慢病毒介导PTEN-Long过表达抑制人脑胶质瘤U251细胞裸鼠移植瘤的生长
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摘要
背景:第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosohataseandtensinhomolguedeletedon chromosome10,PTEN)的翻译变体(PTEN-Long)是新发现的一种肿瘤抑制基因,与多种肿瘤的发展密切相关。目的:探讨PTEN-Long对人脑胶质瘤U251细胞裸鼠异体移植瘤生长的抑制作用及相关分子机制。方法:(1)人脑胶质瘤细胞U251购自国家实验细胞资源共享服务平台北京总部,实验将未感染慢病毒的U251为对照组,感染LV-EGFP的U251为空载组,感染LV-PTEN-Long-EGFP的U251为过表达组,用荧光显微镜、Western blot检测转染效率;(2)实验将购自北京华阜康生物科技股份有限公司的BALB/c-nu雄性裸鼠随机分为3组,将3组细胞分别接种于裸鼠背部皮下,观察裸鼠一般状况及肿瘤生长趋势,分析各组裸鼠皮下肿瘤的体积及质量变化,免疫组织化学染色法检测肿瘤中PTEN-Long,p-Akt及NF-κB p65蛋白表达变化情况。结果与结论:(1)一般状况:PTEN-Long过表达后可抑制裸鼠皮下胶质瘤细胞的增殖,与对照组及空载组相比,过表达组裸鼠肿瘤体积及质量减少(P <0.01);(2)免疫组织化学染色显示:与对照组及空载组相比,过表达组PTEN-Long表达量增加(P<0.05),p-Akt及NF-κBp65表达量降低(P<0.05);(3)结果显示:PTEN-Long可抑制人脑胶质瘤U251细胞裸鼠异体移植瘤的生长,机制可能与下调PI3K-AKT-NF-κB信号通路有关。
BACKGROUND: PTEN-Long is a translational variant of phosohatase and tensin homolgue deleted on chromosome 10(PTEN), which is a newly discovered tumor suppressor gene and is closely related to the development of various tumors. OBJECTIVE: To investigate the inhibition effect of PTEN-Long on the growth of U251 cells(human glioma cells) in nude mice xenografts and related molecular mechanisms. METHODS: Human glioma cell U251 was purchased from the National Infrastructure of Cell Line Resource in Beijing. U251 not infected with lentivirus was used as control group. U251 infected with LV-EGFP was empty group. U251 infected with LV-PTEN-Long-EGFP was overexpressed group. Transfection efficiency was measured by fluorescence microscopy and western blot assay. BALB/c-nu male nude mice purchased from Beijing Huakang Biotechnology Co., Ltd. were randomly divided into three groups, and the three groups of cells were inoculated subcutaneously into the back of nude mice. The general condition and tumor growth trend were observed. The volume and weight of subcutaneous tumors in nude mice were analyzed. The expression levels of PTEN-Long, p-Akt and NF-κB p65 protein in tumors were detected by immunohistochemical staining. RESULTS AND COUNCLUSION: Overexpression of PTEN-Long inhibited the proliferation of subcutaneous glioma cells in nude mice. Compared with the control group and the empty group, the overexpressed group was in poor condition, tumor volume and weight were decreased(P < 0.01). Immunohistochemical staining showed that the expression of PTEN-Long(P < 0.05) was increased, and the expression levels of p-Akt and NF-κB p65 were decreased(P < 0.05). In summary, overexpression of PTEN-Long inhibits the growth of U251 cells in nude mice, and the mechanism may be related to down-regulation of PI3K-AKT-NF-κB signaling pathway.
