抑制GATA5蛋白可否促进HELA细胞中癌干细胞因子sox2、c-myc及CD44的表达
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摘要
背景:GATA5蛋白在多种癌症发生过程中被抑制表达,有研究显示GATA5降低表达或被甲基化后失去功能,不能有效促进干细胞分化,维持细胞的正常功能。宫颈癌HELA细胞表达GATA5蛋白,在宫颈癌中抑制GATA5表达是否可促进其恶性转化还是未知。目的:分析GATA5蛋白在宫颈癌HELA细胞中被进一步抑制表达后,其恶性转化情况及与癌干细胞形成相关因子sox2,c-myc及CD44的表达情况。方法:转入CMV-siRNA-gata5,使GATA5在HELA细胞中被进一步抑制表达后,(1)RT-PCR检测c-myc mRNA,sox2 mRNA的表达;(2)MTT法检测HELA细胞的增殖能力;(3)检测HELA细胞的集落形成和软琼脂单克隆形成能力;(4)Western blot检测细胞GATA5、c-myc、sox2蛋白表达变化;(5)激光共聚焦检测细胞的CD44蛋白定位及蛋白表达变化。结果与结论:用CMV-siRNA-gata5抑制HELA细胞中的GATA5表达后,HELA细胞的增殖能力、单克隆形成及集落形成能力均增强;同时发现其癌干细胞因子c-myc,sox2,CD44的表达增强。提示GATA5蛋白被抑制表达后,HELA细胞的恶行性为增加,其机制可能是GATA5被抑制后,促进了癌干细胞因子sox2,c-myc,CD44等的表达。
BACKGROUND:GATA5, as a tumor suppressor gene, plays a role in inhibiting the development of cancer.GATA5 can be methylated in gastrointestinal tumors and then lose its function. GATA5 can be also methylated during hepatocarcinogenesis, and then cannot promote the differentiation of stem cells and maintain the normal function. Cervical cancer HELA cells express GATA5 protein, in which whether GATA5 plays a tumor suppressor function is still unknown.OBJECTIVE:To analyze the expression of sox2, c-myc and CD44 related to cancer stem cell formation after suppressing the expression of GATA5 in cervical cancer HELA cells.METHODS:Transfection of CMV-siRNA-gata5 further inhibited the expression of GATA5 in cervical cancer HELA cells. The expression of c-myc mRNA and sox2 mRNA were then detected by RT-PCR. The proliferation of HELA cells was detected by MTT. The colony formation and soft agar monoclonal formation of HELA cells were observed The expression of GATA5, c-myc and sox2 was detected by western blot. The expression and location of CD44 was detected by laser confocal microscope.RESULTS AND CONCLUSION:After suppressing the expression of GATA5 in cervical cancer HELA cells by CMV-siRNA-gata5, the proliferation, cell monoclonal formation and colony formation of HELA cells were enhanced The expression of c-myc, sox2 and CD44 was also strengthened. These findings indicate that the malignant activity of HELA cells is increased by inhibiting the GATA5 protein expression. The mechanism may be that inhibition of GATA5 promotes the expression of cancer stem cell factors sox2, c-myc, and CD44.
BACKGROUND:GATA5, as a tumor suppressor gene, plays a role in inhibiting the development of cancer.GATA5 can be methylated in gastrointestinal tumors and then lose its function. GATA5 can be also methylated during hepatocarcinogenesis, and then cannot promote the differentiation of stem cells and maintain the normal function. Cervical cancer HELA cells express GATA5 protein, in which whether GATA5 plays a tumor suppressor function is still unknown.OBJECTIVE:To analyze the expression of sox2, c-myc and CD44 related to cancer stem cell formation after suppressing the expression of GATA5 in cervical cancer HELA cells.METHODS:Transfection of CMV-siRNA-gata5 further inhibited the expression of GATA5 in cervical cancer HELA cells. The expression of c-myc mRNA and sox2 mRNA were then detected by RT-PCR. The proliferation of HELA cells was detected by MTT. The colony formation and soft agar monoclonal formation of HELA cells were observed The expression of GATA5, c-myc and sox2 was detected by western blot. The expression and location of CD44 was detected by laser confocal microscope.RESULTS AND CONCLUSION:After suppressing the expression of GATA5 in cervical cancer HELA cells by CMV-siRNA-gata5, the proliferation, cell monoclonal formation and colony formation of HELA cells were enhanced The expression of c-myc, sox2 and CD44 was also strengthened. These findings indicate that the malignant activity of HELA cells is increased by inhibiting the GATA5 protein expression. The mechanism may be that inhibition of GATA5 promotes the expression of cancer stem cell factors sox2, c-myc, and CD44.
引文
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[25]Zhu H,Mitsuhashi N,Klein A,et al.The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix.Stem Cells.2010;24(4):928-935.
[26]Yang X,Sarvestani SK,Moeinzadeh S,et al.Effect of CD44binding peptide conjugated to an engineered inert matrix on maintenance of breast cancer stem cells and tumorsphere formation.Plos One.2013;8(3):e59147.
[27]Hong I,Hong SW,Chang YG,et al.Expression of the Cancer Stem Cell Markers CD44 and CD133 in Colorectal Cancer:An Immunohistochemical Staining Analysis.Ann Coloproctol.2015;31(3):84-91.
[28]Moghbeli M,Moghbeli F,Forghanifard MM,et al.Cancer stem cell detection and isolation.Med Oncol.2014;31(9):69.
