基质细胞衍生因子-1基因转染对人胃癌细胞株SGC7901增殖、耐药的影响及其机制
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摘要
目的观察基质细胞衍生因子-1(SDF-1)基因转染对人胃癌细胞株SGC7901增殖、耐药的影响,并探讨其可能作用机制。方法取对数生长期SGC7901细胞分为实验组、阴性对照组、空白对照组,实验组、阴性对照组分别转染pc DNA3. 1-SDF-1质粒、pc DNA3. 1质粒(空白质粒),空白对照组不做任何处理。质粒转染48 h时分别采用RT-PCR法、Western bloting法检测细胞SDF-1 mRNA及蛋白,质粒转染24、48、72、96 h时MTT法测算三组细胞增殖能力(以光密度OD值表示),质粒转染48 h时观察三组细胞对奥沙利铂的耐药性(以奥沙利铂对细胞的半数抑制浓度IC50表示),质粒转染48 h时流式细胞仪测算三组细胞凋亡率,质粒转染48 h时Western blotting法检测三组细胞上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)及波形蛋白(Vimentin)、微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)自噬相关因子(Beclin 1)。结果与空白对照组、阴性对照组比较,培养48 h时实验组细胞SDF-1 mRNA及蛋白相对表达量均升高(P均<0. 05)。与空白对照组、阴性对照组比较,培养24、48、72、96 h实验组细胞OD值均升高(P均<0. 05)。质粒转染48 h时奥沙利铂对实验组、空白对照组、阴性对照组细胞的IC50值分别为28. 039±3. 011、10. 403±1. 253、10. 612±1. 044(μg/m L),与空白对照组、阴性对照组比较,奥沙利铂对实验组细胞的IC50值升高(P均<0. 05)。质粒转染48 h时实验组、空白对照组、阴性对照组细胞凋亡率分别为39. 10%±2. 26%、53. 90%±3. 19%、51. 30%±3. 37%,与空白对照组、阴性对照组比较,实验组细胞凋亡率降低(P均<0. 05)。质粒转染48 h时倒置显微镜下可见实验组细胞存在上皮-间质转化(EMT)现象,加入奥沙利铂后实验组细胞自噬活动增加;与空白对照组、阴性对照组比较,实验组细胞E-cadhein蛋白相对表达量降低,N-cadhein、Vimentin、LC3-Ⅱ、Beclin 1蛋白相对表达量均升高(P均<0. 05)。结论 SDF-1基因过表达可促进SGC7901细胞增殖,显著提高SGC7901细胞对奥沙利铂的耐药性,可能与SDF-1促进EMT、诱导细胞自噬增加进而抑制细胞凋亡有关。
Objective To observe the effects of stroma cell-derived factor-1( SDF-1) gene transfection on the proliferation and drug resistance of human gastric cancer cell line SGC7901 and to explore its possible mechanism. Methods SGC7901 cells in the logarithmic growth phase were divided into the experimental group,negative control group,and blank control group. The cells in the experimental group and negative control group were transfected with pc DNA3. 1-SDF-1 plasmid and pc DNA3. 1 plasmid( blank plasmid),respectively,and the blank control group did not receive any treatment. After 48 h of plasmid transfection,SDF-1 mRNA and protein levels were detected by RT-PCR and Western bloting,respectively. The proliferation ability of the three groups was measured by MTT at 24,48,72 and 96 h after plasmid transfection( expressed by optical density). The drug resistance of the three groups to oxaliplatin was observed at 48 h after plasmid transfection( expressed by the 50% inhibitory concentration IC50 of oxaliplatin). The apoptosis rates of the three groups were measured by flow cytometry at 48 h after plasmid transfection. After 48-hour plasmid transfection,Western blotting was used to detect epithelial cadherin( E-cadherin),neurocadherin( N-cadherin) and vimentin( Vimentin),microtubuleassociated protein 1 light chain 3 Ⅱ( LC3-Ⅱ),autophagy-related factor( Beclin 1) in the three groups. Results The relative mRNA and protein expression levels of SDF-1 in the experimental group were significantly higher than those in the blank control group and negative control group at 48 h( both P < 0 05). Compared with the blank control group and the negative control group,the OD values of the experimental group increased at 24,48,72 and 96 h( all P < 0 05). At 48 h after plasmid transfection,the IC50 values of oxaliplatin in the experimental group,blank control group and negative control group were( 28. 039 ± 3. 011),( 10. 403 ± 1. 253) and( 10. 612 ± 1. 044) μ g/m L,respectively. The experimental group had higher IC50 value of oxaliplatin than the blank control group and negative control group( P < 0 05). At 48 h after plasmid transfection,the apoptosis rates of the experimental group,blank control group,and negative control group were 39. 10% ± 2. 26%,53. 90% ± 3. 19%,and 51. 30% ± 3. 