CXCL6在肝细胞肝癌中上调并促进肝癌细胞的增殖与侵袭

发布时间：2018-06-11 09:57

本文选题：CXCL6 + 肝细胞癌　；参考：《山东大学》2016年博士论文

【摘要】：研究背景原发性肝癌中约90%为肝细胞癌(简称肝癌),居全世界恶性肿瘤发病率第5位,其病死率在各种癌症中居第2位。2014年世界卫生组织WHO的统计资料显示：全世界肝癌的发病率还存在上升趋势。中国作为HBV(乙型肝炎病毒)高感染地区,肝癌的发病率占全世界的50%以上。肝癌在中国于危害人们生命健康方面属于前三大恶性肿瘤之一,给社会带来沉重负担。HCC(原发性肝细胞肝癌)的的特点是生长速度快,易于转移和生存率低。目前为止,早期诊断和及时手术治疗是肝癌患者最有效的治疗措施。然而,术后复发和转移是导致肝癌术后患者较差预后的关键问题。原发性肝癌的复发和转移的临床诊断目前主要依靠血清学检测和影像学方法,包括B超的定期复查,术前血清甲胎蛋白(AFP)和术后AFP的对比检测,并观察其变化等。Nobuoka等研究发现,与术前相比,AFP水平没有显著降低的肝癌患者较术后甲胎蛋白显著降低的患者的复发率是明显增高的。AFP检测持续阳性提示残余肿瘤活性增强的可能。一般来讲,AFP对复发性肝癌的早期诊断的检测率比B超低,B超的早期诊断检出率比CT低。这三种方法的组合运用将是提高早期诊断率的有效方法。另外肝癌切除术后常规检查中包括胸部正侧位X线片及全身骨显像ECT。目前尚无明确证据显示正电子发射计算机断层显像(PET-CT)对于肝癌复发转移有早期诊断价值。AFP以外,近年来发现DKK1对诊断原发性肝细胞肝癌有重要价值,DKK1有望成为新的肝癌肿瘤标志物。但上述检测HCC转移复发的方法显然难以满足目前理想的早诊早治的临床需要。因此,对肝癌转移复发机肿瘤复发相关的分子标志物及积极有效的抗复发治疗是当前肝癌诊疗中的重点。迫切需要找到新的治疗靶点来应对HCC的肿瘤的进展转移。炎症作为肿瘤的一大特征近年来已为大家所公认。在过去的研究中,人们发现炎症与肿瘤的发生、发展密切相关,深刻影响了肿瘤的生长,侵袭和转移过程。趋化因子及其受体是炎症微环境的重要组成部分。已有大量文献研究证实,在促进肿瘤的发生、进展和侵袭的过程中,趋化因子及其受体起到了显著的作用。在肿瘤侵袭过程中,癌症细胞由肿瘤起源趋化因子诱导的趋化作用是至关重要的一步。趋化因子在促进肝癌进展中所发挥的作用也得到了越来越多的关注。趋化因子为可溶性小蛋白,是细胞因子超家族中的一类,是结构相似、由多类细胞产生、具有趋化细胞迁移作用的一类因子,其相对分子质量较小,多为8～10kD。其三级结构是由半胱氨酸(cys)残基组合而成。残基包含4个,位置比较保守。根据位于其分子N端的半胱氨酸(cys)数目,另外再结合其前两个半胱氨酸的三维排序方式不同,趋化因子家族可以分为CC(CCL1～CCL28)、 CXC(CXCL1～CXCL17)、C(XCL1～XCL2)和CX3C(CX3L1)4类不同的亚家族。目前为止,相关文献对趋化因子的报道总计有50余种。趋化因子受体是一些G蛋白耦联的七跨膜域受体,表达于一些特定的细胞表面。它们为一种糖蛋白,只含一条肽链,由大约330个氨基酸组成。7个跨膜区域,将分子划分为4个部分,包含游离的细胞外N-端、3个细胞外环、3个细胞内环及C-端。趋化因子可以在多种细胞活动过程中产生,并且与相应的受体相结合,进而调节机体的各种功能。二者的相互作用,可以调控生物组织及器官之间的各类免疫细胞的定向迁移过程。因此,趋化因子和受体的相互作用在免疫细胞分化,发育和定向迁移的调节中有着重要作用。既往研究显示,在某些肿瘤细胞中趋化因子及其受体高表达和肿瘤转移具有高度相关性。肿瘤相关的趋化因子不仅可以促进肿瘤细胞增殖,抑制肿瘤细胞凋亡,还在肿瘤细胞的迁移过程发挥重要作用,同时可以趋化淋巴细胞浸润肿瘤组织,参与肿瘤血管生成和肿瘤细胞器官选择性转移。CXCL6也称为粒细胞趋化蛋白2(GCP-2),属于ELR+趋化因子。类似于其他ELR+趋化因子,CXCL6促进肿瘤的生长作用在动物实验中得到验证。此外,CXCL6已经被证明是在几个实体肿瘤进展中的重要媒介。很多研究表明,CXCL6的高表达与肿瘤的发生、发展及其侵袭转移有关。CXCL6被发现在实体肿瘤上调,包括小细胞肺癌,黑色素瘤和结直肠癌。在常见的肝癌细胞株中,MHCC97H或HCCLM3细胞具有较高的转移能力,而HepG2或SMMC7721具有较低的转移能力。在MHCC97H或HCCLM3细胞中,CXCL6水平显著高于其在HepG2或SMMC7721细胞中的水平。此结果表明CXCL6可能促进肝癌细胞侵袭转移。然而,对CXCL6在肝癌侵袭转移中所起作用的进一步的研究尚不充分。此外,CXCL6对预测肝癌患者的预后的意义尚不完全清楚。目的：通过本研究通过相关实验旨在进一步探索与验证CXCL6对肝癌细胞的增殖与侵袭的作用。分析CXCL6在临床肝癌标本中的表达与肿瘤大小及血管侵犯、总体生存率(OS)以及无复发生存率(RFS)等预后相关指标的联系。研究比较CXCL6在MHCC97L、MHCC97H、HCCLM3、HepG2和SMMC7721经典肝癌细胞株中表达的差异。并通过靶向沉默CXCL6的MHCC97H和HCCLM3细胞系进行了体外细胞增殖和侵袭实验,同时以CXCL6质粒慢病毒转染的SMMC-7721细胞来比较CXCL6表达水平的高低对HCC细胞侵袭能力的影响。并进一步研究MMP9被敲除后,CXCL6对HCC增殖及侵袭能力影响的改变。期待能为HCC复发及转移的早期诊断和治疗提供预测因子及治疗靶点。材料与方法：1.我们收集了从2005年到2011年196份肝癌样本,随访至2013年12月。采用免疫组化法检测了CXCL6在肝癌组织芯片的表达,其中包括212例HCC样本。完整的随访数据包括患者的年龄、性别、肿瘤大小、肝硬化程度、局部侵袭情况,TNM分期,年龄,肿瘤卫星灶,血管侵犯,血栓形成。我们使用SPSS16.0软件分析CXCL6表达与肝细胞癌患者的临床病理特征参数之间的相关性。此外,应用SPSS软件通过乘积极限法(Kaplan-Meier法)来分析CXCL6和肝细胞癌患者的总体生存期和无复发生存期的相关性。2.应用实时定量聚合酶链反应(PCR)和Western blot(免疫印迹)方法比较了CXCL6在HCC及配对癌旁的正常肝组织的表达水平差异。3.通过应用Lipofectamine2000试剂转染小干扰RNA(siRNA)到MHCC97H和HCCLM3细胞评估CXCL6的作用。应用qPCR和免疫印迹法检测的siRNA在肝癌细胞中的敲除效果。同时我们设计了体外侵袭实验以验证沉默CXCL6后MHCC97H和HCCLM3细胞的侵袭能力。4.以siRNA靶向沉默CXCL6的MHCC97H和HCCLM3细胞系进行了体外细胞增殖实验。5.通过慢病毒转染法进行表达检测进一步探讨CXCL6在HCC细胞中作用。6.MMP9在被过表达CXCL6质粒或者过表达对照质粒转染的肝癌细胞中被用RNA干扰的方法敲除,以检测MMP9敲除后,CXCL6在SMMC-7721细胞的促侵袭作用的变化情况。实验结果：1.我们采用免疫组化法检测了CXCL6在肝癌组织芯片的表达,其中包括212例HCC样本。