[29]Maddox J,Shakya A,South S,et al.Transcription factor Oct1 is a somatic and cancer stem cell determinant.Plos Genetics.2012;8(11):e1003048.
[2]Feng D,Peng C,Li C,et al.Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri.Oncology Reports.2009;22(5):1129-1134.
[3]Todaro M,Lombardo Y,Stassi G.Evidences of cervical cancer stem cells derived from established cell lines.Cell Cycle.2010;9(7):1231-1240.
[4]Shu J,Zhang K,Zhang M,et al.GATA family members as inducers for cellular reprogramming to pluripotency.Cell Res.2015;25(2):169-180.
[5]Molkentin JD,Lin Q,Duncan SA,et al.Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis.Genes Dev.1997;11(8):1061-1072.
[6]Laverriere AC,Macneill C,Mueller C,et al.GATA-4/5/6,a subfamily of three transcription factors transcribed in developing heart and gut.J Biol Chem.1994;269(37):23177-23184.
[7]Akiyama Y,Watkins N,Suzuki H,et al.GATA-4 and GATA-5transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer.Mol Cell Biol.2003;23(23):8429-8439.
[8]Wen X,Akiyama Y,Zhou J,et al.Loss of transcription factor GATA-4/-5 by hypermethylation correlated with progression of gastric carcinomas.Cancer Res.2007;67:2873-2873.
[9]Lei X,Yan G,Zhang A,et al.Loss of GATA5 expression due to gene promoter methylation induces growth and colony formation of hepatocellular carcinoma cells.Oncol Lett.2016;11(1):861-869.
[10]Liu P,Zhou TF,Qiu BA,et al.Methylation-Mediated Silencing of GATA5 Gene Suppresses Cholangiocarcinoma Cell Proliferation and Metastasis.Transl Oncol.2018;11(3):585-592.
[11]Pei Y,Qin Y,Yuan S,et al.GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3.Oncotarget.2016;7(47):77890-77901.
[12]Lin B,Liu K,Wang W,et al.Expression and bioactivity of humanα-fetoprotein in a Bac-to-Bac system.Biosci Rep.2017;37(1).pii:BSR20160161.
[13]Sun Y,Huang YH,Huang FY,et al.3′-epi-12β-hydroxyfroside,a new cardenolide,induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.Theranostics.2018;8(7):2044-2060.
[14]Li M,Li H,Li C,et al.Cytoplasmic alpha-fetoprotein functions as a co-repressor in RA-RAR signaling to promote the growth of human hepatoma Bel 7402 cells.Cancer Lett.2009;285(2):190-199.
[15]Li M,Zhu M,Li W,et al.Alpha-fetoprotein receptor as an early indicator of HBx-driven hepatocarcinogenesis and its applications in tracing cancer cell metastasis.Cancer Lett.2013;330(2):170-180.
[16]Gandarillas A,Watt FM.c-Myc promotes differentiation of human epidermal stem cells.Genes Dev.1997;11(21):2869-2882.
[17]Satoh Y,Matsumura I,Tanaka H,et al.Roles for c-Myc in self-renewal of hematopoietic stem cells.J Biol Chem.2004;279(24):24986-24993.
[18]Martinez-Fernandez A,Nelson TJ,Ikeda Y,et al.c-MYCindependent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells.J Cardiovasc Transl Res.2010;3(1):13-23.
[19]Ellis P,Fagan BM,Magness ST,et al.SOX2,a persistent marker for multipotential neural stem cells derived from embryonic stem cells,the embryo or the adult.Dev Neurosci.2004;26(2-4):148-165.
[20]Giorgetti A,Montserrat N,Aasen T,et al.Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2.Cell Stem Cell.2009;5(4):353-357.
[21]Park SB,Seo KW,So AY,et al.SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC.Cell Death Differ.2012;19(3):534-545.
[22]Xiang R,Liao D,Cheng T,et al.Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer.Br J Cancer.2011;104(9):1410-1417.
[23]Leis O,Eguiara A,Lopezarribillaga E,et al.Sox2 expression in breast tumours and activation in breast cancer stem cells.Oncogene.2012;31(11):1354-1365.
[24]Bourguignon LY,Peyrollier K,Xia W,et al.Hyaluronan-CD44interaction activates stem cell marker Nanog,Stat-3-mediated MDR1 gene expression,and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells.J Biol Chem.2008;283(25):17635-17651.
[25]Zhu H,Mitsuhashi N,Klein A,et al.The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix.Stem Cells.2010;24(4):928-935.
[26]Yang X,Sarvestani SK,Moeinzadeh S,et al.Effect of CD44binding peptide conjugated to an engineered inert matrix on maintenance of breast cancer stem cells and tumorsphere formation.Plos One.2013;8(3):e59147.
[27]Hong I,Hong SW,Chang YG,et al.Expression of the Cancer Stem Cell Markers CD44 and CD133 in Colorectal Cancer:An Immunohistochemical Staining Analysis.Ann Coloproctol.2015;31(3):84-91.
[28]Moghbeli M,Moghbeli F,Forghanifard MM,et al.Cancer stem cell detection and isolation.Med Oncol.2014;31(9):69.
[29]Maddox J,Shakya A,South S,et al.Transcription factor Oct1 is a somatic and cancer stem cell determinant.Plos Genetics.2012;8(11):e1003048.