37%,respectively. The apoptosis rate in the experimental group was significantly lower than those in the blank control group and negative control group( all P < 0 05). At 48 h after plasmid transfection,epithelialinterstitial transformation( EMT) was observed under inverted microscope in the experimental group,and the autophagy activity in the experimental group increased after the addition of oxaliplatin. Compared with the blank control group and the negative control group,the relative expression of E-cadhein protein in the experimental group was lower,while the relative expression of N-cadhein,Vimentin,LC3-Ⅱ and Beclin 1 protein was higher. Conclusion Overexpression of SDF-1 gene can promote the proliferation of SGC7901 cells and significantly increase the resistance of SGC7901 cells to oxaliplatin,which may be related to the promotion of EMT,the increase of autophagy and the inhibition of apoptosis.
Objective To observe the effects of stroma cell-derived factor-1( SDF-1) gene transfection on the proliferation and drug resistance of human gastric cancer cell line SGC7901 and to explore its possible mechanism. Methods SGC7901 cells in the logarithmic growth phase were divided into the experimental group,negative control group,and blank control group. The cells in the experimental group and negative control group were transfected with pc DNA3. 1-SDF-1 plasmid and pc DNA3. 1 plasmid( blank plasmid),respectively,and the blank control group did not receive any treatment. After 48 h of plasmid transfection,SDF-1 mRNA and protein levels were detected by RT-PCR and Western bloting,respectively. The proliferation ability of the three groups was measured by MTT at 24,48,72 and 96 h after plasmid transfection( expressed by optical density). The drug resistance of the three groups to oxaliplatin was observed at 48 h after plasmid transfection( expressed by the 50% inhibitory concentration IC50 of oxaliplatin). The apoptosis rates of the three groups were measured by flow cytometry at 48 h after plasmid transfection. After 48-hour plasmid transfection,Western blotting was used to detect epithelial cadherin( E-cadherin),neurocadherin( N-cadherin) and vimentin( Vimentin),microtubuleassociated protein 1 light chain 3 Ⅱ( LC3-Ⅱ),autophagy-related factor( Beclin 1) in the three groups. Results The relative mRNA and protein expression levels of SDF-1 in the experimental group were significantly higher than those in the blank control group and negative control group at 48 h( both P < 0 05). Compared with the blank control group and the negative control group,the OD values of the experimental group increased at 24,48,72 and 96 h( all P < 0 05). At 48 h after plasmid transfection,the IC50 values of oxaliplatin in the experimental group,blank control group and negative control group were( 28. 039 ± 3. 011),( 10. 403 ± 1. 253) and( 10. 612 ± 1. 044) μ g/m L,respectively. The experimental group had higher IC50 value of oxaliplatin than the blank control group and negative control group( P < 0 05). At 48 h after plasmid transfection,the apoptosis rates of the experimental group,blank control group,and negative control group were 39. 10% ± 2. 26%,53. 90% ± 3. 19%,and 51. 30% ± 3. 37%,respectively. The apoptosis rate in the experimental group was significantly lower than those in the blank control group and negative control group( all P < 0 05). At 48 h after plasmid transfection,epithelialinterstitial transformation( EMT) was observed under inverted microscope in the experimental group,and the autophagy activity in the experimental group increased after the addition of oxaliplatin. Compared with the blank control group and the negative control group,the relative expression of E-cadhein protein in the experimental group was lower,while the relative expression of N-cadhein,Vimentin,LC3-Ⅱ and Beclin 1 protein was higher. Conclusion Overexpression of SDF-1 gene can promote the proliferation of SGC7901 cells and significantly increase the resistance of SGC7901 cells to oxaliplatin,which may be related to the promotion of EMT,the increase of autophagy and the inhibition of apoptosis.