通过分析178可配对组织中,我们发现,在71.91%(128/178)HCC患者中CXCL6水平升高,但是在14.04%肝癌患者(25/178)表达下调。2.统计分析发现CXCL6的表达与肿瘤大小及血管侵犯(其是肝癌侵袭的重要临床病理学指标)密切相关。此外,我们发现,较高的CXCL6表达与较差的总体生存率(OS)以及无复发生存率(RFS)相关。肝癌患者低CXCL6表达患者比高表达患者预后较好。3.在HCC组织中CXCL6 mRNA和蛋白表达水平显著高于其在配对的正常肝脏组织的表达水平。研究结果表明CXCL6在肝癌组织中与相应的正常肝脏组织相比是上调的。4. CXCL6在MHCC97L,MHCC97H和HCCLM3细胞株中高表达,但在HepG2和SMMC7721细胞株中低表达。此外,在肝癌细胞系中CXCL6 mRNA表达水平通常与蛋白表达状态一致。5.体外侵袭实验表明CXCL6沉默显著下调了MHCC97H和HCCLM3细胞的侵袭能力。6.以siRNA靶向沉默CXCL6的MHCC97H和HCCLM3细胞系进行了体外细胞增殖实验结果表明,沉默CXCL6在体外显著抑制肝癌细胞增殖。7.与对照组相比,被CXCL6质粒慢病毒转染的SMMC-7721细胞具有更高的侵袭和转移能力。8. CXCL6在SMMC-7721细胞的促侵袭作用在MMP9被敲除后受抑制。结论：1.CXCL6在肝癌组织的阳性表达率显著高于非肿瘤肝脏组织,在具有高转移能力的肝癌细胞中高表达；CXCL6的表达呈与肝癌的恶性潜能正相关；CXCL6可能在促进肝癌细胞的运动中发挥作用。2.CXCL6高表达促进了HCC细胞的增殖和侵袭。3.CXCL6在肝癌细胞中促侵袭效应可能使通过激活或作用MMP9起作用。4.CXCL6将来可能作为HCC一个潜在的独立预后因子及治疗靶标。
[Abstract]:About 90% of the primary hepatocellular carcinoma (HCC) in the background of primary liver cancer (HCC) is the fifth most malignant tumor in the world. The fatality rate of the second.2014 year WHO WHO in various cancers shows that the incidence of liver cancer in the world is still rising. China is a high infection area of HBV (hepatitis B virus). The incidence of liver cancer accounts for more than 50% of the world's world. Liver cancer is one of the top three malignant tumors in China, which endangers people's life and health. The heavy burden of.HCC (primary hepatocellular carcinoma) is characterized by rapid growth, easy transfer and low survival rate. Early diagnosis and timely surgical treatment are liver cancer. The most effective treatment for patients. However, postoperative recurrence and metastasis is a key problem in the poor prognosis of patients with liver cancer. The clinical diagnosis of the recurrence and metastasis of primary liver cancer is mainly based on serological and imaging methods, including regular reexamination of B-ultrasound, preoperative AFP and postoperative AFP. .Nobuoka and other studies found that the recurrence rate of patients with significant decrease in AFP levels compared with pre operation compared with preoperatively, the recurrence rate of patients with significantly lower alpha fetoprotein after operation is a significant increase in the possibility of enhanced residual tumor activity by.AFP detection. Generally speaking, the detection rate of early diagnosis of recurrent liver cancer by AFP The early diagnosis rate of B-ultrasound is lower than that of CT. The combination of these three methods will be an effective method to improve the early diagnosis rate. In addition, there is no clear evidence that positron emission computed tomography (PET-CT) has no clear evidence for the recovery of liver cancer in the routine examination of liver cancer after resection of liver cancer. In addition to the early diagnostic value of.AFP, DKK1 has been found to be of great value in the diagnosis of primary hepatocellular carcinoma in recent years. DKK1 is expected to be a new tumor marker for hepatocellular carcinoma. However, the method of detecting the recurrence of HCC metastasis