摘要
背景与目的：
在世界范围内,宫颈癌是妇女癌症中仅次于乳腺癌而位居第二的恶性肿瘤。在我国宫颈癌是最常见的妇科恶性肿瘤,且发生率有继续增加和年轻化趋势。持续性的高危型HPV感染是宫颈癌的发病的主要病因,而环境因素和人体的基因易感性在宫颈癌的发生中起着十分重要的作用。近年来,随着分子遗传学方面的进展,与生物进程有关的基因单核苷酸多态性(SNP)及其特定的组合被认为参与了致癌过程,已成为宫颈癌病因学的研究热点。
SNP系第三代遗传标记,是涉及到DNA序列单个核苷酸变异的一类基因的多态性。是人类基因组遗传变异中最常见一种涉及的形式,占人类基因组遗传多态性的90%以上。标签SNP (tag SNP)是在基因组中具有高度连锁不平衡区域内具有代表性的一个SNP。不需对一染色体区域的所有SNP进行基因型分析而是通过检测tag SNPs即可确定其遗传学变异,从而极大的提高检出效率,大大提高关联分析的有效性。
RAD21基因是与粟酒裂殖酵母RAD21(S.Pombe RAD21)高度相似的人类同系物,其编码的蛋白质是一种参与DNA的双链断裂修复及在有丝分裂期间确保染色单体的粘合的核磷蛋白。目前已证实,RAD21基因是从酿酒酵母到人类都高度保守的粘连蛋白复合体Cohesin的一个组成部分,并且在有丝分裂过程中RAD21的缺失可以导致染色体非整倍性的发生。众所周知,宫颈癌是一个对放、化疗治疗都十分敏感的恶性肿瘤,同时放化疗是宫颈癌患者的标准治疗之一已有研究发现,RAD21基因表达沉默可提高乳腺癌等恶性肿瘤的化疗敏感性和放疗敏感性。这为恶性肿瘤包括宫颈癌在内的治疗开辟了新的思路。最新的研究表明RAD21基因的多态性与乳腺癌和胰腺癌的发生危险性及预后有关。
我们前期的研究已经表明RAD21基因在宫颈癌中呈现高表达,随着宫颈上皮内瘤变程度的加深,RAD21基因表达量增高。虽然RAD21基因在宫颈癌中呈现高表达,然而目前国内外关于RAD21基因位点多态性与宫颈癌发生之间的相关性研究尚未曾发现有报道。本研究以常规病理确诊的宫颈癌患者和同期经检查排除了宫颈病变的妇女为研究对象,采用病例对照研究方法,从遗传学角度上,检测RAD21基因多态性(SNP)在汉族妇女宫颈癌患者的分布特点,探索RAD21基因多态性与宫颈癌发生之间的关联性,以便进一步发现与宫颈癌发病相关的因素,为宫颈癌的治疗及易感人群的筛查提供更多的生物学手段。
材料与方法：
采用以医院为基础的病例对照研究。选取2010年3月～2011年9月在郑州大学第二附属医院就诊并经病理确诊的120例宫颈癌患者作为研究对象的病例组,同时选择经宫颈细胞学或阴道镜检查等排除了宫颈恶性肿瘤或癌前病变的120例患者作为研究对象的对照组。所有研究对象均为汉族妇女。收集其外周血2m1作为研究标本。所有研究对象采血前均未行手术、放疗或化疗,临床资料完整。病例组中鳞癌106病,腺癌14例,年龄30～71岁,平均年龄为48.9岁；对照组,年龄28-68岁,平均年龄为47.5岁。2组患者年龄经统计学比较无显著性差异。
收集的全部血液标本在经过抗凝剂(柠檬酸钠)处理后,置于-20℃冰箱保存备用。血液基因组小量DNA提取试剂盒提取抗凝血标本的DNA。应用基质辅助激光解吸附电离飞行时间质谱(MALDI-TOF)检测技术对RAD21基因tagSNP位点进行检测。Haploview和SPSS17.0统计软件包统计分析正常宫颈和宫颈癌妇女中RAD21基因多态性的分布特点,从而初步筛选与宫颈癌相关的RAD21基因的SNP位点。
结果：
1、本研究所检测的所有SNP位点其观察值和期望值吻合良好(P>0.001),符合Hardy-Weinberg平衡定律。
2、RAD21基因中,单个SNP位点rs2289937等位基因C在病例组中出现的频率为93.2%,显著高于对照组中的86.7%(χ2=5.637,P=0.0176,OR=2.115,95%CI=1.127-3.969)：
3、在对危险型基因型和非危险型基因型进行频数比较时,病例组rs4570的危险等位基因型(CC+CT)频率(χ2=5.042,P=0.025,OR=1.810,95%C1=1.076-3.043)和rs4579555的危险等位基因型(CC+CT)频率(χ2=5.042,P=0.025,OR=1.810,95%CI=1.076-3.043)显著高于对照组；
4、根据本研究样本数据绘制RAD21基因SNP的连锁不平衡(LD)布局图,并推算出一个连锁不平衡相关区域和该相关区域内的6个单体型。其中,单体型1(H1, TTTCAGGCGC)和单体型6(H6, TTTTAGGCGC)的分布频率在宫颈癌组和正常对照组之间存在显著性差异(P<0.05)。
结论：
RAD21基因遗传多态性与汉族妇女宫颈癌的发生有关；rs4570的等位基因型(CC+CT)和rs4579555的等位基因型(CC+CT)可增加汉族妇女患宫颈癌的风险；单体型H1(TTTCAGGCGC)和单体型H6(TTTTAGGCGC)增加宫颈癌发生的危险性。
Background and Purpose:
Cervical cancer is the second most common type of cancer in women worldwide, after breast cancer, and the most common gynecologic malignant tumor in our country. Persistent HPV infection is considered as a major cause of cervical cancer, and however, environmental factors and human genetic susceptibility also play an important role in the occurrence of cervical cancer. In recent years, along with the progress in molecular genetics, gene single nucleotide polymorphisms(SNPs) and their specific combinations related to biological processes are widely thought to participate in the carcinogenic process, becoming a hot subject of research in etiology of cervical cancer.
