shRNA抑制NANOG基因表达对肾癌细胞A498侵袭力的影响
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摘要
目的研究抑制NANOG基因表达对肾癌细胞侵袭能力的影响。方法以人肾癌细胞系A498细胞为研究对象,转染NANOG短发夹RNA(NANOG-shRNA)抑制A498细胞NANOG基因表达。通过实时定量PCR(qRT-PCR)检测金属基质蛋白酶-2(MMP-2)、金属基质蛋白酶-9(MMP-9)和金属蛋白酶组织抑制剂-1(TIMP-1)表达水平的变化,划痕实验和侵袭实验检测A498细胞侵袭力的变化。结果 qRT-PCR检测结果显示A498细胞转染NANOGshRNA后,其MMP-2与MMP-9基因的表达水平与对照组相比,分别下降了65%和60%,差异均有统计学意义(P<0.05),侵袭实验显示A498细胞转染NANOG-shRNA后,细胞的侵袭能力下降,与对照组相比,穿膜细胞数下降62.5%,孔底部细胞数减少48%,差异有统计学意义(P<0.05),划痕实验结果显示A498细胞转染NANOG-shRNA后,细胞划痕愈合程度较对照组显著降低,差异有统计学意义(P<0.05)。结论抑制NANOG基因表达后,A498细胞的侵袭能力下降,可能与MMP-2及MMP-9的表达水平下降有关。
Objective To study the effect of inhibiting the expression of NANOG gene on the invasion ability of human renal cancer cells A498. Methods Human renal cancer cells A498 was cultivated and transfected with shRNA to silence NANOG gene. Quantitative real time polymerase chain reaction(qRT-PCR)were performed to detect the level of metal matrix proteinase-2(MMP-2), metal matrix proteinase-9(MMP-9)and tissue inhibitors of metalloproteinase-1(TIMP-1).Wound-healing assay and invasion test was performed to determine the invasion capability of A498 cell. Results qRT-PCR showed that the levels of MMP-2 and MMP-9 gene expression in A498 cells transfected with NANOG-shRNA decreased by65% and 60% when compared with control groups(P<0.05),respectively. Transwell assay showed that the invasion ability of A498 cells transfected with NANOG-shRNA decreased when compared with the control group. The number of A498 cells invaded the outer side of the membrane decreased by about 62.5%(P<0.05),and the number of A498 cells invaded to the bottom of well decreased by about 48%(P<0.05). The results of wound healing assay showed that the wound healing rate of A498 cells transfected with NANOG-shRNA was significantly reduced when compared with the control group(P<0.05).Conclusion Inhibition of NANOG gene expression led to the decrease of invasive ability of A498,which was involved with down-regulation of MMP-2 and MMP-9.
Objective To study the effect of inhibiting the expression of NANOG gene on the invasion ability of human renal cancer cells A498. Methods Human renal cancer cells A498 was cultivated and transfected with shRNA to silence NANOG gene. Quantitative real time polymerase chain reaction(qRT-PCR)were performed to detect the level of metal matrix proteinase-2(MMP-2), metal matrix proteinase-9(MMP-9)and tissue inhibitors of metalloproteinase-1(TIMP-1).Wound-healing assay and invasion test was performed to determine the invasion capability of A498 cell. Results qRT-PCR showed that the levels of MMP-2 and MMP-9 gene expression in A498 cells transfected with NANOG-shRNA decreased by65% and 60% when compared with control groups(P<0.05),respectively. Transwell assay showed that the invasion ability of A498 cells transfected with NANOG-shRNA decreased when compared with the control group. The number of A498 cells invaded the outer side of the membrane decreased by about 62.5%(P<0.05),and the number of A498 cells invaded to the bottom of well decreased by about 48%(P<0.05). The results of wound healing assay showed that the wound healing rate of A498 cells transfected with NANOG-shRNA was significantly reduced when compared with the control group(P<0.05).Conclusion Inhibition of NANOG gene expression led to the decrease of invasive ability of A498,which was involved with down-regulation of MMP-2 and MMP-9.
