TFPI-2基因CpG岛在甲状腺结节性疾病中的甲基化状态及意义
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摘要
目的：
甲状腺癌是一种较常见的恶性肿瘤,乳头状甲状腺癌约占甲状腺癌的60%。乳头状甲状腺癌和甲状腺腺瘤、结节性甲状腺肿均表现为甲状腺内结节性肿块,早期临床表现相似。甲状腺腺瘤、结节性甲状腺肿为良性甲状腺病变,10～18%的良性甲状腺病变会转化成恶性病变。浸润和转移是导致甲状腺癌治疗失败和患者死亡的主要原因。因此,寻找乳头状甲状腺癌浸润和转移相关的分子标志非常重要。抑癌基因的失活被认为是肿瘤发生与演进过程中的重要机制。基因启动子区域CpG岛的甲基化是导致基因失活最常见的表观遗传机制。寻找可用于肿瘤诊断、浸润和转移判断及预测良性结节恶性变的表观遗传学分子标志已成为肿瘤研究的热点,以期为临床诊治提供理论依据。
基质金属蛋白酶(matrix metalloproteinases, MMPs)和跨膜丝氨酸蛋白酶(transmembrane proteaseserine4, TMPRSS4)可以降解细胞外基质(extracellular matrix， ECM)中成分,促进肿瘤细胞的侵袭的和转移,而肿瘤的浸润和转移与患者愈后密切相关。组织因子途径抑制物-2(tissue factor pathway inhibitor-2, TFPI-2)可抑制TMPRSS和MMPs对ECM的破坏作用,从而抑制肿瘤的浸润和转移。TFPI-2基因具有典型的管家基因特征,在大多数人类组织中表达,但在起源于这些组织的肿瘤中,常因启动子区CpG岛高甲基化而表达降低或不表达,因此,TFPI-2基因被认为可能是肿瘤抑制因子。本研究拟应用结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌组织标本及人乳头状甲状腺癌TPC-1细胞和人髓样甲状腺癌TT细胞为研究对象,探讨TFPI-2基因在结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌组织中的表达差异,探讨该基因启动子区甲基化状态对其表达的影响及在乳头状甲状腺癌转移中的作用。以期为甲状腺结节性疾病的鉴别诊断和乳头状甲状腺癌转移的判断提供表观遗传学标志,为甲状腺疾病良性结节恶性变的早期发现提供线索。
本论文分三部分：
第一部分甲状腺结节性疾病中TFPI-2的表达及其在乳头状甲状腺癌中与TMPRSS4和MMP-1表达的关系
方法
1.采用免疫组织化学技术检测30例结节性甲状腺肿、20例甲状腺腺瘤和40例乳头状甲状腺癌组织(淋巴结转移患者18例,无淋巴结转移者22例)中TFPI-2蛋白的表达情况及在40例乳头状甲状腺癌组织中TFPI-2、 TMPRSS4和MMP-1蛋白的表达情况,分析TFPI-2在三种不同甲状腺结节性疾病中蛋白表达差异及其在乳头状甲状腺癌中与TMPRSS4和MMP-1蛋白表达的相关性,分析乳头状甲状腺癌中TFPI-2、 TMPRSS4和MMP-1蛋白表达与淋巴结转移的关系。
2.统计学分析：所有数据均经SPSS14.0软件进行统计分析,计数资料采用列联表分析(crosstabs)，相关性分析采用Spearman相关分析,检验水准为a=0.05。
结果
1.免疫组化结果显示：结节性甲状腺肿组织中TFPI-2蛋白的阳性表达率为83.3%,甲状腺腺瘤组织中TFPI-2蛋白的阳性表达率为65.0%,甲状腺乳头状癌组织中TFPI-2蛋白的阳性表达率为52.5%,组间相比差异有显著性,X2=7.221,P=0.027；TFPI-2蛋白在伴转移乳头状甲状腺癌组织中的表达低于无转移甲状腺乳头状癌组织中TFPI-2蛋白的表达,差异有显著性X2=4.821,P=0.028；TMPRSS4和MMP-1蛋白在伴转移乳头状甲状腺癌组织中的表达均高于无转移甲状腺乳头状癌组织中的表达,差异有显著性,X2值分别为4.835和6.852；P值分别为0.028和0.009。
2.相关分析显示：乳头状甲状腺癌中TFPI-2蛋白与TMPRSS、MMP-1蛋白的表达均呈负相关关系,相关系数分别为-0.383和-0.339,相关系数检验P值分别为0.015和0.032。
第二部分甲状腺结节性疾病中TFPI-2基因启动子区CpG岛甲基化状态及意义
方法
1.采用甲基化特异性聚合酶链反应(methylation specific-polymerase chain reaction， MSP)方法检测30例结节性甲状腺肿、20例甲状腺腺瘤和40例乳头状甲状腺癌中TTFPI-2基因启动子区甲基化状态,分析结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌中TFPI-2基因启动子区CpG岛甲基化状态与TFPI-2蛋白表达的关系,分析乳头状甲状腺癌中TFPI-2基因启动子区甲基化状态与淋巴结转移的关系。
2.统计学分析：所有数据均经SPSS14.0软件进行统计分析,计数资料采用列联表分析(crosstabs),相关性分析采用Spearman相关分析,检验水准为a=0.05。
结果
1. MSP结果显示：结节性甲状腺肿组织中TFPI-2基因启动子区甲基化率为16.7%,甲状腺腺瘤组织中TFPI-2基因启动子区甲基化率为25.0%,乳头状甲状腺癌组织中TFPI-2基因启动子区甲基化率为50.0%。组间相比差异有显著性,X2=9.722,P=0.008。乳头状甲状腺癌(伴转移)组织中TFPI-2基因启动子区甲基化率为88.9%,乳头状甲状腺癌(无转移)组织中TFPI-2基因启动子区甲基化率为18.2%,组间相比差异有显著性,X2=19.798,P=0.000。
2.相关分析显示：结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌中TFPI-2基因启动子区CpG岛甲基化状态与TFPI-2蛋白表达均呈负相关关系,r分别为：-0.760、-0.545、-0.651,P值分别为：0.000、0.020、0.000。
第三部分甲状腺癌细胞系中TFPI-2基因启动子区CpG岛甲基化状态及其对TFPI-2、TMPRSS4和MMPl表达的影响
方法
1.以人乳头状甲状腺癌TPC-1细胞和人髓样甲状腺癌TT细胞为研究对象,采用MSP检测5-氮杂胞苷(5-aza-deoxycytidine，5-aza-dc)处理与未处理组TPC-1和TT细胞中TFPI-2基因启动子区甲基化状态。
2.采用Western blotting方法检测5-aza-dc处理与未处理组TPC-1和TT细胞中TFPI-2、TMPRSS4和MMP1的蛋白表达。分析5-aza-dc对TPC-1和TT细胞中TFPI-2基因启动子区甲基化状态的影响及甲基化状态与TFPI-2、 TMPRSS4和MMP1蛋白表达的关系。
3.统计学分析：应用SPSS14.0统计软件进行统计学处理,计量资料采用(x±s)表示；计量资料采用两样本均数t检验。检验水准为α=0.05。
结果
1.MSP结果显示：甲状腺癌TPC-1和TT细胞中TFPI-2基因启动子区处于甲基化高状态,而5-aza-dc处理后TPC-1和TT细胞中TFPI-2基因启动子区呈去甲基化状态。
2.Western blotting结果显示,5-aza-dc处理组TPC-1和TT细胞中TFPI-2蛋白表达织高于5-aza-dc未处理组,差异有统计学意义,t值分别为：32.704和18.432,P值分别为：0.000和0.000；而5-aza-dc处理组TPC-1和TT细胞中TMPRSS4和MMP1蛋白低于5-aza-dc未处理组,P值均小于0.01。
结论
1.TFPI-2基因启动子区CpG岛高甲基化可能是乳头状甲状腺癌中TFPI-2失活的重要机制