引文
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[21]Zhao Z,Han F,Yang S,et al. Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells:involvement of the AKT-mTOR signaling pathway[J].Cancer Lett,2015,358(1):17-26.
[2]王玮,孙哲,邓靖宇,等.基于多中心大样本数据库的胃癌外科治疗相关数据的整合与分析[J].中华胃肠外科杂志,2016,19(2):179-185.
[3]施敏,张俊,朱正纲.从全程管理角度看进展期胃癌围手术期治疗的布局与策略[J].中华胃肠外科杂志,2018,21(10):1093-1098.
[4]徐晓武,杨小敏,胡经纬,等.动力相关蛋白1介导的自噬对胃癌MKN-45细胞奥沙利铂敏感性的影响[J].中华普通外科杂志,2017,32(9):774-777.
[5]欧阳卓,孙晋萍,田秀兰,等.基质细胞衍生因子-1和趋化因子受体-4在子宫内膜异位症患者病灶中的表达及意义[J].中华医学杂志,2018,98(23):1854-1858.
[6]Yu X,Shi W,Zhang Y,et al. CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer[J]. Sci Rep,2017,7:42226.
[7]胡媛媛,陶善东,马晶晶,等. Ibrutinib逆转SDF-1α/CXCR4介导的急性淋巴细胞白血病耐药机制[J].中国实验血液学杂志,2017,25(3):754-760.
[8]邹芳,张志华,张玉妥,等.肿瘤相关成纤维细胞通过分泌基质细胞衍生因子1调节肺癌A549细胞的化疗耐药性[J].中华肿瘤杂志,2017,39(5):339-343.
[9]杨勇,陈小伍,朱达坚,等.上皮-间充质转化在姜黄素逆转结肠癌伊立替康耐药中的作用[J].广东医学,2017,38(3):367-370.
[10]吕晓业,王健,李山虎,等. SDF-1/CXCR4轴在胃癌中的研究进展[J].生物技术通讯,2017,28(2):159-161.
[11]Yadav SS,Prasad SB,Prasad CB,et al. CXCLl2 is a key regulator in tumor microenvironment of cervica I cancer:an in vitro study[J]. Clin Exp Metastasis,2016,33(5):431-439.
[12]Yin X,Liu Z,Zhu P,et al. CXCL12/CXCR4 promotes proliferation,migration,and invasion of adamantinomatous craniopharyngiomas via PI3K/AKT signal pathway[J]. J Cell Biochem,2019,120(6):9724-9736.
[13]Lin Y,Ma Q,Li L,et al. The CXCL12-CXCR4 axis promotes migration,invasiveness,and EMT in human papillary thyroid carcinoma B-CPAP cells via NF-κB signaling[J]. Biochem Cell Biol,2018,96(5):619-626.
[14]肖磊,黄昌浩,袁伟杰,等.胃癌组织YAP1、E-cadherin、N-cadherin表达及临床意义[J].中国普通外科杂志,2018,27(4):442-448.
[15]Liao AY,Shi RR,Jiang YL,et al. SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma[J]. Mol Neurobiol,2016,53(1):210-215.
[16]文俏程,陈志康,陈子华,等.胃癌细胞奥沙利铂耐药与上皮-间质转化关系研究[J].中国普通外科杂志,2015,24(9):1269-1274.
[17]郑翔,邓兵. TGF-β1通过诱导自噬促进胃癌SGC7901细胞侵袭[J].现代肿瘤医学,2017,25(14):2198-2202.
[18]赵雪峰,魏敬妙,李勇,等.丹参酮ⅡA诱导体外胃癌细胞的自噬[J].中成药,2014,36(1):10-14.
[19]Fu Z,Zhang P,Luo H,et al. CXCL12 modulates the radiosensitivity of cervical cancer by regulating CD44[J]. Mol Med Rep,2018,18(6):5101-5108.
[20]王娅南,魏亚宁,徐芳,等. miR-551b在人胃癌组织中的表达及对胃癌细胞凋亡的影响[J].华中科技大学学报(医学版),2017,46(4):386-391.
[21]Zhao Z,Han F,Yang S,et al. Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells:involvement of the AKT-mTOR signaling pathway[J].Cancer Lett,2015,358(1):17-26.