引文
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[14]Siu MK,Wong ES,Chan HY,et al.Overexpression of NANOGin gestational trophoblastic diseases:effect on apoptosis,cell invasion,and clinical outcome[J].Am J Pathol,2008,173(4):1165-1172.
[15]Borrull A,Ghislin S,Deshayes F,et al.Nanog and Oct4overexpression increases motility and transmigration of melanoma cells[J].J Cancer Res Clin Oncol,2012,138(7):1145-1154.
[16]艾威,刘宗来,舒峰,等.miR-193a靶向调控HOTAIR对前列腺癌细胞活力及侵袭能力的影响[J].热带医学杂志,2019,19(1):9-12,59.
[2]Jeter CR,Yang T,Wang J,et al.Concise review:NANOG in cancer stem cells and tumor development:an update and outstanding questions[J].Stem Cells,2015,33(8):2381-2390.
[3]Gagnon JA,Obbad K,Schier AF.The primary role of zebrafish nanog is in extra-embryonic tissue[J].Development,2018,145(1).pii:dev147793.
[4]Shahini A,Choudhury D,Asmani M,et al.NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings[J].Stem Cell Res,2018,26:55-66.
[5]Liang C,Zhang K,Ge H,et al.Prognostic and clinicopathological value of Nanog in hepatocellular carcinoma:a meta-analysis[J].Clin Chim Acta,2018,477:24-31.
[6]Qin S,Li Y,Cao X,et al.NANOG regulates epithelialmesenchymal transition and chemoresistance in ovarian cancer[J].Biosci Rep,2017,37(1).pii:BSR20160247.
[7]Nagata T,Shimada Y,Sekine S,et al.KLF4 and NANOG are prognostic biomarkers for triple-negative breast cancer[J].Breast Cancer,2017,24(2):326-335.
[8]Hu J,Liu J,Chen A,et al.Ino80 promotes cervical cancer tumorigenesis by activating Nanog expression[J].Oncotarget,2016,7(44):72250-72262.
[9]Chang CV,Araujo RV,Cirqueira CS,et al.Differential expression of stem cell markers in human adamantinomatous craniopharyngioma and pituitary adenoma[J].Neuroendocrinology,2017,104(2):183-193.
[10]Sun C,Feng SB,Cao ZW,et al.Up-regulated expression of matrix metalloproteinases in endothelial cells mediates platelet microvesicle-induced angiogenesis[J].Cell Physiol Biochem,2017,41(6):2319-2332.
[11]Jurado Acosta A,Rys?J,Szabo Z,et al.Transcription factor PEX1 modulates extracellular matrix turnover through regulation of MMP-9 expression[J].Cell Tissue Res,2017,367(2):369-385.
[12]Yin X,Zhang BH,Zheng SS,et al.Coexpression of gene Oct4and Nanog initiates stem cell characteristics in hepatocellular carcinoma and promotes epithelial-mesenchymal transition through activation of Stat3/Snail signaling[J].J Hematol Oncol,2015,8:23.
[13]Sun C,Sun L,Jiang K,et al.NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway[J].Int J Biochem Cell Biol,2013,45(6):1099-1108.
[14]Siu MK,Wong ES,Chan HY,et al.Overexpression of NANOGin gestational trophoblastic diseases:effect on apoptosis,cell invasion,and clinical outcome[J].Am J Pathol,2008,173(4):1165-1172.
[15]Borrull A,Ghislin S,Deshayes F,et al.Nanog and Oct4overexpression increases motility and transmigration of melanoma cells[J].J Cancer Res Clin Oncol,2012,138(7):1145-1154.
[16]艾威,刘宗来,舒峰,等.miR-193a靶向调控HOTAIR对前列腺癌细胞活力及侵袭能力的影响[J].热带医学杂志,2019,19(1):9-12,59.
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