting. The cell apoptosis rate was detected by flow cytometry.Results The expression of FLOT2 protein in BGC823, SGC7901 and MKN28 gastric cancer cells was significantly higher than that in normal gastric mucosal epithelial cells(P<0.05). The expression of FLOT2 protein in BGC823 cells transfected with si-FLOT2 was significantly lower than that in blank group(P<0.05). Compared with the blank group, the apoptosis rate in si-FLOT2 group was significantly increased, and the expressions of NF-κB p65, IKK-β and p-IKK-β proteins were significantly decreased, and the expression of Bax protein was significantly increased(P<0.05).Conclusion FLOT2 gene expression with RNA interference can induce apoptosis in gastric cancer cells. The mechanism may be related to down regulation of NF-κB signaling pathway.
Objective To investigate the effect of FLOT2 gene expression with RNA interference on apoptosis and NF-κB signaling in gastric cancer cells.Methods Western blotting was used to detect the expression of FLOT2 protein in human gastric cancer BGC823, SGC7901 and MKN28 cells. BGC823 cells were divided into blank group, negative group and si-FLOT2 group. siRNA was transfected into cells according to the liposome Lipofectamine~(TM)2000. The cells were transfected for 48 hours, expressions of FLOT2, NF-κB p65, IKK-β, p-IKK-β and Bax proteins were detected by Western blotting. The cell apoptosis rate was detected by flow cytometry.Results The expression of FLOT2 protein in BGC823, SGC7901 and MKN28 gastric cancer cells was significantly higher than that in normal gastric mucosal epithelial cells(P<0.05). The expression of FLOT2 protein in BGC823 cells transfected with si-FLOT2 was significantly lower than that in blank group(P<0.05). Compared with the blank group, the apoptosis rate in si-FLOT2 group was significantly increased, and the expressions of NF-κB p65, IKK-β and p-IKK-β proteins were significantly decreased, and the expression of Bax protein was significantly increased(P<0.05).Conclusion FLOT2 gene expression with RNA interference can induce apoptosis in gastric cancer cells. The mechanism may be related to down regulation of NF-κB signaling pathway.
引文
[1] LIU X,LIU Q,FAN Y,et al.Downregulation of PPP2R5E expression by miR-23a suppresses apoptosis to facilitate the growth of gastric cancer cells [J].FEBS Lett,2014,588(17):3160-3169.DOI:10.1016/j.febslet.2014.05.068.
[2] GUO J Q,LI S J,GUO G X.Long noncoding RNA AFAP1-AS1 promotes cell proliferation and apoptosis of gastric cancer cells via PTEN/p-AKT pathway [J].Dig Dis Sci,2017,62(8):2004-2010.DOI:10.1007/s10620-017-4584-0.
[3] WEN Q,WANG W,CHU S,et al.Flot-2 expression correlates with EGFR levels and poor prognosis in surgically resected non-small cell lung cancer [J].PLoS One,2015,10(7):e0132190.DOI:10.1371/journal.pone.0132190.
[4] 许洪礼,胡萍,张振,等.siRNA干扰FLOT2基因表达对肾细胞癌细胞增殖的影响[J].现代肿瘤医学,2015,23(23):3376-3379.DOI:10.3969/j.issn.1672-4992.2015.23.002.XU H L,HU P,ZHANG Z,et al.Effect of slience of FLOT2 expression by siRNA on proliferation of human renal cell carcinoma cells [J].Modern Oncology,2015,23(23):3376-3379.DOI:10.3969/j.issn.1672-4992.2015.23.002.
[5] XIE G,LI J,CHEN J,et al.Knockdown of flotillin-2 impairs the proliferation of breast cancer cells through modulation of Akt/FOXO signaling [J].Oncol Rep,2015,33(5):2285-2290.DOI:10.3892/or.2015.3826.
[6] 谢定芳.FloT-2在胃癌中的表达及其对胃癌细胞增殖和侵袭能力的影响[D].长沙:中南大学,2013.DOI:10.7666/d.Y2424948.
[7] SASAKI Y,OSHIMA Y,KOYAMA R,et al.Identification of flotillin-2,a major protein on lipid rafts,as a novel target of p53 family members [J].Mol Cancer Res,2008,6(3):395-406.DOI:10.1158/1541-7786.MCR-07-0108.
[8] LI R,YANG H Q,XI H L,et al.Inhibition of CDH17 gene expression via RNA interference reduces proliferation and apoptosis of human MKN28 gastric cancer cells [J].Int J Oncol,2017,50(1):15-22.DOI:10.3892/ijo.2016.3783.
[9] ZHOU T,LI Y,YANG L,et al.Silencing of ANXA3 expression by RNA interference inhibits the proliferation and invasion of breast cancer cells [J].Oncol Rep,2017,37(1):388-398.DOI:10.3892/or.2016.5251.
