CENP-H及Ki67的表达在下咽癌术后复发作用研究
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摘要
下咽癌是头颈肿瘤中较少见的恶性肿瘤之一。临床症状主要表现为咽部不适、吞咽困难、吞咽疼痛、颈部肿块和声音嘶哑等。下咽癌约占头颈肿瘤3-5%,约占全身肿瘤的0.5%。对于世界不同的地区、不同的年龄、不同的性别之间,下咽癌的发病率均有一定程度的差异。已有的资料显示欧洲发病率呈上升的趋势,在中国下咽癌的发病率近年来却趋于平缓。同其他恶性肿瘤一样,下咽癌的病因复杂,但具体病因尚不明确,下咽癌可能与烟酒有密切的关系。临床资料表明,大多数病人有吸烟饮酒史而且日用量很大,有的持续时间相当长。其他因素如营养缺乏、环境污染及病毒感染可能参与其发病过程。
下咽癌治疗的目标当前是彻底根除肿瘤,最大限度地减少并发症,优化生活质量,延长生命。手术加放化疗的综合治疗是公认的主要治疗手段。虽然放化疗手术及其他治疗手段有长足的发展,但由于下咽癌临床诊断多处于晚期阶段,而且由于解剖的的特点,复合病变多见。作为头颈肿瘤中恶性程度很高的肿瘤之一,下咽癌复发率高,国外报道5年的生存率只有30%。因此早期诊断,加强术后的复发的监控,有着十分重要的临床意义。虽然下咽癌的发病机制至今仍不明确,科学工作者及临床肿瘤学家越来越关注肿瘤的基因及生长因子在肿瘤发生发展过程的作用,由此可以探查肿瘤发生的原因,提高肿瘤的早期诊断率,提高生活质量,并改善预后。综上,我们对下咽癌术后病人复发相关指标进行研究,以期为临床的治疗提供一些启迪。
有丝分裂的动力是在动粒蛋白和微管之间产生,有丝分裂监测点的信号通路就在动粒蛋白上进行。动力蛋白既是有丝分裂基本活动所必需,又能使染色体的分裂时产生的差错得以纠正。细胞分裂,姊妹染色体分离,子细胞必须得到数目准确和结构功能完整的染色体。染色体的不等分会导致非整倍体的产生,其后果可能是致命性,也可引起先天发育不良,甚至可导致恶性肿瘤的产生。
动力蛋白分为功能性的和结构性的蛋白。作为功能性的蛋白-CENP-H是动力蛋白复合体的重要组成部分,1991年在鼠中首先发现,分子量为33kDa,拥有环-环样的结构,除心脏,脑组织,肾上腺以外的组织中均有表达,而且增殖旺盛的器官表达相对增强。CENP-H具有重要的生理功能,研究表明CENP-H与CENP-A和CENP-C形成动力蛋白复合体,在有丝分裂中CENP-H是蛋白复合体不可缺少的一部分。在Hela细胞中CENP-H被沉默以后可导致CENP-C的减少,预示着CENP-H在复合体中的确是发挥着重要的作用。进一步研究表明CENP-H与肿瘤发生有一定关系。CENP-H在口腔癌、直肠癌中高表达,促进恶性肿瘤的形成。有趣的是CENP-H的高表达在直肠癌中可诱导多倍体的产生。提示CENP-H在直肠癌形成发展演进中扮演着重要角色。近来研究还表明CENP-H高表达是食道癌、非小细胞肺癌、鼻咽癌预后的指标。
Ki67是核增殖抗原,是研究细胞增殖的重要指标,其主要的特点是除G0以外细胞的各个期均有表达,Ki67表达增强预示细胞增殖活跃。在临床及研究领域内己广泛的应用,尤其是乳腺癌及前列腺癌。研究表明,Ki67在口腔癌中过度表达并可能成为其预后的指标。
综合上述,虽然CENP-H及Ki67在一些癌中作为预后的指标,然而CENP-H及Ki67蛋白在下咽癌中与其预后的相关性至今尚无研究。我们要对两因子在下咽癌中的表达及临床的关系进行探讨。特别是二者的表达在下咽癌术后病人复发的关系进行深入的研究。另在体外沉默CENP-H以后,检测细胞的增殖、凋亡及侵袭功能,探讨CENP-H对下咽癌的影响。
第一部分CENP-H及Ki67蛋白在下咽癌中的表达
目的：
1.通过免疫组化在下咽癌术后病人的癌组织及癌旁正常黏膜组织行CENP-H的蛋白水平表达检测,对癌组织行Ki67检测。进一步探讨两个因子表达与临床相关指标如年龄、性别、组织的分化程度、临床阶段、饮酒与非饮酒之间关系。
2.在下咽癌组织及对应的癌旁正常黏膜中,通过RT-PCR检测CENP-H在下咽癌中的mRNA水平的表达。
材料及方法：
1.从2003年3月至2005年3月在齐鲁医院耳鼻喉科住院的首次接受下咽癌手术治疗的病人标本中选出112例符合标准者为研究对象,与每一位病人签知情同意书；免疫组化法检测CENP-H及Ki67二因子在下咽癌中的蛋白水平表达,对CENP-H设癌旁正常黏膜对照。CENP-H及Ki67二因子在下咽癌中的表达评判标准：0(无表达)；1+(浅染色或细胞着色数目小于20%)；2+(中等染色或细胞着色数目20%-50%)；3+(深染色或细胞着色数目大于50%)。0,+是低表达,2+、3+是高表达。
2.8位下咽癌病人术后的癌组织及癌旁正常黏膜,Trizol方法制备提取总RNA, CENP-H上游引物是5'-TGCAAGAAAAGCAAATCGAA-3',下游引物是5'-ATCCC-AAGATTCCTGCTGTG-3;内参GAPDH上游引物是5'-ACC AC AGTCC ATGCC ATC-AC-3',下游引物是5'-TCCACCACCCTGTGGCTGTA-3'.利用RT-PCR方法检测CENP-H的mRNA水平的表达并定量分析。
结果：
1.在112下咽癌中CENP-H高表达者50例(44.6%),低表达者62例(55.4%)；在黏膜中的高表达11例(9%)。两者的阳性表达率差异有统计学意义(P<0.05).在112下咽癌中Ki67高表达者69例(61.6%),低表达者43例(38.4%)。
2. CENP-H高表达与病变的晚期阶段、饮酒呈密切相关(P=0.012及P=0.048)。Ki67高表达与晚期阶段密切相关。Ki67与晚期阶段具有相关性(P=0.021)