Single nucleotide polymorphisms (SNPs), the third generation of genetic markers, are a type of polymorphisms involving variation of a single base pair of DNA sequence, which are the most common form of the human genome genetic variation and account for more than90%of the human genome polymorphism. A tag SNP is a representative SNP in a region of the genome with high linkage disequilibrium. It is possible to identify genetic variation without genotyping every SNP in a chromosomal region, thereby greatly improving the effectiveness of the correlation analysis.
The RAD21gene is a gene that involved in the repair of DNA double-strand breaks, as well as in chromatid cohesion during mitosis. The protein encoded by the RAD21gene is highly similar to the gene product of Schizosaccharomyces pombe rad21. It has been demonstrated that the RAD21gene is an integral part of cohesion protein complex which is highly conserved from Saccharomyces cerevisiae to human beings. Moreover, the absence of RAD21gene can lead the occurrence of chromosomal aneuploidy in the process of mitosis. As we know, cervical cancer is one of malignant tumors which are sensitive to radiotherapy and chemotherapy, while the radiotherapy and chemotherapy is one of the standard treatments for patients with cervical cancer.Studies have found that the RAD21gene silencing can increase sensitivity to radiotherapy and chemotherapy in breast cancer and other malignant tumors. This opens a new approach for the treatment of malignancies including cervical cancer. The latest research shows that the RAD21gene polymorphism is related to risk and prognosis of breast cancer and pancreatic cancer.
Our previous studies have shown that RAD21gene in cervical carcinoma exhibits significantly higher expression. Nevertheless, the association study on the RAD21gene polymorphism and cervical cancer has not been reported around the world. Accordingly, by using a method of case-control study, this study detected the distribution characteristics of RAD21tag SNPs in Chinese Han women with cervical cancer and genetically explored the correlation of the RAD21gene polymorphisms and cervical cancer. The final goal of current study was to provide more valuable methods for the treatment of cervical cancer and screening susceptible populations.
Materials and Methods:
Using a hospital-based case-control study, we selected120cases of cervical cancer patients confirmed by conventional pathology as case group and120cases of patients with benign uterine diseases as control group. All research subjects came from the department of gynecology and obstetrics of the Second Affiliated Hospital of Zhengzhou University. All of the subjects were women of Han nationality. The specimens of2ml peripheral blood were collected from the research subjects. All of the subjects with complete clinical data were not received operation, radiotherapy or chemotherapy before blood sampling. Case group included106cases of squamous cell carcinoma and14cases of adenocarcinoma, with an average age of48.9years old from30to71; the average age of control group was47.5years old from28to68; no significant difference of women's age was observed between the two groups.
All blood specimens were treated with an anticoagulant (Sodium citrate) and stored at-20℃. Genome DNAs were extracted by using DNA extraction kit and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) was applied to detect the RAD21gene SNP sites. Haploview and SPSS17.0statistical software were used to analyze the distribution characteristics of RAD21gene polymorphisms in case and control women, thus initially screening RAD21gene SNP sites which are associated with the occurrence of cervical cancer.
Results:
1、The observed and expected values of all SNP alleles tested in this study are well fitted and conformed to Hardy-Weinberg equilibrium law (P>0.001)
2、In RAD21gene, the frequency of risk allele C of SNP rs2289937in the case group (93.2%) was significantly higher than that in the control group of86.7%(χ2=5.637, P=0.0176, OR=2.115,95%CI=1.127-3.969).
3、When comparing the frequencies of risk genotypes of SNPs, the risk genotype frequencies of both rs4570(CC+CT,χ2=5.042, P=0.025, OR=1.810,95%CI=1.076-3.043) and rs4579555(CC+CT,χ2=5.042, P=0.025, OR=1.810,95%CI=1.076-3.043) were significantly higher in the case group than those in the control group.
4、Based on our data, linkage disequilibrium plot was produced, and1hyplotype block with6its haplotypes were generated. Among6haplotypes, the frequencies of both hyplotype1(HI, TTTCAGGCGC) and hyplotype6(H6, TTTTAGGCGC) were significantly different between the case group and the control group (all P<0.05).
Conclusions:
The SNPs of RAD21gene might be associated with the occurrence of cervical cancer in Han women; the risk genotypes of rs4570(CC+CT) and rs4579555(CC+CT) may increase the risk of cervical cancer; haplotype H1(TTTCAGGCGC) and hyplotype H6(TTTTAGGCGC) may increase the risk of cervical cancer.
引文
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