RNA interferes with Id2 gene expression to inhibit proliferation and invasion of PC-3 prostate cancer stem cells

doi:10.3969/j.issn.2095-4344.1623

Abstract

Abstract:

BACKGROUND: The Id2 gene is an endogenous negative regulator of basic helix-loop-helix factor, which is involved in the cell proliferation, differentiation and existence. Id2 also shows functional diversity in the progression and infiltration of different types of tumors
OBJECTIVE: To observe the changes of proliferation and invasiveness of PC-3 human prostate cancer stem cells after shRNA-Id2 transfection.
METHODS: PC-3 human prostate cancer stem cells in logarithmic growth phase were harvested to isolate tumor stem cell spheres by serum-free suspension culture. The expression of CD44⁺CD24^- on the surface of tumor stem cells was detected by flow cytometry. The shRNA-Id2 expression vector was constructed and transfected into PC-3 human prostate cancer stem cells. Untransfected PC-3 human prostate cancer stem cells were used as control. At 48 hours after transfection, the expression of Id2 gene and protein in shRNA-Id2 transfected prostate cancer stem cells, NC-shRNA empty vector transfected prostate cancer stem cells and untransfected prostate cancer stem cells were detected by RT-PCR and western blot, respectively. The proliferation and invasion of shRNA-Id2 transfected prostate cancer stem cells and untransfected prostate cancer stem cells were detected by MTT assay and Transwell chamber, respectively. The expressions of E-cadherin, vimentin and Twist were detected by western blot and RT-PCR.
RESULTS AND CONCLUSION: Tumor stem cell spheres were successfully isolated by the serum-free suspension culture. The expression rate of CD44⁺CD24^- on the surface of the third-generation PC-3 human prostate cancer stem cells was (85.69±8.96)%, indicating that the cultured tumor stem cell spheres overexpressed the phenotype of tumor stem cells. At 48 hours after transfection, the expression of Id2 gene and protein was significantly lower in the shRNA-Id2 transfection group than the non-transfection group (P < 0.05), indicating that the expression of Id2 was successfully interfered with the expression of PC-3 prostate cancer stem cells. The invasive ability of the cells in the shRNA-Id2 transfection group was significantly lower than that in the non-transfection group (P < 0.05). Western blot and RT-PCR detection showed that the expression of E-cadherin, an epithelial marker of PC-3 prostate cancer stem cells, in the shRNA-Id2 transfection group was significantly higher than that in non-transfection group (P < 0.05), while the expression of vimentin, a marker of mesenchymal stem cells, and Twist, a transcription factor regulating cell-mesenchymal transformation, in the shRNA-Id2 transfection group was significantly lower than that in the non-transfection group (P < 0.05). These findings indicate that RNA interference with Id2 gene can inhibit the proliferation and invasion of PC-3 prostate cancer stem cells by regulating the expression of E-cadherin, vimentin and Twist.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Prostatic Neoplasms, Neoplastic Stem Cells, RNA Interference, Cell Proliferation, Tissue Engineering

CLC Number:

Zhang Yingying, Wang Yinglei, Meng Lin, Xiao Lin, Li Zhonghai, Zhao Zhankui, Wu Houke. RNA interferes with Id2 gene expression to inhibit proliferation and invasion of PC-3 prostate cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1342-1348.

Figures/Tables 5

References

[1] 韩苏军,张思维,陈万青,等.中国前列腺癌发病现状和流行趋势分析[J]. 临床肿瘤学杂志, 2013,18(4):330-334.

[2] Rycaj K, Tang DG. Molecular determinants of prostate cancer metastasis. Oncotarget. 2017;8(50):88211-88231.

[3] Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer. 2005;5(8):603-614.

[4] Patel D, Morton DJ, Carey J, et al. Inhibitor of differentiation 4 (ID4): From development to cancer. Biochim Biophys Acta. 2015;1855(1):92-103.

[5] Ling F, Kang B, Sun XH. Id proteins: small molecules, mighty regulators. Curr Top Dev Biol. 2014;110:189-216.

[6] 李亚卓,赵坡.Id2与NF-κB/P65在胃癌中的表达及临床病理意义[J].中华医学杂志,2018,98(11):846-850

[7] Gray MJ, Dallas NA, Van Buren G, et al. Therapeutic targeting of Id2 reduces growth of human colorectal carcinoma in the murine liver. Oncogene. 2008;27(57):7192-7200.

[8] Lasorella A, Rothschild G, Yokota Y, et al. Id2 mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice. Mol Cell Biol. 2005;25(9):3563-3574.