3.复合Ki67及CENP-H在下咽癌中表达四种的表现：Ki67高表达/CENP-H高表达(n=38),Ki67高表达/CENP-H D低表达(n=31)ki67低表达/CENP-H高表达(n=12)Ki67低表达/CENP-H低表达(n=31)。CENP-H与Ki67表达在下咽癌中具有正相关(P=0.005Pearson[卡方检验]；P=0.006[Fisher's检验])。
4.8例下咽癌病人中显示CENP-H mRNA水平在下咽癌中高表达,但在癌旁正常粘膜组织中低表达。
结论：
1. CENP-H与Ki67在下咽癌中高表达与临床阶段关系密切。
2.在下咽癌中CENP-H与Ki67表达具有正相关。
第二部分CENP-H与Ki67在下咽癌中的表达与下咽癌术后复发的系
目的：前期研究表明CENP-H或Ki67在下咽癌中的高表达与临床晚期阶段密切相关,我们进一步研究二者是否作为复发的指标。
方法：
对112例下咽癌的病人及病人家属进行面对面的术后随访。在随访中,对病人进行电子喉镜及CT检查,并且与病人及／或家属谈话超过15分钟,记录病人术后的情况。平均随访时间48.5个月(2-74月)。然后用Kaplan-Meier制作出生存曲线并分析其术后复发的关系。Cox分析二者的表达是否为下咽癌术后复发的独立危险因素。
结果：
1. CENP-H与Ki67的表达与下咽癌术后复发差异有显著性统计学意义(分别为P<0.001和P=0.009)
2. CENP-H与Ki67的高表达成为下咽癌术后复发的危险因素(分别为P=0.001和p=0.018)。
结论：
1.我们的研究结果显示,CENP-H与Ki67的表达增强是下咽癌术后复发一个标志。
2. CENP-H与Ki67可能是下咽癌术后复发独立的危险因素。
第三部分在FaDu细胞系中沉默CENP-H对细胞增殖、凋亡及侵袭的影响
目的：在FaDu细胞系中,探讨沉默CENP-H中对细胞增殖、凋亡及侵袭的影响。细胞增殖是通过CCK8实验加以检测,流式细胞仪检测细胞凋亡,侵袭实验检测细胞的侵袭能力。
方法：
1. FaDu细胞系(ATCC)于RPMI1640培养基加上10%胎牛血清(FBS)在37℃5%CO2增湿培养箱中培养,每3天进行传代。当细胞愈合率达40-70%进行转染。
2.在FaDu细胞系中沉默CENP-H48小时后Western Blot检测沉默的效率。流式细胞仪检测CENP-H沉默后FaDu细胞系的凋亡。
3.CCK8检测CENP-H沉默后的FaDu细胞系的增殖情况。
4.侵袭实验检测沉默CENP-H对FaDu细胞的侵袭能力的影响。
结果：
1.瞬时转染的效率约60-80%,Western Blot检测沉默的效果,CENP-H沉默组与PBS组及阴性对照组比较,瞬时转染沉默的效果好。
2.沉默CENP-H之后,PBS组、阴性对照组及沉默组细胞凋亡数分别为12.18%±2.12%,14.38%±3.12%49.17%±7.78%,它们之间的差异具有显著的统计学意义(P<0.01)。
3.沉默组、阴性对照组以及未处理组的吸光值在48小时,72小时分别是0.312±0.031、0.528±0.025、0.531±0.029和0.512±0.041、0.822±0.064、0.823±0.053。沉默CENP-H后,沉默组、阴性对照组以及未处理组48,72小时后细胞的增殖明显受影响,三组比较具有显著的统计学意义(P<0.01)。
4.细胞进入小室的细胞数沉默组与对照组分别是每个高倍视野45±6.45和50±8.73。沉默CENP-H后,对细胞的侵袭能力影响无统计学意义(P>0.05)。
结论：
1. CENP-H在体外对细胞增殖有促进作用并诱导细胞凋亡。
2. CENP-H在体外实验中证实不能增加下咽癌的侵袭能力。
Hypopharyngeal squamous cell cancer (HSCC) is a rare malignancy accounting for approximately0.5%of all human malignancies and about3%to5%of all head and neck cancers. Symptoms of HSCC include sore pharynx, pharyngeal malaise, dysphasia, hoarseness and neck lump etc. The rate of the disease differs in areas gender and age. Increasing people surfer this malignancy at present in Europe, but in China it has a hovering rate. There are many factors which link to hypopharyngeal cancer like some other malignancies. Smoking and alcohol abuse are the two major factors which attract great attention. Most of patients have a long and heavy alcohol consumption and smoking history. Other factors such as environment nutrition and virus infection may play a part. Howerver, the real reasons for the malignancy is uncertain.