BACKGROUND: PTEN-Long is a translational variant of phosohatase and tensin homolgue deleted on chromosome 10(PTEN), which is a newly discovered tumor suppressor gene and is closely related to the development of various tumors. OBJECTIVE: To investigate the inhibition effect of PTEN-Long on the growth of U251 cells(human glioma cells) in nude mice xenografts and related molecular mechanisms. METHODS: Human glioma cell U251 was purchased from the National Infrastructure of Cell Line Resource in Beijing. U251 not infected with lentivirus was used as control group. U251 infected with LV-EGFP was empty group. U251 infected with LV-PTEN-Long-EGFP was overexpressed group. Transfection efficiency was measured by fluorescence microscopy and western blot assay. BALB/c-nu male nude mice purchased from Beijing Huakang Biotechnology Co., Ltd. were randomly divided into three groups, and the three groups of cells were inoculated subcutaneously into the back of nude mice. The general condition and tumor growth trend were observed. The volume and weight of subcutaneous tumors in nude mice were analyzed. The expression levels of PTEN-Long, p-Akt and NF-κB p65 protein in tumors were detected by immunohistochemical staining. RESULTS AND COUNCLUSION: Overexpression of PTEN-Long inhibited the proliferation of subcutaneous glioma cells in nude mice. Compared with the control group and the empty group, the overexpressed group was in poor condition, tumor volume and weight were decreased(P < 0.01). Immunohistochemical staining showed that the expression of PTEN-Long(P < 0.05) was increased, and the expression levels of p-Akt and NF-κB p65 were decreased(P < 0.05). In summary, overexpression of PTEN-Long inhibits the growth of U251 cells in nude mice, and the mechanism may be related to down-regulation of PI3K-AKT-NF-κB signaling pathway.
引文
[1]Stylli SS,Luwor RB,Ware TMB,et al.Mouse models of glioma.J Clin Neurosci.2015;22(4):619-626.
[2]许蓓,李爱群,江高峰.胶质瘤的细胞来源研究进展[J].中国病理生理杂志,2018,34(3):566-571.
[3]Zhao YD,Zhang QB,Chen H,et al.Research on human glioma stem cells in China.Neural Regen Res.2017;12(11):1918-1926.
[4]Hopkins BD,Fine B,Steinbach N,et al.A secreted PTENphosphatase that enters cells to alter signaling and survival.Science.2013;341:399-402.
[5]潘梦武,侯玲玲.PTEN和PTEN-Long的关系及其抑瘤作用[J].生理科学进展,2016,47(5):355-360.
[6]Li J,Yen C,Liaw D,et al.PTEN,a putative protein tyrosine phosphatase gene mutated in human brain,breast,and prostate cancer.Science.1997;275:1943-1947.
[7]Steck PA,Pershouse MA,Jasser SA,et al.Identification of a candidate tumour suppressor gene,MMAC1,at chromosome10q23.3 that is mutated in multiple advanced cancers.Nat Genet.1997;15(4):356-362.
[8]Sun H,Lesche R,Li DM,et al.PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.Proc Natl Acad Sci U S A.1999;96(11):6199-6204.
[9]Fang N,Gu T,Wang Y,et al.Expression of PTEN‐long mediated by CRISPR/Cas9 can repress U87 cell proliferation.J Cell Mol Med.2017;21(12):3337-3346.
[10]Wu Q,Li Z,Liu Q.Treatment with PTEN-Long protein inhibits hepatitis C virus replication.Virology.2017;511:1-8.
[11]Wang H,Zhang P,Lin C,et al.Relevance and therapeutic possibility of pten-long in renal cell carcinoma.PLoS One.2015;10(2):e114250.
[12]Baltimore D.NF-[kappa]B is 25.Nat Immunol.2011;12:683-685.
[13]刘畅,徐玉杰,曹亮,等.NF-κB抑制剂QNZ对人胶质瘤细胞生物学功能的影响[J].现代肿瘤医学,2018,26(5):664-667.
[14]张昀昇,张俊文,米蕊芳,等.TRIM33对人脑胶质瘤U251细胞生物学功能影响的实验研究[J].中华神经外科杂志,2018,34(3):292-298.
[15]马一鸣,夏祥国.YAP蛋白对C6胶质瘤干细胞特性的影响[J].中国组织工程研究,2018,22(21):3335-3340.