2.TFPI-2基因启动子区CpG岛甲基化状态和TFPI-2蛋白表达可能是结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌的鉴别诊断的重要分子标志。
3.TFPI-2基因启动子区甲基化状态、TFPI-2、 TMPRSS4和MMP-1蛋白表达可能与乳头状甲状腺癌的转移存在密切的关系。
4.TFPI-2在乳头状甲状腺癌转移中的作用可能与TFPI-2因基因启动子区高甲基化而表达降低,从而使MMP-1和TMPRSS4表达增高有关。
5.TFPI-2可能成为检测良性结节恶性变的指示因子。
本研究创新点：
甲状腺癌的发病率不断上升,且部分良性结节会发生恶性变,但少有人关注。以结节性甲状腺肿、甲状腺腺瘤、乳头状甲状腺癌和甲状腺癌细胞系为研究对象,从组织和细胞培养等不同角度探讨了TFPI-2基因启动子区甲基化状态在乳头状甲状腺癌转移中的作用及其在乳头状甲状腺癌、结节性甲状腺肿和甲状腺腺瘤的鉴别诊断中的意义。为甲状腺结节性疾病的鉴别诊断和乳头状甲状腺癌转移的判断提供了新的表观遗传学标志物,为甲状腺疾病良性结节恶性变提供了早期检测的线索。
Objectives
Thyroid carcinoma is a common malignant tumor. Papillary thyroid carcinoma accounts for60%of all thyroid carcinoma, and invasion and metastasis are the main reasons of treatment failure and leading to death of patient with thyroid carcinoma. Accordingly, it becomes increasingly concerned to looking for the related specific molecular markers as the invasion and metastasis of papillary thyroid cancer. Papillary thyroid carcinoma, nodular goiter and thyroid adenoma represent an enlarged thyroid gland containing circumscribed nodules within its substance. It is difficult to discriminate these which the clinical manifestation is similar.10-18%out of benign nodules have the possibility of malignant change. Deactivation of cancer suppressor genes is taken for important mechanism in the process of carcinogenesis, and CpG island methylation in the cancer suppressor gene promoter region is the most common change of deactivation. So, it has been the hot topic to search for the epigenetic molecular markers which used to diagnosis, estimating of invasion and metastasis and predicting malignant change, may be it can be used to guiding the treatment. Matrix metalloproteinase can degrade the proteins in extracellular matrix (ECM) and destroyed the barrier for protecting normal tissue. This is the most important reason of invasion and metastasis related to recovery. More and more studies have shown that tissue factor pathway inhibitor-2(TFPI-2) gene promoter was characterized by housekeeping gene, which expressed in most human tissues. But it was down-expression or no-expression in the tumors origining from above organizations, which often dued to promoter CpG island methylation. There for, TFPI-2gene was estimated as a cancer suppressor gene. The studies also showed that TFPI-2could inhibit the combination of transmembrane proteaseserine4(TMPRSS4), matrix metalloproteinases (MMPs) and the extracellular matrix (ECM), reduced the damaging effects of the TMPSS4and MMPs to the ECM, which inhibited tumor invasion and metastasis. In our study, the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma were investigated, so as to provide molecular genetics target for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma, and in order to get the information about detecting the indicator of predicting malignant change earlier..
This study includes the following three parts.
Part1The expression of TFPI-2in thyroid nodular diseases and the relationships between TFPI-2andTMPRSS4, MMP-1in papillary thyroid carcinoma
Methods