The goals of treatment for squamous cell carcinoma of the head and neck include eradication the cancer, minimizing the risk of treatment complications and optimizing quality of life. At present, surgery after or prior to radiotherapy has been regarded as the ideal treatment. Most patients are diagnosed with an initial diagnosis in advanced cancer stage and complicated symptoms. The relapse is so common that the mortality is high despite of the progress with over the past decades. The5-year survival rate has been about only30%despite improvements in surgical and radiochemistry approaches due to advanced stage. Early diagnosis and supervision of relapse after operation may highlight clinical cure rate. The mechanism of hypopharyngeal cancer has been uncertain at present. More and more oncologists and clinical workers have focused on genetic issues and growth factors. By doing so, the early diagnose rate and quality of life and survival rate will be improved. Thus, we decide to study the factors concerning relapse after surgery and provide some meaningful materials.
Mitotic power occurs between microtubule and kinetochore during mitosis. Checkpoint of mitosis path may proceed by kinetochore. Kinetochores have a role in chromosomes separating from each other and controlling the cell cycle during mitosis. Another function for kinetochore is to recorrects the errors during mitosis. Therefor, kinetochores play a fundamental role in accurate cell segregation. During period of cell segregation and chromosomal division, cell will get the right number of chromosome. Aneuploid will occur due to unequal cell segregation, which results in mortality, dysplasia and even cancer.
Kinetochores comprise facultative and constitutive kinetochore proteins. Centromere protein (CENP-H) with molecular weight33Kda, one of constitutive kinetochores, is an essential part of complex, which was initially found in1991. CENP-H, a coiled-coil structural and a nuclear signal protein was found in mouse. It was demonstrated that CENP-H expressed in most organ except heart brain and kidney. Also, it expressed intensely in some proliferative organ. It plays a crucial role in kinetochore organization and function throughout the whole cell cycle. Along with other members, CENP-H forms a functional complex which is required for faithful chromosome segregation. Furthermore, knockdown of CENP-H led to severe mitotic phenotypes and reduced CENP-C level, suggesting that CENP-H plays an important role in the architecture and function of the kinetochore complex. The expression of CENP-H was found upregulated in malignant tumors such as colorectal cancer, suggesting that CENP-H may be involved in tumor development and progression. Other studies demonstrated that CENP-H might be a promising prognostic marker in non-small cell lung cancer, esophageal carcinoma, oral squamous cell carcarcinoma, and nasopharyngeal carcinoma. However, the involvement of CENP-H as a relapse-associated biomarker in HSCC has not been clarified.
Ki67is strongly expressed in proliferating cells and universally regarded as a proliferation marker, because it is present in all active phases but not the resting phase (GO) of the cell cycle during mitosis. The overexpression of Ki67indicates the proliferation of cells. Ki67has been widely studied and utilized in various fields. It has been demonstrated to predict survival and relapse in oral carcinoma.
As mentioned above, CENP-H and Ki67overexpression have been demonstrated in some malignancies, and in some of them it has been correlated with poor patient prognosis, but whether they can be overexpressed in HSCC and serve as predictors of relapse-free survival after primary resection remains unclear. The current study was designed to investigate the expression of CENP-H and Ki67in specimens of HSCC patients, and analyze its association with relapse-free survival. Furthermore, we also examined the roles of CENP-H in cell proliferation and apoptosis and invasion.
Part1CENP-H and Ki67expression in HSCC
Objective
1. CENP-H and Ki67exprssion were investigated in hypopharygeal cancer. CENP-H exprssion was also detected in adjacent normal tissues. We also identified the relationship between the expression of CENP-H and Ki67and clinical various clinical parameters such as age, gender, stage, ect.
