SMOC2在原发性肝细胞癌中的预后预测作用及功能的初步研究

发布时间：2018-04-26 13:20

本文选题：SMOC2 + 原发性肝癌　；参考：《广东药科大学》2017年硕士论文

【摘要】：研究背景及目的肝癌是全球范围内高发的一类恶性肿瘤,其中大约有90%的肝癌为原发性肝癌(HCC)。据全球癌症调查资料显示,肝癌发病率在所有肿瘤中排列第五,因肝癌而死亡的人数在所有恶性肿瘤中高居第三位。在中国,肝癌更是威胁我国公民生命健康的主要肿瘤之一。据中国2015年癌症统计数据显示,肝癌已成为我国实体瘤中第四位高发病率和第三位死因的癌症。目前研究发现原发性肝癌的分子发病机制包括多基因及表观遗传的改变、染色体畸变、基因突变等,但关于HCC具体发病机制仍不明确。因此,探索与HCC发生发展相关的潜在基因并研究其在HCC发生进展过程中的作用,对于人们更好地了解HCC发生发展的分子机制具有重要意义。模块化钙结合蛋白2(SMOC2)属于SPARC蛋白家族成员之一。有研究表明,SMOC2参与调节多种细胞生物学功能,如细胞周期、细胞增殖、迁移等,但关于其在肿瘤中作用的相关研究仍较少。目前,有研究利用微阵列分析技术发现SMOC2在多种肿瘤中呈现低表达,包括卵巢癌、胰腺癌等,由此可推断SMOC2在多数肿瘤中可能为抑癌基因,但目前仍未见关于SMOC2在HCC中的表达、预防诊治意义及作用的研究报道。本研究欲通过检测SMOC2在HCC中的表达情况,结合肝癌患者病历及随访资料来探讨SMOC2表达与HCC患者临床病理参数及预后的关系,并进一步探究SMOC2对HCC发展过程中的细胞生物学功能的影响,从而来明确SMOC2在HCC中的临床诊治、预防意义及生物学功能。研究方法实验方法:采用荧光定量RT-PCR技术检测SMOC2在肝癌组织中的转录水平,利用western blotting和免疫组织化学技术检测SMOC2蛋白在肝癌标本和肝癌细胞中的表达,探讨SMOC2与肝癌患者肿瘤相关指标的关系,并分析其表达情况与患者预后的关系。通过慢病毒转染,构建过表达SMOC2的HepG2和Bel-7402细胞稳株,研究SMOC2过表达后对肝癌细胞的生长活力、运动能力等的影响。统计方法:肝癌组织和癌旁对照组织中的SMOC2在转录水平、蛋白水平上的差异性比较及相关细胞生物学功能指标的差异性比较使用符号秩和检验;卡方检验用于分析SMOC2表达水平与HCC患者临床病理参数间关系;生存分析用于比较SMOC2高表达组与低表达组之间总体生存期及无病进展生存期的差异;单因素、多因素Cox比例风险回归分析用于研究SMOC2表达及临床病理指标对HCC患者总体生存期及无病进展生存期的影响。研究结果1、肝癌标本中SMOC2 mRNA转录水平(n=40,P0.0001)和蛋白表达水平(n=40,P=0.0101)明显低于癌旁对照组织;较正常肝细胞系L02,SMOC2在肝癌细胞系中(Bel-7402,HepG2,QGY-7701,SK-Hep1,SMMC-7721)的表达水平下调。2、免疫组化结果显示SMOC2免疫阳性染色主要处于胞浆中;卡方检验结果表明SMOC2表达水平的高低在肿瘤大小(χ~2=9.156,P=0.002)、肿瘤数量(χ~2=4.896,P=0.027)、TNM分期(χ~2=16.356,P0.001)及是否发生转移(χ~2=6.141,P=0.013)中的分布情况存在着差异;生存分析发现SMOC2高表达组的总生存期(OS,P0.001)和无病进展生存期(DFS,P=0.001)明显长于低表达组;多因素Cox回归分析显示SMOC2表达水平是影响患者预后的独立因素。3、亚组分析结果显示在AFP≤25ng/ml或400ng/ml、肿瘤长径5cm或≥5cm及TNM分期为Ⅰ-Ⅱ的HCC患者中,SMOC2高表达组总生存率及无病进展生存率均比低表达组高(P0.05)。4、体外实验显示:肝癌细胞的SMOC2表达水平上调后,细胞的增殖、克隆形成以及迁移侵袭能力受到抑制,并且会阻碍肝癌细胞周期进程,诱导细胞周期阻滞于G0/G1期。5、小鼠成瘤实验显示皮下注射过表达SMOC2肝癌细胞的裸鼠肿瘤生长速度明显低于对照组(P0.05),皮下注射过表达SMOC2肝癌细胞的裸鼠长出的肿瘤体积及重量也明显小于对照组(P0.05)。结论与HCC发展相关的细胞生物学进程中,SMOC2发挥着抑癌基因的作用。它可作为预测HCC患者预后的有效指标,也可能作为HCC分子靶向治疗的潜在靶点。此外,肝癌患者SMOC2的表达情况还可作为为患者制定三级预防方案的参考依据。
[Abstract]:Background and objective liver cancer is a worldwide type of high incidence of malignant tumors, of which about 90% of the liver cancer is primary liver cancer (HCC). According to the global cancer survey data, the incidence of liver cancer is fifth in all tumors. The number of deaths caused by liver cancer is third in all malignant tumors. In China, liver cancer is a threat. According to China's 2015 cancer statistics, liver cancer has become one of the fourth high incidence and third causes of death in China's solid tumors. The molecular pathogenesis of primary liver cancer includes polygene and epigenetic changes, chromosomal aberrations and gene mutations. The specific pathogenesis of HCC is still unclear. Therefore, exploring the potential genes associated with the development of HCC and studying its role in the progress of HCC are of great significance for people to better understand the molecular mechanism of HCC development. Modularized calcium binding protein 2 (SMOC2) is one of the members of the SPARC family. SMOC2 is involved in regulating a variety of cellular biological functions, such as cell cycle, cell proliferation, migration, etc. but there are still few related studies on its role in the tumor. At present, microarray analysis techniques have been used to detect the low expression of SMOC2 in a variety of tumors, including nesting and pancreatic cancer, and thus infer that SMOC2 is in most of the tumors. It may be a tumor suppressor gene, but there is still no study on the expression of SMOC2 in HCC, the significance and the role of prevention and treatment. This study intends to explore the relationship between the expression of SMOC2 in HCC, the case history and the follow-up data of the patients with liver cancer, to explore the relationship between the expression of SMOC2 and the clinical pathophysiological parameters and prognosis of the patients with HCC, and to further explore the SMOC2 The effect of HCC on the cell biological function in the process of development, so as to clarify the clinical diagnosis, the preventive significance and the