Snail转录因子与ER-α36介导的雌激素信号在胃癌肿瘤细胞成球作用中的研究
|
摘要
目的探讨Snail转录因子与ER-α36介导的雌激素信号在胃癌肿瘤细胞中的成球作用。方法应用无血清的培养液DMEM/F12悬浮培养胃癌细胞系SGC7901及SGC7901/ER-α36重组细胞系High36和Low36。测定肿瘤细胞球体中Snail+细胞的含量,运用Western blot检测技术鉴定Snail和相关蛋白在肿瘤细胞球体中的表达。给予不同浓度的雌激素刺激,观察不同浓度雌激素对肿瘤细胞球体的影响,检测雌激素处理后Snail蛋白的表达等。结果超低吸附培养皿无血清悬浮培养肿瘤微球体的效果优于普通培养皿,大约培养1周可形成肿瘤细胞球体,且形成的数量最多,这些肿瘤细胞球体中的细胞可以稳定传代继续形成新的微球体,同时,Snail+细胞含量升高,并且其蛋白在肿瘤细胞球体中的表达增高。ER-α36高表达的High36细胞系经无血清悬浮培养1周后形成的肿瘤微球体数增加。0.1μmol浓度的ER刺激胃癌细胞株细胞,可以诱导Snail、ER-α36表达上调,而1μmol浓度的ER刺激却作用相反(P﹤0.05)。结论低浓度的ER可以诱导胃癌细胞株细胞Snail转录因子与ER-α36高表达,Snail转录因子与ER-α36高表达与肿瘤细胞的成球作用关系密切。
Objective To analyze the effect and mechanism of Snail transcription factor and ER-α36 mediated estrogen signaling in the formation of gastric cancer spheroids.Method We used serum-free DMEM/F12 suspension medium to culture gastric cancer cell line SGC7901 and recombinant cell lines High36 and Low36 of SGC7901.The content of Snail + cells in spheroids were determined,and western blot was utilized to evaluate the expression of Snail and related proteins in gastric cancer spheroids.Estrogen at different concentrations was applied to stimulate the cells,and the effect on tumor cells,as well as the Snail protein expression after treatment were observed and detected.Result Ultra-low adsorption serum-free suspension culture media was practically better than normal medium when used in culturing gastric cancer spheroids,and abundant spheroids formed in about 1 week,these cells could steadily produce new spheroids in subcultures,meanwhile,the Snail + cells increased,and the protein expression elevated in gastric cancer spheroids.After 1 week of suspension culture using serum-free media,the spheroids formed by High36 cells with high expression of ER-α36 increased 0.1 μmol of ER stimulation could induce the upregulation of Snail and ER-α36,while 1μmol of ER stimulation would lead to opposite effect(P<0.05).Conclusion Low concentration of ER may induce the high expression of Snail transcription factor and ER-α36,indicating a close relationship between the Snail transcription factor,high expression of ER-α36 and the formation of gastric cancer spheroids.
Objective To analyze the effect and mechanism of Snail transcription factor and ER-α36 mediated estrogen signaling in the formation of gastric cancer spheroids.Method We used serum-free DMEM/F12 suspension medium to culture gastric cancer cell line SGC7901 and recombinant cell lines High36 and Low36 of SGC7901.The content of Snail + cells in spheroids were determined,and western blot was utilized to evaluate the expression of Snail and related proteins in gastric cancer spheroids.Estrogen at different concentrations was applied to stimulate the cells,and the effect on tumor cells,as well as the Snail protein expression after treatment were observed and detected.Result Ultra-low adsorption serum-free suspension culture media was practically better than normal medium when used in culturing gastric cancer spheroids,and abundant spheroids formed in about 1 week,these cells could steadily produce new spheroids in subcultures,meanwhile,the Snail + cells increased,and the protein expression elevated in gastric cancer spheroids.After 1 week of suspension culture using serum-free media,the spheroids formed by High36 cells with high expression of ER-α36 increased 0.1 μmol of ER stimulation could induce the upregulation of Snail and ER-α36,while 1μmol of ER stimulation would lead to opposite effect(P<0.05).Conclusion Low concentration of ER may induce the high expression of Snail transcription factor and ER-α36,indicating a close relationship between the Snail transcription factor,high expression of ER-α36 and the formation of gastric cancer spheroids.
引文
[1]张雷,何流,龙佩,等.胃癌应用CA724、CEA、CA242、CA199肿瘤标志物联合检验的价值分析[J].辽宁医学杂志,2017,31(2):1-2.
[2]黎伟超,牛晓敏,何慧清,等.自体外周血造血干细胞移植对恶性淋巴瘤患者T细胞亚群的影响[J].辽宁医学杂志,2015,29(1):9-10.
[3]黄辉,潘志坚.胃癌干细胞在胃癌侵袭、转移中及血管形成中的作用和机制[J].中国组织工程研究,2016,20(14):2021-2026.
[4]Deng H,Zhang XT,Wang ML,et al.ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells[J].PLo S One,2014,9(2):e88034.
[5]Kume K,Haraguchi M,Hijioka H,et al.The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells[J].Biochem Biophys Res Commun,2013,430(3):889-894.
[6]Hwang WL,Yang MH,Tsai ML,et al.SNAIL regulates interleukin-8 expression,stem cell-like activity,and tumorigenicity of human colorectal carcinoma cells[J].Gastroenterology,2011,141(1):279-291.