[16]叶园英,黄煜,颜莉莉,等.人子宫内膜癌裸鼠皮下移植瘤模型的建立[J].山东医药,2016,56(35):28-30.
[17]汪柳旭,朱凤军,廖建湘,等.U87胶质瘤裸鼠皮下移植瘤模型建立[J].中国实用神经疾病杂志,2018,21(3):239-242.
[18]张玉辉,李伟,李香香,等.基质金属蛋白酶-26在人脑胶质瘤血管新生中的作用[J].临床与实验病理学杂志,2017,33(6):623-628.
[19]赵瑞君,谢春伟,陈戈,等.PTP1B在ER阳性乳腺癌中的表达及临床预后意义[J].临床与实验病理学杂志,2016,32(5):492-495.
[20]Baker SJ.PTEN enters the nuclear age.Cell.2007;128(1):25-28.
[21]Lewis C.Cantley.The phosphoinositide 3-kinase pathway.Science.2002;296(5573):1655-1657.
[22]Maehama T,Dixon JE.The tumor suppressor,PTEN/MMAC1,dephosphorylates the lipid second messenger,phosphatidylinositol 3,4,5-trisphosphate.J Biol Chem.1998;273(22):13375-13378.
[23]Huang ZR,Chen HY,Hu ZZ,et al.PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve.Neural Regen Res.2018;13(1):135-144.
[24]Cully M,You H,Levine AJ,et al.Beyond PTEN mutations:the PI3K pathway as an integrator of multiple inputs during tumorigenesis.Nat Rev Cancer.2006;6:184-192.
[25]Leslie NR,Downes CP.PTEN function:how normal cells control it and tumour cells lose it.Biochem J.2004;382:1-11.
[26]Muskan G,Gurcharan K.Aqueous extract from theWithania somniferaleaves as a potential anti-neuroinflammatory agent:a mechanistic study.J Neuroinflammation.2016;13(1):193.
[27]Hou YC,Chiu WC,Yeh CL,et al.Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/mTOR pathway in lung epithelial cells.Am J Physiol Lung Cell Mol Physiol.2012;302:L174-183.
[28]Legg K.PTEN goes paracrine.Nat Cell Biol.2013;15(8):894.
[29]Masson GR,Perisic O1,Burke JE,et al.The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity.Biochem J.2016;473(Pt 2):135-144.
[30]Malaney P,Uversky VN,DavéV.The PTEN Long N-tail is intrinsically disordered:increased viability for PTEN therapy.Mol Biosyst.2013;9(11):2877-2888.
[31]Xiao M,An Y,Wang F,et al.A chimeric protein PTEN-L-p53enters U251 cells to repress proliferation and invasion.Exp Cell Res.2018;369(2):234-242.
[32]Lavictoire SJ,Gont A,Julian L M,et al.Engineering PTEN-Lfor cell-mediated delivery.Mol Ther Methods Clin Dev.2018;9:12-22.
[33]Liu P,Liao L,Xu W,et al.Adherence,virological outcome,and drug resistance in Chinese HIV patients receiving first-line antiretroviral therapy from 2011 to 2015.Medicine(Baltimore).2018;97(50):e13555.
[34]Salehi B,Kumar NVA,?ener B,et al.Medicinal plants used in the treatment of human immunodeficiency virus.Int J Mol Sci.2018;19(5):E1459.
[35]Adeyanju K,Bend JR,Rieder MJ,et al.HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells.Virol J.2018;15(1):82.
[36]Rasmussen TA,McMahon JH,Chang JJ,et al.The effect of antiretroviral intensification with dolutegravir on residual virus replication in HIV-infected individuals:a randomised,placebo-controlled,double-blind trial.Lancet HIV.2018;5(5):e221-e230.
[37]Wilmshurst JM,Hammond CK,Donald K,et al.NeuroAIDS in children.Handb Clin Neurol.2018;152:99-116.