[10] LIU R,XIE H,LUO C,et al.Identification of FLOT2 as a novel target for microRNA-34a in melanoma [J].J Cancer Res Clin Oncol,2015,141(6):993-1006.DOI:10.1007/s00432-014-1874-1.
[11] BERGER T,UEDA T,ARPAIA E,et al.Flotillin-2 deficiency leads to reduced lung metastases in a mouse breast cancer model [J].Oncogene,2013,32(41):4989-4994.DOI:10.1038/onc.2012.499.
[12] ZHAO L,LIN L,PAN C,et al.Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β [J].Oncotarget,2015,6(12):9781-9793.DOI:10.18632/oncotarget.3382.
[13] 李莉娜,肖鹏,王云峰,等.Flot-2高表达促进食管鳞状细胞癌的生长和侵袭[J].中国组织化学与细胞化学杂志,2017,26(2):160-164.DOI:10.16705/j.cnki.1004-1850.2017.02.011.LI L N,XIAO P,WANG Y F,et al.High expression of Flot-2 promotes the development and invasion of esophageal squamous cell carcinoma [J].Chinese Journal of Histochemistry and Cytochemistry,2017,26(2):160-164.DOI:10.16705/j.cnki.1004-1850.2017.02.011.
[14] HUANG H,DU T,XU G,et al.Matrine suppresses invasion of castration-resistant prostate cancer cells by downregulating MMP-2/9 via NF-κB signaling pathway [J].Int J Oncol,2017,50(2):640-648.DOI:10.3892/ijo.2016.3805.
[15] LIU Y,LI G,LIU C,et al.RSF1 regulates the proliferation and paclitaxel resistance via modulating NF-κB signaling pathway in nasopharyngeal carcinoma [J].J Cancer,2017,8(3):354-362.DOI:10.7150/jca.16720.
[16] 易蓓,袁海汀,许永会,等.NF-κB抑制剂PDTC对人骨髓瘤U266细胞增殖和凋亡的影响[J].中国病理生理杂志,2017,33(7):1177-1183.DOI:10.3969/j.issn.1000-4718.2017.07.004.YI B,YUAN H T,XU Y H,et al.Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells [J].Chinese Journal of Pathophysiology,2017,33(7):1177-1183.DOI:10.3969/j.issn.1000-4718.2017.07.004.
[17] 赵雪峰,魏敬妙,李勇,等.丹参酮ⅡA对胃癌细胞的抑制作用及其与NF-κB信号通路的关系[J].中国普通外科杂志,2014,23(4):483-487.DOI:10.7659/j.issn.1005-6947.2014.04.016.ZHAO X F,WEI J M,LI Y,et al.Inhibitory effect of tanshinone ⅡA on gastric cancer cells and involvement of NF-κB signaling pathway [J].Chinese Journal of General Surgery,2014,23(4):483-487.DOI:10.7659/j.issn.1005-6947.2014.04.016.
[18] LIN B,LI D,ZHANG L.Oxymatrine mediates Bax and Bcl-2 expression in human breast cancer MCF-7 cells [J].Pharmazie,2016,71(3):154-157.
[19] LIN C H,WU M R,LI C H,et al.Periodic exposure to smartphone-mimic low-luminance blue light induces retina damage through Bcl-2/BAX-dependent apoptosis [J].Toxicol Sci,2017,157(1):196-210.DOI:10.1093/toxsci/kfx030.
[20] WANG Y,LIU L,LIU X,et al.Shugoshin1 enhances multidrug resistance of gastric cancer cells by regulating MRP1,Bcl-2,and Bax genes [J].Tumor Biol,2013,34(4):2205-2214.DOI:10.1007/s13277-013-0758-3.
[2] GUO J Q,LI S J,GUO G X.Long noncoding RNA AFAP1-AS1 promotes cell proliferation and apoptosis of gastric cancer cells via PTEN/p-AKT pathway [J].Dig Dis Sci,2017,62(8):2004-2010.DOI:10.1007/s10620-017-4584-0.
[3] WEN Q,WANG W,CHU S,et al.Flot-2 expression correlates with EGFR levels and poor prognosis in surgically resected non-small cell lung cancer [J].PLoS One,2015,10(7):e0132190.DOI:10.1371/journal.pone.0132190.
[4] 许洪礼,胡萍,张振,等.siRNA干扰FLOT2基因表达对肾细胞癌细胞增殖的影响[J].现代肿瘤医学,2015,23(23):3376-3379.DOI:10.3969/j.issn.1672-4992.2015.23.002.XU H L,HU P,ZHANG Z,et al.Effect of slience of FLOT2 expression by siRNA on proliferation of human renal cell carcinoma cells [J].Modern Oncology,2015,23(23):3376-3379.DOI:10.3969/j.issn.1672-4992.2015.23.002.
