Lentivirus-mediated overexpression of PTEN-Long inhibits the growth of human glioma U251 cell in nude mice xenografts

doi:10.3969/j.issn.2095-4344.1140

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Glioma, Models, Animal, Lentivirus Infections, Tissue Engineering

CLC Number:

Geng Lianting¹, Li Chunhui², Shan Xiaosong², Zheng Yabei¹, Zeng Zhaomu¹, Zheng Kebin² . Lentivirus-mediated overexpression of PTEN-Long inhibits the growth of human glioma U251 cell in nude mice xenografts[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(11): 1743-1748.

Figures/Tables 2

2.1 实验动物数量分析纳入裸鼠12只，均进入结果分析，无脱失值。 2.2 携带PTEN-Long慢病毒转染的检测结果当感染复数为50时，荧光显微镜下显示细胞的荧光表达率均为80%以上，见图1。相同条件下进行实验重复3次，取所得3组数据进行统计分析。 Western blot分析显示3组PTEN-Long蛋白表达量(吸光度值)差异有显著性意义(P < 0.01)，过表达组(0.59±0.55)表达量高于对照组(0.28±0.04)与空载组(0.23±0.03)；P < 0.01)。对照组与空载组差异无显著性意义(P=0.19)。各组p-Akt表达量差异有显著性意义(P < 0.01)，与对照组(0.46±0.53)、空载组(0.44±0.04)相比，过表达组p-Akt(0.09±0.05)表达量降低(P < 0.01)。各组Akt及PTEN蛋白表达量差异无显著性意义，见图2。 2.3 PTEN-Long抑制裸鼠皮下移植瘤的生长接种肿瘤细胞约1周后可见开始形成肿瘤组织，肿瘤体积逐渐增加。裸鼠一般状态逐渐变差，过表达组与对照组、空载组相比较明显。接种约第4周时，对照组有出现肿瘤坏死的表现，可能主要因为肿瘤生长速度过快造成。研究发现，随着时间的进展，过表达组肿瘤体积增长速度缓于对照组与空载组，肿瘤体积生长变化曲线记录的是各组在不同时间的肿瘤体积的平均值。接种第28天取瘤后测量体积，各组肿瘤体积差异有显著性意义(P < 0.01)，过表达组肿瘤体积明显小于对照组与空载组 (P < 0.01)。各组肿瘤质量差异有显著性意义(P < 0.01)，过表达组肿瘤质量明显小于对照组与空载组(P < 0.01)。结果说明，PTEN-Long可抑制胶质瘤U251细胞体内增殖，见图3。 2.4 PTEN-Long抑制肿瘤组织中p-Akt及NF-κB p65蛋白表达免疫组织化学染色显示，PTEN-Long、p-Akt主要表达于细胞质，偶有表达于细胞核，NF-κB p65主要表达于细胞核，偶有细胞质内表达，染色后阳性结果显示为中度染色或强染色，见图4。与对照组及空载组相比，过表达组PTEN-Long表达水平明显增加(P < 0.01)，p-Akt和p65表达水平明显降低明显降低(P < 0.05，P < 0.01)，见表1。结果说明，PTEN-Long可能通过抑制PI3K-Akt-NF-κB信号通路抑制胶质瘤细胞增殖。"

References

[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 PTEN phosphatase 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 chromosome 10q23. 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, Davé V. 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-p53 enters 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-L for 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 Weizsäcker 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.