肝X受体和5-氮杂-2’-脱氧胞苷通过上调SOCS3而分别抑制肝癌和肾癌细胞增殖的实验研究

发布时间：2019-01-27 09:42

【摘要】：研究背景：细胞因子信号抑制蛋白3(suppressor of cytokine signaling3，SOCS3)是一种重要的诱导性抗肿瘤分子,可诱导表达于包括肝细胞在内的多种组织细胞。其由N端可变区、中央核心SH2结构区和C末端的SOCS盒区组成。SOCS3作为JAK-STAT信号通路的负调控因子，在抑制肿瘤中生长和促进肿瘤细胞凋亡中发挥着重要的作用。一方面SOCS3可以诱导P21的表达进而抑制细胞的增殖；另一方面过表达SOCS3后，可以下调Bcl-2同时上调Casepase3，进而促进肿瘤细胞的凋亡。但是目前对于研究Socs3基因表达调控的领域较少。肝X受体(liver X receptor，LXR)为配体依赖的转录因子，是核受体的一种，作为一种多功能转录因子，LXR广泛参与调节物质(糖、脂和胆固醇)代谢以及抗炎、神经退行性疾病如阿尔茨海默病。近年研究表明，抗肿瘤作用是LXR的一项新功能，其在抑制不同种类的肿瘤细胞系增殖方面有着重要的作用如肝细胞癌，前列腺癌，乳腺癌，结肠癌，宫颈癌，淋巴癌，卵巢癌等，但是其具体的抗肿瘤机制仍不是十分清楚。既然LXR和SOCS3都具有抗肿瘤的功能，而且SOCS3在肿瘤细胞中处于地表达或者不表达状态，那么LXR能否通过促进SCOS3的表达进而发挥抗肿瘤的功能，目前尚无报道。作为甲基化转移酶抑制剂，5-氮杂-2＇-脱氧胞苷能使肿瘤细胞周期阻滞在G2期，但这一过程具体的分子机制仍不清楚。研究目的：研究LXR调控SOCS3表达的分子机制以及SOCS3在抑制肝细胞癌(hepatocellular carcinoma，HCC)细胞系增殖中的作用以及5-氮杂-2＇-脱氧胞苷导致肿瘤细胞周期阻滞的分子机制。研究方法： (1)用LXR的两种激动剂TO901317和GW3965处理两种肝癌细胞系HepG2和Hep3B，提取m-RNA、逆转录成c-DNA、进而通过RT-PCR和QRT-PCR检测Socs3基因的表达情况；提取细胞内总蛋白，通过Western blot检测SCOS3以及CylinD1、P21和P27的表达情况。 (2)在核受体网站上预测SOCS3的启动子区是否存在LXR的结合位点，构建SOCS3上游启动子区的重组质粒，进而用双荧光素酶报告基因检测LXR激动剂对SOCS3启动子活性的影响。 (3)通过CCK-8和流式细胞技术检测LXR激动剂对肝癌细胞系HepG2细胞增殖和细胞周期的影响。 (4)用siRNA干扰肝癌细胞系SOCS3后，检测LXR激动剂对肝癌细胞系HepG2和Hep3B细胞增殖、周期的影响以及用WB检测相应的细胞周期蛋白cyclin D1，p21和p27的表达情况。 (5)对裸鼠腋下进行异种移植肝癌细胞系HepG2，当肿瘤形成后，对荷瘤鼠腹腔注射TO901317(25mg/kg/d)，观察TO901317对肿瘤生长的影响以及用QRT-PCR,WB以及免疫组化检测SOCS3mRNA和蛋白水平的变化。 (6)5-氮杂-2＇-脱氧胞苷处理细胞后，CCK-8和流式细胞检测细胞增殖和周期。 (7)半定量PCR检测SOCS3和甲基化转移酶的mRNA水平，WB检测SOCS3和p21的水平。 (8)干扰SOSS3后，WB检测5-氮杂-2＇-脱氧胞苷对p21蛋白水平的影响。研究结果： (1) LXR的两种激动剂TO901317和GW3965能够剂量依赖的上调SOCS3蛋白和m-RNA的水平。 (2) LXR激动剂不能上调SOCS3的启动子活性,导致SOCS3mRNA上调的原因是因为LXR激动剂增加了SOCS3mRNA的稳定性。 (3) TO901317能够剂量依赖的抑制肝癌细胞系HepG2增殖并且将细胞周期阻滞在G1期。 (4)干扰SOCS3的表达后削弱了LXR抗肿瘤细胞增殖的效应。 (5)在裸鼠肝细胞癌皮下异种移植瘤模型中，TO901317能够抑制荷瘤鼠HCC的生长以及上调SOCS3蛋白和mRNA的水平。 (6)5-氮杂-2＇-脱氧胞苷能够使肾癌细胞周期抑制在G2期，抑制细胞增殖。 (7)5-氮杂-2＇-脱氧胞苷能降低DNA甲基化转移酶1/3a的mRNA水平和提高SOCS3的mRNA水平，也能提高SOCS3和p21的蛋白水平。 (8)在干扰SOCS3后，5-氮杂-2＇-脱氧胞苷诱导p21蛋白水平增加的作用减弱。结论： (1) LXR激动剂通过增强SOCS3mRNA的稳定性从而导致SOCS3m-RNA和蛋白的水平的上调。 (2)在肝癌细胞系HepG2和Hep3B中，LXR激动剂能够通过上调SOCS3,从而降低cyclin D1的蛋白水平，升高p21和p27的蛋白水平，使细胞阻滞在G1期，最终抑制细胞的增殖。 (3)5-氮杂-2＇-脱氧胞苷能恢复SOCS3的表达，，从而诱导p21的蛋白水平升高，最终使肾癌细胞周期阻滞在G2期。
[Abstract]:Study Background: The cytokine signal-inhibiting protein 3 (SOCS3) is an important inducible anti-tumor molecule, which can be induced to be expressed in a variety of tissues, including liver cells. a cell comprising an N-terminal variable region, a central core SH2 structure region and a C-terminal SOCS box region group, As a negative regulator of JAK-STAT signal pathway, SOCS3 plays an important role in inhibiting the growth of tumor and promoting the apoptosis of tumor cells. On the one hand, SOCS3 can induce the expression of P21 and then inhibit the proliferation of cells. On the other hand, after the expression of SOCS3, Bcl-2 can be down-regulated while the Casepase3 is up-regulated, thus promoting the growth of tumor cells. However, in the field of research on the regulation of the expression of Socs3 gene, Low. The liver X receptor (LXR) is a ligand-dependent transcription factor, a kind of nuclear receptor, as a multi-function transcription factor, LXR is widely involved in the regulation of metabolism of substances (sugar, fat and cholesterol) and anti-inflammatory, neurodegenerative diseases such as Alzheimer's disease. Recent studies have shown that the anti-tumor effect is a new function of LXR, which plays an important role in inhibiting the proliferation of different types of tumor cell lines, such as hepatocellular carcinoma, prostate cancer, breast cancer, colon cancer, cervical cancer, lymphoma, and ovarian cancer. and the like, but the specific anti-tumor mechanism is not very clear. If LXR and SOCS3 both have anti-tumor function, and SOCS3 is in the expression or non-expression state in tumor cells, the