[7]Qu BL,Yu W,Huang YR,et al.6-OH-BDE-47 promotes human lung cancer cells epithelial mesenchymal transition via the AKT/Snail signal pathway[J].Environ Toxicol Pharmacol,2015,39(1):271-279.
[8]Hwang WL,Yang MH,Tsai ML,et al.SNAIL regulates interleukin-8 expression,stem cell-like activity,and tumorigenicity of human colorectal carcinoma cells[J].Gastroenterology,2011,141(1):279-291.
[9]Sato F,Sato H,Jin D,et al.Smad3 and Snail show circadian expression in human gingival fibroblasts,human mesenchymal stem cell,and in mouse liver[J].Biochem Biochem Biophys Res Commun,2012,419(2):441-446.
[10]吴丽霞,王绪明,张尚昆,等.胃癌细胞的无血清悬浮培养与初步鉴定[J].世界华人消化杂志,2014,22(11):1531-1536.
[11]王绪明,刘晶晶,邓昊,等.ER-α36对胃癌SGC7901细胞在裸鼠体内生长的影响[J].世界华人消化杂志,2011,19(28):2919-2924.
[12]邹丰,王绪明,刘丽江.ER-α36和mi R-143介导胃癌细胞的侵袭[J].中国病理生理杂志,2012,28(12):2167-2171.
[13]Wang X,Deng H,Zou F,et al.ER-α36-mediated gastric cancer cell proliferation via the c-Src pathway[J].Oncol Lett,2013,6(2):329-335.
[14]宋伟.RNAi抑制Snail表达对于胃癌细胞上皮-间充质转化以及体外侵袭的研究[D].武汉:华中科技大学,2009.
[15]Huang JW,Guan BZ,Yin LH,et al.Effects of estrogenrelated receptor alpha(ERRα)on proliferation and metastasis of human lung cancer A549 cells[J].J Huazhong Univ Sci Technolog Med Sci,2014,34(6):875-881.
[16]Lam SS,Mak AS,Yam JW,et al.Targeting estrogenrelated receptor alpha inhibits epithelial-to-mesenchymal transition and stem cell properties of ovarian cancer cells[J].Mol Ther,2014,22(4):743-751.
[2]黎伟超,牛晓敏,何慧清,等.自体外周血造血干细胞移植对恶性淋巴瘤患者T细胞亚群的影响[J].辽宁医学杂志,2015,29(1):9-10.
[3]黄辉,潘志坚.胃癌干细胞在胃癌侵袭、转移中及血管形成中的作用和机制[J].中国组织工程研究,2016,20(14):2021-2026.
[4]Deng H,Zhang XT,Wang ML,et al.ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells[J].PLo S One,2014,9(2):e88034.
[5]Kume K,Haraguchi M,Hijioka H,et al.The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells[J].Biochem Biophys Res Commun,2013,430(3):889-894.
[6]Hwang WL,Yang MH,Tsai ML,et al.SNAIL regulates interleukin-8 expression,stem cell-like activity,and tumorigenicity of human colorectal carcinoma cells[J].Gastroenterology,2011,141(1):279-291.
[7]Qu BL,Yu W,Huang YR,et al.6-OH-BDE-47 promotes human lung cancer cells epithelial mesenchymal transition via the AKT/Snail signal pathway[J].Environ Toxicol Pharmacol,2015,39(1):271-279.
[8]Hwang WL,Yang MH,Tsai ML,et al.SNAIL regulates interleukin-8 expression,stem cell-like activity,and tumorigenicity of human colorectal carcinoma cells[J].Gastroenterology,2011,141(1):279-291.
[9]Sato F,Sato H,Jin D,et al.Smad3 and Snail show circadian expression in human gingival fibroblasts,human mesenchymal stem cell,and in mouse liver[J].Biochem Biochem Biophys Res Commun,2012,419(2):441-446.
[10]吴丽霞,王绪明,张尚昆,等.胃癌细胞的无血清悬浮培养与初步鉴定[J].世界华人消化杂志,2014,22(11):1531-1536.
[11]王绪明,刘晶晶,邓昊,等.ER-α36对胃癌SGC7901细胞在裸鼠体内生长的影响[J].世界华人消化杂志,2011,19(28):2919-2924.
[12]邹丰,王绪明,刘丽江.ER-α36和mi R-143介导胃癌细胞的侵袭[J].中国病理生理杂志,2012,28(12):2167-2171.
[13]Wang X,Deng H,Zou F,et al.ER-α36-mediated gastric cancer cell proliferation via the c-Src pathway[J].Oncol Lett,2013,6(2):329-335.
[14]宋伟.RNAi抑制Snail表达对于胃癌细胞上皮-间充质转化以及体外侵袭的研究[D].武汉:华中科技大学,2009.
[15]Huang JW,Guan BZ,Yin LH,et al.Effects of estrogenrelated receptor alpha(ERRα)on proliferation and metastasis of human lung cancer A549 cells[J].J Huazhong Univ Sci Technolog Med Sci,2014,34(6):875-881.
[16]Lam SS,Mak AS,Yam JW,et al.Targeting estrogenrelated receptor alpha inhibits epithelial-to-mesenchymal transition and stem cell properties of ovarian cancer cells[J].Mol Ther,2014,22(4):743-751.