biological function of SMOC2 in HCC. The method of research is to detect the transcriptional level of SMOC2 in the liver cancer tissues by fluorescence quantitative RT-PCR technique, and to detect SMOC2 protein by Western blotting and immunohistochemical technique. The relationship between SMOC2 and the tumor related indexes of HCC patients and the relationship between the expression of HCC and the prognosis of the patients were analyzed. The expression of HepG2 and Bel-7402 cells expressing SMOC2 were constructed by lentivirus transfection. The effects of SMOC2 overexpression on the growth activity and exercise ability of hepatoma cells were studied. Statistical methods: the difference in the transcriptional level, the protein level and the related cell biological function indexes of the SMOC2 in the liver cancer tissue and the paracancerous control tissue were compared with the sign rank sum test, and the chi square test was used to analyze the relationship between the SMOC2 expression level and the clinical pathophysiological parameters of the HCC patients; the survival analysis was used to compare the high SMOC2 level. The difference between the overall life period and the progression free survival period between the expression group and the low expression group; the single factor, multi factor Cox proportional risk regression analysis was used to study the effect of SMOC2 expression and clinicopathological indexes on the overall survival and the progression free survival of HCC patients. Results 1, the SMOC2 mRNA transcriptional level (n=40, P0.0001) in the specimens of liver cancer. The expression level of protein (n=40, P=0.0101) was significantly lower than that of the paracancerous control tissue, and the expression level of SMOC2 in the hepatocellular carcinoma cell line (Bel-7402, HepG2, QGY-7701, SK-Hep1, SMMC-7721) was down regulated by L02, and the immunohistochemical results showed that SMOC2 immunoreactive staining was mainly in the cytoplasm. The chi square test showed that the level of SMOC2 expression was high. There were differences in the tumor size (chi ~2=9.156, P=0.002), the number of tumor (x ~2=4.896, P=0.027), TNM staging (chi ~2=16.356, P0.001) and the occurrence of metastasis (chi ~2=6.141, P=0.013). The survival analysis found that the total survival time (OS, P0.001) and the progression free survival period of the high expression group of SMOC2 were significantly longer than those of low expression groups. Multiple factor Cox regression analysis showed that the expression of SMOC2 was an independent factor affecting the prognosis of the patients.3. The results of subgroup analysis showed that in AFP < 25ng/ml or 400ng/ml, the total survival rate and the progression free survival rate of SMOC2 high expression group were higher than those of low expression group (P0.05) in the AFP < 25ng/ml or 400ng/ml. The proliferation, clone formation and migration and invasion ability of hepatoma cells were inhibited, and the cell cycle progression was inhibited and the cell cycle was blocked at G0/G1.5. The tumor growth rate of nude mice subcutaneously expressed by subcutaneous injection of SMOC2 liver cancer cells was significantly lower than that of the control group, after the SMOC2 expression level of hepatoma cells was up-regulated. (P0.05) the tumor size and weight of the nude mice expressed by subcutaneous injection of SMOC2 hepatoma cells were also significantly smaller than those of the control group (P0.05). Conclusion SMOC2 plays a role in the tumor suppressor gene in the process of cell biology related to the development of HCC. It can be used as an effective indicator for predicting the prognosis of HCC patients, and may also be the potential for targeting therapy of HCC molecules. In addition, the expression of SMOC2 in patients with liver cancer can also be used as a reference for patients to develop a three level prevention plan.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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