ability of LXR to play an anti-tumor function by promoting the expression of SCOS3 is not yet reported. As a methyltransferase inhibitor, the 5-nitroza-2 '-donor cell can block the cell cycle of the tumor in the G2 phase, but the molecular mechanism of this process is still unclear. Objective: To study the molecular mechanism of the expression of SOCS3 by LXR and the role of SOCS3 in the inhibition of the proliferation of the human hepatocellular carcinoma (HCC) cell line and the division of the cell cycle of the tumor. a sub-mechanism. Methods: (1) Two kinds of liver cancer cell lines HepG2 and Hep3B were treated with two agonists TO901317 and GW3965 of LXR, and m-RNA and reverse transcription c-DNA were extracted, and then the expression of the Socs3 gene was detected by RT-PCR and QRT-PCR, and the total protein in the cells was extracted, and the SCOS3 was detected by Western blot. CylinD1, P21 and P and (2) predicting the binding site of the LXR in the promoter region of the SOCS3 on the nuclear receptor website, constructing a recombinant plasmid of the upstream promoter region of the SOCS3, 3) Detection of LXR Agonist by CCK-8 and Flow Cytometry in the Treatment of Hepatocarcinoma Cell Line HepG2 The effect of LXR agonist on the proliferation and cycle of HepG2 and Hep3B cells of the liver cancer cell line HepG2 and Hep3B was detected by siRNA, and the corresponding cell cycle protein cyclin D1 was detected by WB. and the expression of p21 and p27. (5) Xenograft hepatoma cell line HepG2 was carried out on the underarm of nude mice. After the tumor was formed, the tumor-bearing mice were injected with TO901317 (25mg/ kg/ day), and the effect of TO901317 on the growth of the tumor was observed and the SO-PCR, WB and immunohistochemistry were used to detect the SO. Changes in the level of CS3 mRNA and protein. (6) After 5-nitroza-2'-yeast cell-treated cells, CC K-8 and flow cytometry for cell proliferation and cycle. (7) Semi-quantitative PCR to detect the mRNA of SOCS3 and methyltransferase Level, WB detects levels of SOCS3 and p21. (8) After interference with SOSS3, WB detects 5-N-to-2 '-The effect of cytoskeleton on the level of p21 protein. Results: (1) Two agonists of LXR, TO901317 and GW3965 Up-regulation of the level of SOCS3 protein and m-RNA can be dose-dependent. (2) LXR agonists cannot upregulate the promoter activity of SOCS3, resulting in an up-regulation of SOCS3 mRNA The reason is that the LXR agonist increases the stability of the SOCS3 mRNA. (3) The TO901317 can be dose-dependent The inhibition of the proliferation of the liver cancer cell line HepG2 and the cell cycle arrest in the G1 phase. (4) The effect of the proliferation of LXR anti-tumor cells was weakened after interfering with the expression of SOCS3. (5) In the subcutaneous xenograft model of nude mice, TO90131 7 can inhibit the growth of the tumor-bearing mouse HCC and increase the level of the SOCS3 protein and the mRNA.) 5-aza-2'-pyocyanin can inhibit the cell cycle of the kidney cancer in the G2 phase and inhibit cell proliferation. (7) 5-aza-2 '-pyocyanin can reduce the mRN of the DNA methyltransferase 1/ 3a. The level of the mRNA of SOCS3 and the level of the protein of SOCS3 can be increased, and the level of the protein of SOCS3 and p21 can be improved. (8)) in dry Conclusion: (1) LXR Agonists increase the level of SOCS3-RNA and protein by enhancing the stability of SOCS3 mRNA. (2) In the liver cancer cell lines HepG2 and Hep3B, LXR agonists can reduce cyc by up-regulation of SOCS3. lin D1 protein level, increase the protein level of p21 and p27, block the cell in G1 phase, and finally inhibit cell proliferation. (3) 5-nitroza-2'
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.7;R737.11

【相似文献】