2. RT-PCR was examined with cancer and normal tissues in hypopharygeal cacner.
Methods We enrolled112patients with primary HSCC who underwent surgical resection as initial treatment at the Department of Otolaryngology-Head and Neck Surgery, Qilu Hospital, Shandong University, between March2003and March2005. Patients were enrolled if they had received a diagnosis of HSCC by postoperative pathologic examination and showed negative surgical margins after resection. Postoperative radiation was routinely recommended to reduce relapse risk. Patients with preoperative radiotherapy and/or chemotherapy were excluded. The study was approved by the Investigation and Ethical Committee of Qilu Hospital according to the Standards of the Declaration of Helsinki.
Immunohistochemistry of CENP-H and Ki67expression involved112hypopharyngeal tissues or normal tissues. Labeled nuclei were reported as a percentage of total tumor cells counted in the samples and graded as0(negative staining);1+(low staining or<20%);2+(intermediate staining or20%-50%); and3+(strong staining or>50%).0and+were considered low expression and2+and3+high expression.
RT-PCR
Total RNA was extracted by use of RNAiso Plus (TaKaRa Biotechnology, Dalian, China). The primer sequences were for CENP-H, forward,5'-TGCAAGAAAAGCAAATCGAA-3', and reverse,5'-ATCCC-AAGATTCCTGCTGTG-3'; and GAPDH, forward,5'-ACCACAGTCCATGCCATC-AC-3', and reverse,5'-TCCACCACCCTGTGGCTGTA-3'as a normalization control. Amplification of transcripts involved a two-step RT-PCR system (Invitrogen). The optimal number of PCR cycles was35cycles. The samples were from8patients.
Results
1. CENP-H and Ki67protein immunostaining was mainly observed in nuclei of HSCC cells of the112specimens:50cases (44.6%) showed upregulated CENP-H expression (62specimens showed low CENP-H expression). But in normal tissues,9cases showed upregulated CENP-H expression. It was statisticly significant between them (P<0.05).69cases (61.6%) showed upregulated Ki67overexpression.
2. Four different coexpressing phenotypes are as following:Ki67high expression/CENP-H high expression (n=38); Ki67high expression/CENP-H low expression (n=31); Ki671ow expression/CENP-H high expression (n=12); Ki67low expression/CENP-H low expression (n=31). CENP-H expression was associated with Ki67expression.(Pearson chi-square P=0.005; Fisher's Exact Test P=0.006).
3. CENP-H and Ki67protein immunostaining were associated with advanced stage.Moreover, CENP-H protein immunostaining were associated with alcohol history.
4. RT-PCR analysis of hypopharyngeal tumor tissues and adjacent normal epithelia showed that CENP-H overexpression at mRNA level were shown caner tissues and but not normal epithelia.
Conclusion:
1. The high expression of CENP-H and Ki67in hypopharyngeal cancer was significantly assocated with advanced stage.
2. There is significant association between Ki67and CENP-H expression in hypopharyngeal cancer.
Part2. Role of Centromere Protein H and Ki67in Relapse-freeSurvival of Patients after Primary Surgery for Hypo pharyngeal Cancer
Objective
The overexpressions of CENP-H and Ki67in HSCC were shown in our previous study. The results prompted us to furtherly invitigate the overexpressions of CENP-H and Ki67will be prognostic for hypopharygeal cancer relapse after primary resection.
Methods
After resection, we took on face-face follow-up with patients and/or their relatives. During follow-up, electronic laryngoscopy examination was performed. Patients underwent CT to identify relapse or not. Relapse-free survival was defined as the interval between the date of surgery and the date of diagnosis of relapse. Face-to-face conversation with patients and/or relatives was over15min in the follow-up. The follow-up ended April2010. The median follow-up was48.5months (range2-74months).
Results
1. CENP-H and Ki67expression are associated with relapse-free survival of patients after primary surgery for hypopharyngeal cancer (P<0.001and P=0.009).
2. Increased CENP-H and Ki67protein levels were independent predictors of relapse-free survival (P=0.001and P=0.018, respectively).
Conclusion
1. Upregulated CENP-H and Ki67levels were significantly associated with short relapse-free survival in HSCC.
2. These factors might be predictors of a relapsing phenotype in HSSC.
Part3Knockdown of CENP-H in cells effects on proliferation and apoptosis and invasion
Objective:Tests were used to explore whether CENP-H can impair cell proliferation and stimulate apoptosis or invasion in FaDu cell lines after knockdown of CENP-H.
Methods:
1. The Lipofectamin2000(Invitrogen) was used on transient transfections of siRNA cells in our study, and transfection was carried out by siRNA to knockdown of CENP-H in FaDu cell lines.
2. Western blot was used to check the efficiency of knockdown of CENP-H.
3. Flow cytometer was carried out to examine apoptosis.
4. CCK8ws used to check cell proliferation.
5. Invasion test was performed to identify the changes of cell motoricity.
Results:
1. As shown in the picture, the efficiency of silence was perfect.
2. Compared to the control groups the number of cell apoptosis increased significantly (P<0.01) The number of apoptosis was12.18%±2.12%for he control groups,14.38%±3.12%for negative group and49.17%±7.78%for treated group.
