沉默转导重排基因对抑制乳腺癌细胞增殖和迁移的影响
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摘要
目的探讨转导重排基因(rearranged during transfection,RET)在乳腺癌中的作用机制。方法选取2015年6月至2016年6月在北京大学深圳医院确诊的乳腺癌女性患者,采用实时荧光定量PCR对60例乳腺癌组织和20例癌旁组织中RET mRNA的相对表达量进行检测;经RET-siRNA转染乳腺癌细胞株MCF-7和乳腺癌内分泌耐药细胞株MCF-7/Aro后,MTT检测细胞增殖作用,Transwell细胞迁移实验评价细胞迁移能力,Western-blot和RT-PCR检测RET及磷脂酰肌醇-3-羟激酶(PI3K)、丝氨酸/苏氨酸激酶1(AKT1)、雷帕霉素靶蛋白(mTOR)、重组人丝裂原活化激酶(MEK)1和MEK2的相对表达情况。结果乳腺癌组织中RET mRNA的相对表达量高于癌旁组织(2.70±1.37vs.1.29±0.51),差异有统计学意义(P<0.05)。沉默RET后,MCF-7和MCF-7/Aro增殖、迁移能力受抑制,PI3K/AKT/mTOR通路相关蛋白表达量下调。结论 RET-siRNA能有效抑制RET基因的表达,抑制乳腺癌细胞增殖侵袭,其作用机制可能是通过下调PI3K/AKT/mTOR信号通路相关蛋白,进而抑制乳腺癌细胞增殖。
Objective To investigate the mechanism of rearranged during transfection(RET)in breast cancer.Methods The female patients with breast cancer treated in Shenzhen Hospital of Peking University from June of 2015 to June of 2016 were selected as the research objects,and the relative expression of RET mRNA in the breast cancer tissues of 60 cases and the adjacent tissues of 20 cases was detected by real-time fluorescence quantitative PCR.After the breast cancer cell line MCF-7 and the breast cancer endocrine resistant cell line MCF-7/Aro were transfected with RET-siRNA,the MTT assay was used to detect the cell viability,the transwell cell migration assay was used to evaluate the cell migration,and the Western blot and RT-PCR methods were used to detect the relative expression level of RET and the relative expression levels of phosphatidylinositol-3-hydroxyltryptin(PI3 K),serine/threonine kinase 1(AKT1),rapamycin target protein(mTOR),MEK1 and MEK2 in the PI3 K/AKT/mTOR pathway.Results The relative expression level of RET mRNA in the breast cancer tissues was higher than that in adjacent tissues(2.70±1.37 vs.1.29±0.51)and the difference was statistically significant(P<0.05).After the silencing of RET,the proliferation and migration of MCF-7 and MCF-7/Aro were inhibited and the expression level of the protein related with the PI3 K/AKT/mTOR pathway was down-regulated.Conclusion RET-siRNA can effectively inhibit the expression of RET genes and the proliferation and invasion of breast cancer cells.The mechanism of RET-siRNA may inhibit the proliferation of breast cancer cells by downregulating the protein related with the PI3 K/AKT/mTOR signaling pathway.
Objective To investigate the mechanism of rearranged during transfection(RET)in breast cancer.Methods The female patients with breast cancer treated in Shenzhen Hospital of Peking University from June of 2015 to June of 2016 were selected as the research objects,and the relative expression of RET mRNA in the breast cancer tissues of 60 cases and the adjacent tissues of 20 cases was detected by real-time fluorescence quantitative PCR.After the breast cancer cell line MCF-7 and the breast cancer endocrine resistant cell line MCF-7/Aro were transfected with RET-siRNA,the MTT assay was used to detect the cell viability,the transwell cell migration assay was used to evaluate the cell migration,and the Western blot and RT-PCR methods were used to detect the relative expression level of RET and the relative expression levels of phosphatidylinositol-3-hydroxyltryptin(PI3 K),serine/threonine kinase 1(AKT1),rapamycin target protein(mTOR),MEK1 and MEK2 in the PI3 K/AKT/mTOR pathway.Results The relative expression level of RET mRNA in the breast cancer tissues was higher than that in adjacent tissues(2.70±1.37 vs.1.29±0.51)and the difference was statistically significant(P<0.05).After the silencing of RET,the proliferation and migration of MCF-7 and MCF-7/Aro were inhibited and the expression level of the protein related with the PI3 K/AKT/mTOR pathway was down-regulated.Conclusion RET-siRNA can effectively inhibit the expression of RET genes and the proliferation and invasion of breast cancer cells.The mechanism of RET-siRNA may inhibit the proliferation of breast cancer cells by downregulating the protein related with the PI3 K/AKT/mTOR signaling pathway.
