RNA干扰沉默信息调节因子2同源蛋白1阻滞前列腺癌PC3细胞周期

摘要: 目的观察沉默信息调节因子2同源蛋白1（SIRT1）小干扰RNA（siRNA）对前列腺癌PC3细胞生长增殖、细胞周期和P21、P27细胞周期调节蛋白及视网膜母细胞瘤（retinoblastoma，Rb）蛋白表达变化影响，探讨SIRT1在前列腺癌发生中的可能作用机制。方法体外培养PC3细胞，分空白对照（Mock）组、发夹siRNA对照（scramble siRNA）组和SIRT1 siRNA转染组。RT-PCR和Western blot方法检测SIRT1 siRNA的转染效率；MTT方法测定细胞生长抑制率；流式细胞术检测细胞周期；Western blot方法检测P21、P27蛋白表达和Rb蛋白的磷酸化状态。结果与Mock组和scramble siRNA组比较，SIRT1 siRNA组SIRT1 mRNA和蛋白表达降低，明显抑制PC3细胞增殖，阻滞在G1期。P21和P27蛋白表达增加，Rb蛋白磷酸化受到抑制。结论 SIRT1 siRNA可抑制前列腺癌PC3细胞增殖、阻滞细胞周期，其机制可能与改变P21、P27细胞周期蛋白表达和Rb蛋白的磷酸化状态相关。
- SIRT1 /
- siRNA /
- 前列腺癌 /
- PC3细胞 /
- 细胞周期
Abstract: Objective To observe the effects of double-stranded small interfering RNA (siRNA) of the silent mating-type information regulation 2 homolog 1 (SIRT1) on the cell proliferation, cell cycle progression, and expression levels of the cell cycle negative regulators. These regulators include P21, P27, and phosphorylated retinoblastoma (PRb) proteins present in prostate cancer PC3 cells. This work further aims to explore the possible underlying mechanism for such effects. Methods PC3 cells were cultured in vitro and then randomly divided into the mock group, scramble siRNA transfected group, and SIRT1 siRNA-transfected group. SIRT1 siRNA efficiency was examined through reverse transcription polymerase chain reaction and Western blot analysis. The inhibitory rate of PC3 cell growth was determined through a methyl thiazolyl tetrazolium assay, and the cell cycle was investigated with the use of flow cytometry. The P21 and P27 protein expression levels and PRb status were determined by Western blot assay. Results Compared with those of the mock and scramble siRNA groups, the expression levels of SIRT1 mRNA and protein significantly decreased in SIRT1 siRNA-transfected cells. In addition, the inhibitory rate of PC3 cell growth was markedly increased, and the cell cycle of the PC3 cells was arrested at the G₁ stage. The expression levels of negative cell cycle regulators, including P21 and P27 protein levels increased, whereas Rb protein phosphorylation was inhibited in SIRT1 siRNA-transfected PC3 cells. Conclusion SIRT1 RNA interference inhibits PC3 cell growth and arrests cell cycle progression through the upregulation of the P21 and P27 proteins and the inhibition of Rb protein phosphorylation.
- SIRT1 /
- siRNA /
- prostate cancer /
- PC3 cells /
- cell cycle

HTML全文

图 1 PC3细胞转染SIRT1 siRNA后SIRT1 mRNA和蛋白表达水平

Figure 1. Expression levels of SIRT1 mRNA and protein in SIRT1 siRNA-transfected PC3 cells

A: Electrophoresis of SIRT1 mRNA expression; B: Western blot of SIRT1 protein expression.
Lane 1: Mock group; Lane 2: Scramble siRNA group; Lane 3: SIRT1 siRNA group

下载: 全尺寸图片幻灯片

图 2 MTT方法检测转染SIRT1 siRNA后的PC3细胞增殖

Figure 2. PC3 cell proliferation rate as evaluated using methyl thiazolyl tetrazolium assays

下载: 全尺寸图片幻灯片

图 3 PC3细胞转染SIRT1 siRNA后P21、P27和Rb蛋白表达

Figure 3. Western blot analysis of P21, P27, and phosphorylated Rb protein expression levels in SIRT1 siRNA-transfected PC3 cells

Lane 1:Mock group; Lane 2:scramble siRNA group; Lane 3:SIRT1 siRNA group

下载: 全尺寸图片幻灯片

参考文献(15)