is obviously difficult to meet the clinical requirements of the ideal early diagnosis and early treatment. Molecular markers and active and effective anti relapse therapy are the focus of current diagnosis and treatment of liver cancer. New therapeutic targets are urgently needed to cope with the progression of HCC tumor. In recent years, inflammation is recognized as a major feature of cancer. In the past, people found that inflammation and tumor development are closely related. It has a profound influence on the growth, invasion and metastasis of tumor. Chemokines and their receptors are important components of the inflammatory microenvironment. A large number of literature studies have shown that chemokines and their receptors play a significant role in promoting the occurrence, progression and invasion of tumors. Chemokine induced chemotaxis of tumor origin chemokine is a crucial step. Chemokine plays an increasingly important role in promoting the progression of liver cancer. Chemokine is a soluble small protein, a kind of cytokine superfamily, which is similar in structure, produced by multiple types of cells, and has chemotactic cell migration. One class of factors, whose relative molecular weight is smaller, is 8 to 10kD., and the tertiary structure is composed of cysteine (Cys) residues. The residue contains 4, and the position is conservative. According to the number of cysteine (Cys) located at the N end of its molecule, the chemokine family can be divided into C, which is in addition to the three dimensional ordering method of the former 2.5 cystine. 4 subfamilies of C (CCL1 to CCL28), CXC (CXCL1 to CXCL17), C (XCL1 to XCL2) and CX3C (CX3L1). So far, there are more than 50 reports on chemokines. Chemokine receptor is a seven transmembrane domain receptor coupled with some G protein, expressed on a specific cell surface. They are a glycoprotein containing only one polypeptide chain. Approximately 330 amino acids are composed of.7 transmembrane regions that divide molecules into 4 parts, including free extracellular N-, 3 extracellular rings, 3 cell rings and C- ends. Chemokines can be produced during a variety of cell activities and are combined with corresponding receptors to regulate the various functions of the body. The interaction of the two is possible. The interaction between chemokines and receptors plays an important role in the regulation of immune cell differentiation, development and directional migration. Previous studies have shown high expression of chemokines and their receptors and tumor metastasis in some tumor cells. Correlation. Tumor related chemokine can not only promote tumor cell proliferation, inhibit tumor cell apoptosis, but also play an important role in tumor cell migration, but also chemotactic lymphocyte infiltrating tumor tissue, and participate in tumor angiogenesis and tumor cell organ selective transfer.CXCL6, also known as granulocyte chemoattractant protein 2 (G CP-2) belongs to ELR+ chemokine. Similar to other ELR+ chemokines, CXCL6 