3. The observed values were0.312±0.031,0.528±0.025and0.531±0.029at48hour after treatment for knockdown group, the control group and the negatitive group respectively. The observed value was0.823±0.0530.822±0.064and0.512±0.041after72hour. Knockdown of CENP-H significantly impaired cell proliferation after48hour and72hour (P<0.01)
4. The number of cells per field was for the knockdown group45±6.45and50±8.73for the control one which were shown in our test. There was no significant influence on invasion by knockdown of CENP-H.(P>0.05)
Conclusion:
1. CENP-H might improve cancer cell proliferation and stop cell apoptosis.
2. CENP-H might not improve cancer cell invasion ability.
下咽癌治疗的目标当前是彻底根除肿瘤,最大限度地减少并发症,优化生活质量,延长生命。手术加放化疗的综合治疗是公认的主要治疗手段。虽然放化疗手术及其他治疗手段有长足的发展,但由于下咽癌临床诊断多处于晚期阶段,而且由于解剖的的特点,复合病变多见。作为头颈肿瘤中恶性程度很高的肿瘤之一,下咽癌复发率高,国外报道5年的生存率只有30%。因此早期诊断,加强术后的复发的监控,有着十分重要的临床意义。虽然下咽癌的发病机制至今仍不明确,科学工作者及临床肿瘤学家越来越关注肿瘤的基因及生长因子在肿瘤发生发展过程的作用,由此可以探查肿瘤发生的原因,提高肿瘤的早期诊断率,提高生活质量,并改善预后。综上,我们对下咽癌术后病人复发相关指标进行研究,以期为临床的治疗提供一些启迪。
有丝分裂的动力是在动粒蛋白和微管之间产生,有丝分裂监测点的信号通路就在动粒蛋白上进行。动力蛋白既是有丝分裂基本活动所必需,又能使染色体的分裂时产生的差错得以纠正。细胞分裂,姊妹染色体分离,子细胞必须得到数目准确和结构功能完整的染色体。染色体的不等分会导致非整倍体的产生,其后果可能是致命性,也可引起先天发育不良,甚至可导致恶性肿瘤的产生。
动力蛋白分为功能性的和结构性的蛋白。作为功能性的蛋白-CENP-H是动力蛋白复合体的重要组成部分,1991年在鼠中首先发现,分子量为33kDa,拥有环-环样的结构,除心脏,脑组织,肾上腺以外的组织中均有表达,而且增殖旺盛的器官表达相对增强。CENP-H具有重要的生理功能,研究表明CENP-H与CENP-A和CENP-C形成动力蛋白复合体,在有丝分裂中CENP-H是蛋白复合体不可缺少的一部分。在Hela细胞中CENP-H被沉默以后可导致CENP-C的减少,预示着CENP-H在复合体中的确是发挥着重要的作用。进一步研究表明CENP-H与肿瘤发生有一定关系。CENP-H在口腔癌、直肠癌中高表达,促进恶性肿瘤的形成。有趣的是CENP-H的高表达在直肠癌中可诱导多倍体的产生。提示CENP-H在直肠癌形成发展演进中扮演着重要角色。近来研究还表明CENP-H高表达是食道癌、非小细胞肺癌、鼻咽癌预后的指标。
Ki67是核增殖抗原,是研究细胞增殖的重要指标,其主要的特点是除G0以外细胞的各个期均有表达,Ki67表达增强预示细胞增殖活跃。在临床及研究领域内己广泛的应用,尤其是乳腺癌及前列腺癌。研究表明,Ki67在口腔癌中过度表达并可能成为其预后的指标。
综合上述,虽然CENP-H及Ki67在一些癌中作为预后的指标,然而CENP-H及Ki67蛋白在下咽癌中与其预后的相关性至今尚无研究。我们要对两因子在下咽癌中的表达及临床的关系进行探讨。特别是二者的表达在下咽癌术后病人复发的关系进行深入的研究。另在体外沉默CENP-H以后,检测细胞的增殖、凋亡及侵袭功能,探讨CENP-H对下咽癌的影响。
第一部分CENP-H及Ki67蛋白在下咽癌中的表达
目的：
1.通过免疫组化在下咽癌术后病人的癌组织及癌旁正常黏膜组织行CENP-H的蛋白水平表达检测,对癌组织行Ki67检测。进一步探讨两个因子表达与临床相关指标如年龄、性别、组织的分化程度、临床阶段、饮酒与非饮酒之间关系。
2.在下咽癌组织及对应的癌旁正常黏膜中,通过RT-PCR检测CENP-H在下咽癌中的mRNA水平的表达。
材料及方法：
1.从2003年3月至2005年3月在齐鲁医院耳鼻喉科住院的首次接受下咽癌手术治疗的病人标本中选出112例符合标准者为研究对象,与每一位病人签知情同意书；免疫组化法检测CENP-H及Ki67二因子在下咽癌中的蛋白水平表达,对CENP-H设癌旁正常黏膜对照。CENP-H及Ki67二因子在下咽癌中的表达评判标准：0(无表达)；1+(浅染色或细胞着色数目小于20%)；2+(中等染色或细胞着色数目20%-50%)；3+(深染色或细胞着色数目大于50%)。0,+是低表达,2+、3+是高表达。
2.8位下咽癌病人术后的癌组织及癌旁正常黏膜,Trizol方法制备提取总RNA, CENP-H上游引物是5'-TGCAAGAAAAGCAAATCGAA-3',下游引物是5'-ATCCC-AAGATTCCTGCTGTG-3;内参GAPDH上游引物是5'-ACC AC AGTCC ATGCC ATC-AC-3',下游引物是5'-TCCACCACCCTGTGGCTGTA-3'.利用RT-PCR方法检测CENP-H的mRNA水平的表达并定量分析。
结果：