引文
[1]王乐,张玥,石菊芳,等.中国女性乳腺癌疾病负担分析[J].中华流行病学杂志,2016,37(7):970-976.
[2]Boulay A,Breuleux M,Stephan C,et al.The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer[J].Cancer Res,2008,68(10):3743-3751.
[3]Califano D,Rizzo C,D'Alessio A,et al.Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12cells[J].J Biol Chem,2000,275(25):19297-19305.
[4]Mai K T,Landry D C,Thomas J,et al.Ret oncogene protein expression in papillary thyroid carcinoma and related lesions[J].Tumori,2001,87(3):166-172.
[5]Takahashi M,Ritz J,Cooper G M.Activation of a novel human transforming gene,ret,by DNA rearrangement[J].Cell,1985,42(2):581-588.
[6]de Groot J W,Links T P,Plukker J T,et al.RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors[J].Endocr Rev,2006,27(5):535-560.
[7]Morrison P J,Atkinson A B.Genetic aspects of familial thyroid cancer[J].Oncologist,2009,14(6):571-577.
[8]Bolk S,Pelet A,Hofstra R M,et al.A human model for multigenic inheritance:phenotypic expression in Hirschsprung disease requires both the RET gene and a new9q31locus[J].Proc Natl Acad Sci USA,2000,97(1):268-273.
[9]de Groot J W,Links T P,Plukker J T,et al.RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors[J].Endocr Rev,2006,27(5):535-560.
[10]Borrello M G,Alberti L,Fischer A,et al.Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1oncogene[J].Proc Natl Acad Sci USA,2005,102(41):14825-14830.
[11]Boulay A,Breuleux M,Stephan C,et al.The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer[J].Cancer Res,2008,68(10):3743-3751.
[12]Boulay A,Rudloff J,Ye J,et al.Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer[J].Clin Cancer Res,2005,11(14):5319-5328.
[13]Plaza-Menacho I,Morandi A,Robertson D,et al.Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance[J].Oncogene,2010,29(33):4648-4657.
[2]Boulay A,Breuleux M,Stephan C,et al.The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer[J].Cancer Res,2008,68(10):3743-3751.
[3]Califano D,Rizzo C,D'Alessio A,et al.Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12cells[J].J Biol Chem,2000,275(25):19297-19305.
[4]Mai K T,Landry D C,Thomas J,et al.Ret oncogene protein expression in papillary thyroid carcinoma and related lesions[J].Tumori,2001,87(3):166-172.
[5]Takahashi M,Ritz J,Cooper G M.Activation of a novel human transforming gene,ret,by DNA rearrangement[J].Cell,1985,42(2):581-588.
[6]de Groot J W,Links T P,Plukker J T,et al.RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors[J].Endocr Rev,2006,27(5):535-560.
[7]Morrison P J,Atkinson A B.Genetic aspects of familial thyroid cancer[J].Oncologist,2009,14(6):571-577.
[8]Bolk S,Pelet A,Hofstra R M,et al.A human model for multigenic inheritance:phenotypic expression in Hirschsprung disease requires both the RET gene and a new9q31locus[J].Proc Natl Acad Sci USA,2000,97(1):268-273.
[9]de Groot J W,Links T P,Plukker J T,et al.RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors[J].Endocr Rev,2006,27(5):535-560.
[10]Borrello M G,Alberti L,Fischer A,et al.Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1oncogene[J].Proc Natl Acad Sci USA,2005,102(41):14825-14830.
[11]Boulay A,Breuleux M,Stephan C,et al.The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer[J].Cancer Res,2008,68(10):3743-3751.
[12]Boulay A,Rudloff J,Ye J,et al.Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer[J].Clin Cancer Res,2005,11(14):5319-5328.
[13]Plaza-Menacho I,Morandi A,Robertson D,et al.Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance[J].Oncogene,2010,29(33):4648-4657.