promotes tumor growth in animal experiments. In addition, CXCL6 has been proved to be an important medium in the progress of several solid tumors. Many studies have shown that the high expression of CXCL6 is associated with the occurrence, development and invasion of the tumor in relation to the occurrence of.CXCL6 being transmitted. Now solid tumors are up-regulated, including small cell lung cancer, melanoma and colorectal cancer. In common hepatocellular carcinoma cell lines, MHCC97H or HCCLM3 cells have higher metastatic ability, while HepG2 or SMMC7721 has a lower metastatic ability. In MHCC97H or HCCLM3 cells, the level of CXCL6 is significantly higher than that in HepG2 or SMMC7721 cells. The results suggest that CXCL6 may promote the invasion and metastasis of hepatoma cells. However, further research on the role of CXCL6 in the invasion and metastasis of liver cancer is not sufficient. Furthermore, the significance of CXCL6 for predicting the prognosis of liver cancer patients is not completely clear. Objective: through this study, the purpose of this study was to further explore and verify the CXCL6's effect on liver cancer The effect of cell proliferation and invasion. Analysis of the relationship between CXCL6 expression in clinical liver cancer specimens and tumor size and vascular invasion, total survival rate (OS) and non relapse rate (RFS). Comparison of the differences in the expression of CXCL6 in MHCC97L, MHCC97H, HCCLM3, HepG2 and SMMC7721 classical hepatocellular carcinoma cell lines. The cell proliferation and invasion experiments of the MHCC97H and HCCLM3 cell lines of CXCL6 were carried out in vitro, and the effect of CXCL6 expression level on the invasion ability of HCC cells was compared with the SMMC-7721 cells transfected with CXCL6 plasmid lentivirus. The effect of CXCL6 on the proliferation and invasion of HCC was further studied. The early diagnosis and treatment of HCC recurrence and metastasis provide predictive factors and therapeutic targets. Materials and methods: 1. we collected 196 liver cancer samples from 2005 to 2011 and followed up to December 2013. The expression of CXCL6 in the tissue microarray of liver cancer was detected by immunohistochemistry, including 212 cases of HCC. Complete follow-up data included patients. Age, sex, tumor size, degree of liver cirrhosis, local invasion, TNM staging, age, tumor satellite, vascular invasion, thrombosis. We use SPSS16.0 software to analyze the correlation between CXCL6 expression and the clinicopathological parameters of patients with hepatocellular carcinoma. In addition, the SPSS software is used by the product limit method (Kaplan-Meier method). Analysis of the correlation between the overall survival time and the non recurrent survival period of CXCL6 and hepatocellular carcinoma patients.2. application real time quantitative polymerase chain reaction (PCR) and Western blot (immunoblotting) method compared CXCL6 expression levels in normal liver tissues near HCC and paired cancerous carcinoma by