1.在112下咽癌中CENP-H高表达者50例(44.6%),低表达者62例(55.4%)；在黏膜中的高表达11例(9%)。两者的阳性表达率差异有统计学意义(P<0.05).在112下咽癌中Ki67高表达者69例(61.6%),低表达者43例(38.4%)。
2. CENP-H高表达与病变的晚期阶段、饮酒呈密切相关(P=0.012及P=0.048)。Ki67高表达与晚期阶段密切相关。Ki67与晚期阶段具有相关性(P=0.021)
3.复合Ki67及CENP-H在下咽癌中表达四种的表现：Ki67高表达/CENP-H高表达(n=38),Ki67高表达/CENP-H D低表达(n=31)ki67低表达/CENP-H高表达(n=12)Ki67低表达/CENP-H低表达(n=31)。CENP-H与Ki67表达在下咽癌中具有正相关(P=0.005Pearson[卡方检验]；P=0.006[Fisher's检验])。
4.8例下咽癌病人中显示CENP-H mRNA水平在下咽癌中高表达,但在癌旁正常粘膜组织中低表达。
结论：
1. CENP-H与Ki67在下咽癌中高表达与临床阶段关系密切。
2.在下咽癌中CENP-H与Ki67表达具有正相关。
第二部分CENP-H与Ki67在下咽癌中的表达与下咽癌术后复发的系
目的：前期研究表明CENP-H或Ki67在下咽癌中的高表达与临床晚期阶段密切相关,我们进一步研究二者是否作为复发的指标。
方法：
对112例下咽癌的病人及病人家属进行面对面的术后随访。在随访中,对病人进行电子喉镜及CT检查,并且与病人及／或家属谈话超过15分钟,记录病人术后的情况。平均随访时间48.5个月(2-74月)。然后用Kaplan-Meier制作出生存曲线并分析其术后复发的关系。Cox分析二者的表达是否为下咽癌术后复发的独立危险因素。
结果：
1. CENP-H与Ki67的表达与下咽癌术后复发差异有显著性统计学意义(分别为P<0.001和P=0.009)
2. CENP-H与Ki67的高表达成为下咽癌术后复发的危险因素(分别为P=0.001和p=0.018)。
结论：
1.我们的研究结果显示,CENP-H与Ki67的表达增强是下咽癌术后复发一个标志。
2. CENP-H与Ki67可能是下咽癌术后复发独立的危险因素。
第三部分在FaDu细胞系中沉默CENP-H对细胞增殖、凋亡及侵袭的影响
目的：在FaDu细胞系中,探讨沉默CENP-H中对细胞增殖、凋亡及侵袭的影响。细胞增殖是通过CCK8实验加以检测,流式细胞仪检测细胞凋亡,侵袭实验检测细胞的侵袭能力。
方法：
1. FaDu细胞系(ATCC)于RPMI1640培养基加上10%胎牛血清(FBS)在37℃5%CO2增湿培养箱中培养,每3天进行传代。当细胞愈合率达40-70%进行转染。
2.在FaDu细胞系中沉默CENP-H48小时后Western Blot检测沉默的效率。流式细胞仪检测CENP-H沉默后FaDu细胞系的凋亡。
3.CCK8检测CENP-H沉默后的FaDu细胞系的增殖情况。
4.侵袭实验检测沉默CENP-H对FaDu细胞的侵袭能力的影响。
结果：
1.瞬时转染的效率约60-80%,Western Blot检测沉默的效果,CENP-H沉默组与PBS组及阴性对照组比较,瞬时转染沉默的效果好。
2.沉默CENP-H之后,PBS组、阴性对照组及沉默组细胞凋亡数分别为12.18%±2.12%,14.38%±3.12%49.17%±7.78%,它们之间的差异具有显著的统计学意义(P<0.01)。
3.沉默组、阴性对照组以及未处理组的吸光值在48小时,72小时分别是0.312±0.031、0.528±0.025、0.531±0.029和0.512±0.041、0.822±0.064、0.823±0.053。沉默CENP-H后,沉默组、阴性对照组以及未处理组48,72小时后细胞的增殖明显受影响,三组比较具有显著的统计学意义(P<0.01)。
4.细胞进入小室的细胞数沉默组与对照组分别是每个高倍视野45±6.45和50±8.73。沉默CENP-H后,对细胞的侵袭能力影响无统计学意义(P>0.05)。
结论：
1. CENP-H在体外对细胞增殖有促进作用并诱导细胞凋亡。
2. CENP-H在体外实验中证实不能增加下咽癌的侵袭能力。
Hypopharyngeal squamous cell cancer (HSCC) is a rare malignancy accounting for approximately0.5%of all human malignancies and about3%to5%of all head and neck cancers. Symptoms of HSCC include sore pharynx, pharyngeal malaise, dysphasia, hoarseness and neck lump etc. The rate of the disease differs in areas gender and age. Increasing people surfer this malignancy at present in Europe, but in China it has a hovering rate. There are many factors which link to hypopharyngeal cancer like some other malignancies. Smoking and alcohol abuse are the two major factors which attract great attention. Most of patients have a long and heavy alcohol consumption and smoking history. Other factors such as environment nutrition and virus infection may play a part. Howerver, the real reasons for the malignancy is uncertain.