[9] Coma S, Amin DN, Shimizu A, et al. Id2 promotes tumor cell migration and invasion through transcriptional repression of semaphorin 3F. Cancer Res. 2010;70(9):3823-3832.

[10] 汪萍,李晓军,贾丽,等.分化抑制因子Id2和Id3在肿瘤细胞中的表达[J].医学研究生报,2007,20(1):20-23.

[11] 汪洋,冯杨焜,吴岩,等.人前列腺癌PC-3细胞系中肿瘤干细胞的富集、鉴定以及生物学特征的初步研究[J].中国男科学杂志, 2018, 32(2):20-29.

[12] 岳黎敏,张莽,程书珍,等. Id2和VEGF的表达与食管鳞癌临床病理特征的关系研究[J].现代中西医结合杂志 2015,24(33): 3662-3664,3684.

[13] 温晓芬,许宏武,Min Chen,等.分化抑制因子2的生物学功能及其在乳腺癌中的研究进展[J].中华乳腺病杂志(电子版), 2016,10(6): 366-369.

[14] 邱建龙,杨维群,徐海刚. Id2在上皮性卵巢肿瘤中的表达及意义[J].临床肿瘤学杂志, 2011,16(12): 1088-1091.

[15] 杨忠信,李晓宇,蔡静,等.Xklp2靶蛋白对肺癌细胞裸鼠成瘤能力的影响及机制研究[J].实用医院临床杂志,2018,15(2):4-8.

[16] 赵泓帝,张振,邵志强,等. 沉默EMMPRIN对肾透明细胞癌增殖的影响[J].现代肿瘤医学,2018,26(7): 992-997.

[17] 鲁保才,卢振民,张爱民,等.干扰性小核糖核酸靶向抑制趋化因子受体4基因表达对鼻咽癌细胞增殖和侵袭的影响[J].新乡医学院学报,2018,35(1):30-34.

[18] 赵振宇,贺华,卢亦成,等.Id2shRNA慢病毒载体的构建及其转染后对胶质瘤细胞增殖和凋亡的影响[J].第二军医大学学报, 2011, 32(6):598-602.

[19] 姚春飞.恶性肿瘤侵袭与转移的病理探讨[J].养生保健指南, 2018, (7):246,276.

[20] 薛无涯,王舰梅,夏天,等.miR-204-5p对结直肠癌细胞增殖、侵袭、迁移及上皮间质转化的影响[J].西南医科大学学报, 2018,41(2): 104-110.

[21] 杜媛鲲,米源,陈阁,等.Gli通过PI3K/AKT途径促进肺腺癌A549细胞侵袭转移的研究[J].河北医科大学学报,2018,39(1):24-28.

[22] 李艳,陈琼霞,王绪明. 敲低Stat5对甲状腺癌TT细胞侵袭及上皮间质转化的影响[J].国际肿瘤学杂志, 2018,45(1):1-4.

[23] Lo UG, Lee CF, Lee MS, et al. The Role and Mechanism of Epithelial-to-Mesenchymal Transition in Prostate Cancer Progression. Int J Mol Sci. 2017;18(10): E2079.

[24] 刘婷,吴俊成,陆伦根,等.STAT3加重TGF-β1诱导的肝癌细胞上皮-间质转化的发生[J].肝脏,2018,23(2):128-132.

[25] Ivanova K, Ananiev J, Aleksandrova E, et al. Expression of E-Cadherin/Beta-Catenin in Epithelial Carcinomas of the Thyroid Gland. Open Access Maced J Med Sci. 2017;5(2): 155-159.

[26] Da C, Wu K, Yue C, et al. N-cadherin promotes thyroid tumorigenesis through modulating major signaling pathways. Oncotarget. 2017;8(5):8131-8142.

[27] Calangiu CM, Simionescu CE, Stepan AE, et al. The expression of CK19, vimentin and E-cadherin in differentiated thyroid carcinomas. Rom J Morphol Embryol. 2014;55(3): 919-925.

[28] 付海丹.宫颈上皮内瘤变组织中转录因子Twist、Snail的表达及与上皮间质转化、细胞增殖的关系[J].海南医学院学报, 2016,22(13): 1339-1342.

[29] 高原,郑小玉. Twist和Snail在子宫腺肌病患者异位内膜组织中的表达及意义[J].临床医学研究与实践,2018,3(14):1-3,10.

[30] Kwok WK, Ling MT, Lee TW, et al. Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res. 2005;65(12):5153-5162.