[1]	Chen J, Li HM, Zhang XN, et al. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase-3 and modulation of Bcl-2 protein family[J]. J Huazhong Univ Sci Technolog Med Sci, 2014, 34(1):125-130. doi: 10.1007/s11596-014-1243-y
[2]	Khan MI, Adhami VM, Lall RK, et al. YB-1 expression promotes epithelial-to-mesenchymal transition in prostate cancer that is inhibited by a small molecule fisetin[J]. Oncotarget, 2014, 5(9): 2462-2474. doi: 10.18632/oncotarget.1790
[3]	Li JP, Cao NX, Jiang RT, et al. Knockdown of GCF2/LRRFIP1 by RNAi causes cell growth inhibition and increased apoptosis in human hepatoma HepG2 cells[J]. Asian Pac J Cancer Prev, 2014, 15 (6):2753-2758. doi: 10.7314/APJCP.2014.15.6.2753
[4]	Civenni G, Malek A, Albino D, et al. RNAi-mediated silencing of Myc transcription inhibits stem-like cell maintenance and tumorigenicity in prostate cancer[J]. Cancer Res, 2013, 73(22):6816-6827. doi: 10.1158/0008-5472.CAN-13-0615
[5]	Abdelmohsen K, Pullmann R Jr, Lal A, et al. Phosphorylation of HuR by Chk2 regulates SIRT1 expression[J]. Mol Cell, 2007, 25(4): 543-557. doi: 10.1016/j.molcel.2007.01.011
[6]	Byles V, Zhu L, Lovaas JD, et al. SIRT1 induces EMT by cooperating with EMT transcription factors and enhances prostate cancer cell migration and metastasis[J]. Oncogene, 2012, 31(43):4619-4629. doi: 10.1038/onc.2011.612
[7]	Yuan H, Su L, Chen WY. The emerging and diverse roles of sirtuins in cancer: a clinical perspective[J]. Onco Targets Ther, 2013, 6: 1399-1416. http://cn.bing.com/academic/profile?id=9e5c03a123d527966daff73c449de460&encoded=0&v=paper_preview&mkt=zh-cn
[8]	Li Y, Wong K, Giles A, et al. Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21[J]. Gastroenterology, 2014, 146(2):539-549. doi: 10.1053/j.gastro.2013.10.059
[9]	Wang RH, Sengupta K, Li C, et al. Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice[J]. Cancer Cell, 2008, 14(4):312-323. doi: 10.1016/j.ccr.2008.09.001
[10]	Hori YS, Kuno A, Hosoda R, et al. Regulation of FOXOs and p53 by SIRT1 modulators under oxidative stress[J]. PLoS One, 2013, 8 (9):e73875. doi: 10.1371/journal.pone.0073875
[11]	Yang Q, Wang B, Gao W, et al. SIRT1 is downregulated in gastric cancer and leads to G₁-phase arrest via NF-κB/Cyclin D1 signaling [J]. Mol Cancer Res, 2013, 11(12):1497-1507. doi: 10.1158/1541-7786.MCR-13-0214
[12]	Kojima K, Ohhashi R, Fujita Y, et al. A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells[J]. Biochem Biophys Res Commun, 2008, 373(3):423-428. http://cn.bing.com/academic/profile?id=22d430fde6520acd1a4409d602cfcdff&encoded=0&v=paper_preview&mkt=zh-cn
[13]	Baptista T, Graça I, Sousa EJ, et al. Regulation of histone H2A. Z expression is mediated by sirtuin 1 in prostate cancer[J]. Oncotarget, 2013, 4(10):1673-1685. doi: 10.18632/oncotarget.1237
[14]	张秀梅, 刘超, 赵颂, 等.9-顺式维甲酸抑制甲状腺鳞癌细胞SW579增殖的作用机制[J].中国肿瘤临床, 2012, 39(11):761-764. doi: 10.3969/j.issn.1000-8179.2012.11.002 Zhang XM, Liu C, Zhao S, et al. Mechanism of 9-cisRA in inhibiting the proliferation of thyroid squamous cell carcinoma cell SW579 [J]. Chin J Clin Oncol, 2012, 39(11):761-764. doi: 10.3969/j.issn.1000-8179.2012.11.002
[15]	Burke JR, Liban TJ, Restrepo T, et al. Multiple mechanisms for E2F binding inhibition by phosphorylation of the retinoblastoma protein C-terminal domain[J]. J Mol Biol, 2014, 426(1):245-255. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=3ae05f33f9df1b878ad38485e9900f7f