The goals of treatment for squamous cell carcinoma of the head and neck include eradication the cancer, minimizing the risk of treatment complications and optimizing quality of life. At present, surgery after or prior to radiotherapy has been regarded as the ideal treatment. Most patients are diagnosed with an initial diagnosis in advanced cancer stage and complicated symptoms. The relapse is so common that the mortality is high despite of the progress with over the past decades. The5-year survival rate has been about only30%despite improvements in surgical and radiochemistry approaches due to advanced stage. Early diagnosis and supervision of relapse after operation may highlight clinical cure rate. The mechanism of hypopharyngeal cancer has been uncertain at present. More and more oncologists and clinical workers have focused on genetic issues and growth factors. By doing so, the early diagnose rate and quality of life and survival rate will be improved. Thus, we decide to study the factors concerning relapse after surgery and provide some meaningful materials.
Mitotic power occurs between microtubule and kinetochore during mitosis. Checkpoint of mitosis path may proceed by kinetochore. Kinetochores have a role in chromosomes separating from each other and controlling the cell cycle during mitosis. Another function for kinetochore is to recorrects the errors during mitosis. Therefor, kinetochores play a fundamental role in accurate cell segregation. During period of cell segregation and chromosomal division, cell will get the right number of chromosome. Aneuploid will occur due to unequal cell segregation, which results in mortality, dysplasia and even cancer.
Kinetochores comprise facultative and constitutive kinetochore proteins. Centromere protein (CENP-H) with molecular weight33Kda, one of constitutive kinetochores, is an essential part of complex, which was initially found in1991. CENP-H, a coiled-coil structural and a nuclear signal protein was found in mouse. It was demonstrated that CENP-H expressed in most organ except heart brain and kidney. Also, it expressed intensely in some proliferative organ. It plays a crucial role in kinetochore organization and function throughout the whole cell cycle. Along with other members, CENP-H forms a functional complex which is required for faithful chromosome segregation. Furthermore, knockdown of CENP-H led to severe mitotic phenotypes and reduced CENP-C level, suggesting that CENP-H plays an important role in the architecture and function of the kinetochore complex. The expression of CENP-H was found upregulated in malignant tumors such as colorectal cancer, suggesting that CENP-H may be involved in tumor development and progression. Other studies demonstrated that CENP-H might be a promising prognostic marker in non-small cell lung cancer, esophageal carcinoma, oral squamous cell carcarcinoma, and nasopharyngeal carcinoma. However, the involvement of CENP-H as a relapse-associated biomarker in HSCC has not been clarified.
Ki67is strongly expressed in proliferating cells and universally regarded as a proliferation marker, because it is present in all active phases but not the resting phase (GO) of the cell cycle during mitosis. The overexpression of Ki67indicates the proliferation of cells. Ki67has been widely studied and utilized in various fields. It has been demonstrated to predict survival and relapse in oral carcinoma.
As mentioned above, CENP-H and Ki67overexpression have been demonstrated in some malignancies, and in some of them it has been correlated with poor patient prognosis, but whether they can be overexpressed in HSCC and serve as predictors of relapse-free survival after primary resection remains unclear. The current study was designed to investigate the expression of CENP-H and Ki67in specimens of HSCC patients, and analyze its association with relapse-free survival. Furthermore, we also examined the roles of CENP-H in cell proliferation and apoptosis and invasion.
Part1CENP-H and Ki67expression in HSCC
Objective
1. CENP-H and Ki67exprssion were investigated in hypopharygeal cancer. CENP-H exprssion was also detected in adjacent normal tissues. We also identified the relationship between the expression of CENP-H and Ki67and clinical various clinical parameters such as age, gender, stage, ect.
2. RT-PCR was examined with cancer and normal tissues in hypopharygeal cacner.
Methods We enrolled112patients with primary HSCC who underwent surgical resection as initial treatment at the Department of Otolaryngology-Head and Neck Surgery, Qilu Hospital, Shandong University, between March2003and March2005. Patients were enrolled if they had received a diagnosis of HSCC by postoperative pathologic examination and showed negative surgical margins after resection. Postoperative radiation was routinely recommended to reduce relapse risk. Patients with preoperative radiotherapy and/or chemotherapy were excluded. The study was approved by the Investigation and Ethical Committee of Qilu Hospital according to the Standards of the Declaration of Helsinki.