[38]Molina JM,Squires K,Sax PE,et al.Doravirine versus ritonavir-boosted darunavir in antiretroviral-naive adults with HIV-1(DRIVE-FORWARD):48-week results of a randomised,double-blind,phase 3,non-inferiority trial.Lancet HIV.2018;5(5):e211-e220.
[39]Krüger R,Borte S,von Weizs?cker K,et al.Positive kappa-deleting recombination excision circles(KREC)newborn screening in a neonate with intrauterine exposure to rituximab.Scand J Immunol.2018;87(1):54-56.
[40]谭麟,张谢,李宏.磷酸酶及张力蛋白同源物与肝细胞肝癌[J].新医学,2017,48(5):293-296.
[2]许蓓,李爱群,江高峰.胶质瘤的细胞来源研究进展[J].中国病理生理杂志,2018,34(3):566-571.
[3]Zhao YD,Zhang QB,Chen H,et al.Research on human glioma stem cells in China.Neural Regen Res.2017;12(11):1918-1926.
[4]Hopkins BD,Fine B,Steinbach N,et al.A secreted PTENphosphatase that enters cells to alter signaling and survival.Science.2013;341:399-402.
[5]潘梦武,侯玲玲.PTEN和PTEN-Long的关系及其抑瘤作用[J].生理科学进展,2016,47(5):355-360.
[6]Li J,Yen C,Liaw D,et al.PTEN,a putative protein tyrosine phosphatase gene mutated in human brain,breast,and prostate cancer.Science.1997;275:1943-1947.
[7]Steck PA,Pershouse MA,Jasser SA,et al.Identification of a candidate tumour suppressor gene,MMAC1,at chromosome10q23.3 that is mutated in multiple advanced cancers.Nat Genet.1997;15(4):356-362.
[8]Sun H,Lesche R,Li DM,et al.PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.Proc Natl Acad Sci U S A.1999;96(11):6199-6204.
[9]Fang N,Gu T,Wang Y,et al.Expression of PTEN‐long mediated by CRISPR/Cas9 can repress U87 cell proliferation.J Cell Mol Med.2017;21(12):3337-3346.
[10]Wu Q,Li Z,Liu Q.Treatment with PTEN-Long protein inhibits hepatitis C virus replication.Virology.2017;511:1-8.
[11]Wang H,Zhang P,Lin C,et al.Relevance and therapeutic possibility of pten-long in renal cell carcinoma.PLoS One.2015;10(2):e114250.
[12]Baltimore D.NF-[kappa]B is 25.Nat Immunol.2011;12:683-685.
[13]刘畅,徐玉杰,曹亮,等.NF-κB抑制剂QNZ对人胶质瘤细胞生物学功能的影响[J].现代肿瘤医学,2018,26(5):664-667.
[14]张昀昇,张俊文,米蕊芳,等.TRIM33对人脑胶质瘤U251细胞生物学功能影响的实验研究[J].中华神经外科杂志,2018,34(3):292-298.
[15]马一鸣,夏祥国.YAP蛋白对C6胶质瘤干细胞特性的影响[J].中国组织工程研究,2018,22(21):3335-3340.
[16]叶园英,黄煜,颜莉莉,等.人子宫内膜癌裸鼠皮下移植瘤模型的建立[J].山东医药,2016,56(35):28-30.
[17]汪柳旭,朱凤军,廖建湘,等.U87胶质瘤裸鼠皮下移植瘤模型建立[J].中国实用神经疾病杂志,2018,21(3):239-242.
[18]张玉辉,李伟,李香香,等.基质金属蛋白酶-26在人脑胶质瘤血管新生中的作用[J].临床与实验病理学杂志,2017,33(6):623-628.
[19]赵瑞君,谢春伟,陈戈,等.PTP1B在ER阳性乳腺癌中的表达及临床预后意义[J].临床与实验病理学杂志,2016,32(5):492-495.