[5] XIE G,LI J,CHEN J,et al.Knockdown of flotillin-2 impairs the proliferation of breast cancer cells through modulation of Akt/FOXO signaling [J].Oncol Rep,2015,33(5):2285-2290.DOI:10.3892/or.2015.3826.
[6] 谢定芳.FloT-2在胃癌中的表达及其对胃癌细胞增殖和侵袭能力的影响[D].长沙:中南大学,2013.DOI:10.7666/d.Y2424948.
[7] SASAKI Y,OSHIMA Y,KOYAMA R,et al.Identification of flotillin-2,a major protein on lipid rafts,as a novel target of p53 family members [J].Mol Cancer Res,2008,6(3):395-406.DOI:10.1158/1541-7786.MCR-07-0108.
[8] LI R,YANG H Q,XI H L,et al.Inhibition of CDH17 gene expression via RNA interference reduces proliferation and apoptosis of human MKN28 gastric cancer cells [J].Int J Oncol,2017,50(1):15-22.DOI:10.3892/ijo.2016.3783.
[9] ZHOU T,LI Y,YANG L,et al.Silencing of ANXA3 expression by RNA interference inhibits the proliferation and invasion of breast cancer cells [J].Oncol Rep,2017,37(1):388-398.DOI:10.3892/or.2016.5251.
[10] LIU R,XIE H,LUO C,et al.Identification of FLOT2 as a novel target for microRNA-34a in melanoma [J].J Cancer Res Clin Oncol,2015,141(6):993-1006.DOI:10.1007/s00432-014-1874-1.
[11] BERGER T,UEDA T,ARPAIA E,et al.Flotillin-2 deficiency leads to reduced lung metastases in a mouse breast cancer model [J].Oncogene,2013,32(41):4989-4994.DOI:10.1038/onc.2012.499.
[12] ZHAO L,LIN L,PAN C,et al.Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β [J].Oncotarget,2015,6(12):9781-9793.DOI:10.18632/oncotarget.3382.
[13] 李莉娜,肖鹏,王云峰,等.Flot-2高表达促进食管鳞状细胞癌的生长和侵袭[J].中国组织化学与细胞化学杂志,2017,26(2):160-164.DOI:10.16705/j.cnki.1004-1850.2017.02.011.LI L N,XIAO P,WANG Y F,et al.High expression of Flot-2 promotes the development and invasion of esophageal squamous cell carcinoma [J].Chinese Journal of Histochemistry and Cytochemistry,2017,26(2):160-164.DOI:10.16705/j.cnki.1004-1850.2017.02.011.
[14] HUANG H,DU T,XU G,et al.Matrine suppresses invasion of castration-resistant prostate cancer cells by downregulating MMP-2/9 via NF-κB signaling pathway [J].Int J Oncol,2017,50(2):640-648.DOI:10.3892/ijo.2016.3805.
[15] LIU Y,LI G,LIU C,et al.RSF1 regulates the proliferation and paclitaxel resistance via modulating NF-κB signaling pathway in nasopharyngeal carcinoma [J].J Cancer,2017,8(3):354-362.DOI:10.7150/jca.16720.
[16] 易蓓,袁海汀,许永会,等.NF-κB抑制剂PDTC对人骨髓瘤U266细胞增殖和凋亡的影响[J].中国病理生理杂志,2017,33(7):1177-1183.DOI:10.3969/j.issn.1000-4718.2017.07.004.YI B,YUAN H T,XU Y H,et al.Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells [J].Chinese Journal of Pathophysiology,2017,33(7):1177-1183.DOI:10.3969/j.issn.1000-4718.2017.07.004.
[17] 赵雪峰,魏敬妙,李勇,等.丹参酮ⅡA对胃癌细胞的抑制作用及其与NF-κB信号通路的关系[J].中国普通外科杂志,2014,23(4):483-487.DOI:10.7659/j.issn.1005-6947.2014.04.016.ZHAO X F,WEI J M,LI Y,et al.Inhibitory effect of tanshinone ⅡA on gastric cancer cells and involvement of NF-κB signaling pathway [J].Chinese Journal of General Surgery,2014,23(4):483-487.DOI:10.7659/j.issn.1005-6947.2014.04.016.
[18] LIN B,LI D,ZHANG L.Oxymatrine mediates Bax and Bcl-2 expression in human breast cancer MCF-7 cells [J].Pharmazie,2016,71(3):154-157.
[19] LIN C H,WU M R,LI C H,et al.Periodic exposure to smartphone-mimic low-luminance blue light induces retina damage through Bcl-2/BAX-dependent apoptosis [J].Toxicol Sci,2017,157(1):196-210.DOI:10.1093/toxsci/kfx030.
[20] WANG Y,LIU L,LIU X,et al.Shugoshin1 enhances multidrug resistance of gastric cancer cells by regulating MRP1,Bcl-2,and Bax genes [J].Tumor Biol,2013,34(4):2205-2214.DOI:10.1007/s13277-013-0758-3.