Immunohistochemistry of CENP-H and Ki67expression involved112hypopharyngeal tissues or normal tissues. Labeled nuclei were reported as a percentage of total tumor cells counted in the samples and graded as0(negative staining);1+(low staining or<20%);2+(intermediate staining or20%-50%); and3+(strong staining or>50%).0and+were considered low expression and2+and3+high expression.
RT-PCR
Total RNA was extracted by use of RNAiso Plus (TaKaRa Biotechnology, Dalian, China). The primer sequences were for CENP-H, forward,5'-TGCAAGAAAAGCAAATCGAA-3', and reverse,5'-ATCCC-AAGATTCCTGCTGTG-3'; and GAPDH, forward,5'-ACCACAGTCCATGCCATC-AC-3', and reverse,5'-TCCACCACCCTGTGGCTGTA-3'as a normalization control. Amplification of transcripts involved a two-step RT-PCR system (Invitrogen). The optimal number of PCR cycles was35cycles. The samples were from8patients.
Results
1. CENP-H and Ki67protein immunostaining was mainly observed in nuclei of HSCC cells of the112specimens:50cases (44.6%) showed upregulated CENP-H expression (62specimens showed low CENP-H expression). But in normal tissues,9cases showed upregulated CENP-H expression. It was statisticly significant between them (P<0.05).69cases (61.6%) showed upregulated Ki67overexpression.
2. Four different coexpressing phenotypes are as following:Ki67high expression/CENP-H high expression (n=38); Ki67high expression/CENP-H low expression (n=31); Ki671ow expression/CENP-H high expression (n=12); Ki67low expression/CENP-H low expression (n=31). CENP-H expression was associated with Ki67expression.(Pearson chi-square P=0.005; Fisher's Exact Test P=0.006).
3. CENP-H and Ki67protein immunostaining were associated with advanced stage.Moreover, CENP-H protein immunostaining were associated with alcohol history.
4. RT-PCR analysis of hypopharyngeal tumor tissues and adjacent normal epithelia showed that CENP-H overexpression at mRNA level were shown caner tissues and but not normal epithelia.
Conclusion:
1. The high expression of CENP-H and Ki67in hypopharyngeal cancer was significantly assocated with advanced stage.
2. There is significant association between Ki67and CENP-H expression in hypopharyngeal cancer.
Part2. Role of Centromere Protein H and Ki67in Relapse-freeSurvival of Patients after Primary Surgery for Hypo pharyngeal Cancer
Objective
The overexpressions of CENP-H and Ki67in HSCC were shown in our previous study. The results prompted us to furtherly invitigate the overexpressions of CENP-H and Ki67will be prognostic for hypopharygeal cancer relapse after primary resection.
Methods
After resection, we took on face-face follow-up with patients and/or their relatives. During follow-up, electronic laryngoscopy examination was performed. Patients underwent CT to identify relapse or not. Relapse-free survival was defined as the interval between the date of surgery and the date of diagnosis of relapse. Face-to-face conversation with patients and/or relatives was over15min in the follow-up. The follow-up ended April2010. The median follow-up was48.5months (range2-74months).
Results
1. CENP-H and Ki67expression are associated with relapse-free survival of patients after primary surgery for hypopharyngeal cancer (P<0.001and P=0.009).
2. Increased CENP-H and Ki67protein levels were independent predictors of relapse-free survival (P=0.001and P=0.018, respectively).
Conclusion
1. Upregulated CENP-H and Ki67levels were significantly associated with short relapse-free survival in HSCC.
2. These factors might be predictors of a relapsing phenotype in HSSC.
Part3Knockdown of CENP-H in cells effects on proliferation and apoptosis and invasion
Objective:Tests were used to explore whether CENP-H can impair cell proliferation and stimulate apoptosis or invasion in FaDu cell lines after knockdown of CENP-H.
Methods:
1. The Lipofectamin2000(Invitrogen) was used on transient transfections of siRNA cells in our study, and transfection was carried out by siRNA to knockdown of CENP-H in FaDu cell lines.
2. Western blot was used to check the efficiency of knockdown of CENP-H.
3. Flow cytometer was carried out to examine apoptosis.
4. CCK8ws used to check cell proliferation.
5. Invasion test was performed to identify the changes of cell motoricity.
Results:
1. As shown in the picture, the efficiency of silence was perfect.
2. Compared to the control groups the number of cell apoptosis increased significantly (P<0.01) The number of apoptosis was12.18%±2.12%for he control groups,14.38%±3.12%for negative group and49.17%±7.78%for treated group.
3. The observed values were0.312±0.031,0.528±0.025and0.531±0.029at48hour after treatment for knockdown group, the control group and the negatitive group respectively. The observed value was0.823±0.0530.822±0.064and0.512±0.041after72hour. Knockdown of CENP-H significantly impaired cell proliferation after48hour and72hour (P<0.01)
4. The number of cells per field was for the knockdown group45±6.45and50±8.73for the control one which were shown in our test. There was no significant influence on invasion by knockdown of CENP-H.(P>0.05)
Conclusion:
1. CENP-H might improve cancer cell proliferation and stop cell apoptosis.
2. CENP-H might not improve cancer cell invasion ability.
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