[20]Baker SJ.PTEN enters the nuclear age.Cell.2007;128(1):25-28.
[21]Lewis C.Cantley.The phosphoinositide 3-kinase pathway.Science.2002;296(5573):1655-1657.
[22]Maehama T,Dixon JE.The tumor suppressor,PTEN/MMAC1,dephosphorylates the lipid second messenger,phosphatidylinositol 3,4,5-trisphosphate.J Biol Chem.1998;273(22):13375-13378.
[23]Huang ZR,Chen HY,Hu ZZ,et al.PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve.Neural Regen Res.2018;13(1):135-144.
[24]Cully M,You H,Levine AJ,et al.Beyond PTEN mutations:the PI3K pathway as an integrator of multiple inputs during tumorigenesis.Nat Rev Cancer.2006;6:184-192.
[25]Leslie NR,Downes CP.PTEN function:how normal cells control it and tumour cells lose it.Biochem J.2004;382:1-11.
[26]Muskan G,Gurcharan K.Aqueous extract from theWithania somniferaleaves as a potential anti-neuroinflammatory agent:a mechanistic study.J Neuroinflammation.2016;13(1):193.
[27]Hou YC,Chiu WC,Yeh CL,et al.Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/mTOR pathway in lung epithelial cells.Am J Physiol Lung Cell Mol Physiol.2012;302:L174-183.
[28]Legg K.PTEN goes paracrine.Nat Cell Biol.2013;15(8):894.
[29]Masson GR,Perisic O1,Burke JE,et al.The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity.Biochem J.2016;473(Pt 2):135-144.
[30]Malaney P,Uversky VN,DavéV.The PTEN Long N-tail is intrinsically disordered:increased viability for PTEN therapy.Mol Biosyst.2013;9(11):2877-2888.
[31]Xiao M,An Y,Wang F,et al.A chimeric protein PTEN-L-p53enters U251 cells to repress proliferation and invasion.Exp Cell Res.2018;369(2):234-242.
[32]Lavictoire SJ,Gont A,Julian L M,et al.Engineering PTEN-Lfor cell-mediated delivery.Mol Ther Methods Clin Dev.2018;9:12-22.
[33]Liu P,Liao L,Xu W,et al.Adherence,virological outcome,and drug resistance in Chinese HIV patients receiving first-line antiretroviral therapy from 2011 to 2015.Medicine(Baltimore).2018;97(50):e13555.
[34]Salehi B,Kumar NVA,?ener B,et al.Medicinal plants used in the treatment of human immunodeficiency virus.Int J Mol Sci.2018;19(5):E1459.
[35]Adeyanju K,Bend JR,Rieder MJ,et al.HIV-1 tat expression and sulphamethoxazole hydroxylamine mediated oxidative stress alter the disulfide proteome in Jurkat T cells.Virol J.2018;15(1):82.
[36]Rasmussen TA,McMahon JH,Chang JJ,et al.The effect of antiretroviral intensification with dolutegravir on residual virus replication in HIV-infected individuals:a randomised,placebo-controlled,double-blind trial.Lancet HIV.2018;5(5):e221-e230.
[37]Wilmshurst JM,Hammond CK,Donald K,et al.NeuroAIDS in children.Handb Clin Neurol.2018;152:99-116.
[38]Molina JM,Squires K,Sax PE,et al.Doravirine versus ritonavir-boosted darunavir in antiretroviral-naive adults with HIV-1(DRIVE-FORWARD):48-week results of a randomised,double-blind,phase 3,non-inferiority trial.Lancet HIV.2018;5(5):e211-e220.
[39]Krüger R,Borte S,von Weizs?cker K,et al.Positive kappa-deleting recombination excision circles(KREC)newborn screening in a neonate with intrauterine exposure to rituximab.Scand J Immunol.2018;87(1):54-56.
[40]谭麟,张谢,李宏.磷酸酶及张力蛋白同源物与肝细胞肝癌[J].新医学,2017,48(5):293-296.