using Lipofectamine2000 reagent to transfect small interference RNA (siRNA). MHCC97H and HCCLM3 cells were used to evaluate the role of CXCL6. The effects of qPCR and Western blot on the knockout effect of siRNA in liver cancer cells were used. Meanwhile, we designed an in vitro invasion experiment to verify the invasion ability of MHCC97H and HCCLM3 cells after the silence CXCL6. The proliferation experiment.5. was detected by the expression of lentivirus transfection. The effect of CXCL6 in HCC cells was knocked out by RNA interference in the hepatoma cells transfected by overexpressed CXCL6 plasmids or over expressed control plasmids to detect the change of CXCL6 in SMMC-7721 cells after MMP9 knockout. Results: 1. we used immunohistochemical method to detect the expression of CXCL6 in the tissue microarray of liver cancer, including 212 cases of HCC. Through the analysis of 178 paired tissues, we found that the level of CXCL6 increased in 71.91% (128/178) HCC patients, but the expression of CXCL6 in 14.04% liver cancer patients (25/178) expression and analysis of.2. found the expression of CXCL6 and the tumor The size and vascular invasion are closely related to the important clinicopathological indicators of the invasion of liver cancer. In addition, we have found that higher CXCL6 expression is associated with poor overall survival (OS) and no relapse rate (RFS). The prognosis of low CXCL6 expression in patients with liver cancer is better than that of high expression patients.3. in HCC tissues, CXCL6 mRNA and protein expression water The results showed that CXCL6 was up to up.4. CXCL6 in MHCC97L, MHCC97H and HCCLM3 cell lines, but was low in the HepG2 and SMMC7721 cell lines, and the CXCL6 mRNA expression in the liver cancer cell lines. .5. in vitro invasiveness test showed that CXCL6 silencing significantly lowered the invasiveness of MHCC97H and HCCLM3 cells.6. with siRNA targeting the MHCC97H and HCCLM3 cell lines silenced CXCL6 in vitro. The results showed that silent CXCL6 significantly inhibited the proliferation of hepatoma cells in vitro and the control group. SMMC-7721 cells transfected by CXCL6 plasmid lentivirus had higher invasiveness and metastasis ability,.8. CXCL6 was inhibited after MMP9 knockout in SMMC-7721 cells. Conclusion: the positive expression rate of 1.CXCL6 in liver cancer tissues is significantly higher than that of non tumor liver tissues, and high expression in hepatoma cells with high metastasis ability; C The expression of XCL6 is positively related to the malignant potential of liver cancer; CXCL6 may play a role in promoting the movement of hepatoma cells,.2.CXCL6 high expression promotes the proliferation of HCC cells and invade the invasion effect of.3.CXCL6 in the liver cancer cells, which may cause.4.CXCL6 to act as a potential independent preconditioning of HCC in the future by activating or acting on MMP9. Post factor and therapeutic target.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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