1. The expressions of TFPI-2, TMPRSS4and MMP-1protein were detected by immunohistochemistry (IHC) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid cancer. The differences of the expression of TFPI-2in three thyriod nodular disease were analyzed, and the correlations between TFPI-2and TMPRSS4, MMP-1expressions in papillary thyroid carcinoma were analyzed. In papillary thyroid carcinoma, the relationships between TFPI-2, TMPRSS4, MMP-1expression and lymphatic metastasis in papillary thyroid carcinoma were analyzed.
2. Statistical analysis:All the data were statistically analyzed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P＜0.05level.
Results
1. The results from IHC revealed that:the positive expression rate of TFPI-2protein was83.3%in nodular goiter,65.0%in thyroid adenoma and52.5%in thyroid carcinoma, respectively. There was significant difference among the groups, χ2=7.221, P=0.027; The expression of TFPI-2protein was significantly lower in papillary thyroid carcinoma with metastasis than that in papillary thyroid carcinoma without metastasis χ2=4.821, P=0.028; The expressions of TMPRSS4and MMP-1protein were significantly higher in papillary thyroid carcinoma with lymphatic metastasis than that in papillary thyroid carcinoma without lymphatic metastasis. The χ2value is4.835and6.852and the P value is0.028and0.009, respectively.
2. The data from correlation analysis showed that there were significant negative correlation of expression between TFPI-2and TMPRSS4, MMP-1in papillary thyroid carcinoma. The correlation coefficient r is-0.383and-0.339, respectively, and the corresponding P value is0.015and0.032, respectively.
Part2The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2in thyroid nodular diseases
Methods
1. The methylation status of TFPI-2gene promoter region was detected by methylation-specific PCR (MSP) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid carcinoma. The relationship between the methylation status of TFPI-2gene promoter region and the expression of TFPI-2in thyroid nodular diseases, and the effects caused by the methylation status of TFPI-2gene promoter region on TFPI-2and the relationship between the methylation status and lymphatic metastasis in papillary thyroid carcinoma was analyzed.
2. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P<0.05level.
Results
1. The results from MSP indicated that①the methylation rate of TFPI-2gene promoter region was16.7%in nodular goiter,25.0%in thyroid adenoma,50.0%in papillary thyroid carcinoma, respectively. There was significant difference among the groups.χ2=9.722, P=0.008.②The methylation rate of TFPI-2gene promoter region was88.9%in papillary thyroid carcinoma with lymphatic metastasis,18.2%in papillary thyroid carcinoma without lymphatic metastasis, respectively. There was significant difference between the groups.χ2=19.798, P=0.000.
2. The data from correlation analysis suggested that there were significant negative correlation between the methylation status of TFPI-2gene promoter region and TFPI-2expression in nodular goiter, thyroid adenoma, and papillary thyroid carcinoma (P＜0.05or0.01). r value is-0.760,-0.545,-0.651,and P value is0.000、0.020、0.000, respectively.
Part3The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2, TMPRSS4and MMP-1in thyroid carcinoma cells
Methods
1. Taken human papillary thyroid carcinoma TPC-1cells and human medullary thyroid carcinoma TT cells as study targets, the methylation status of TFPI-2gene promoter region was detected by MSP in TPC-1cells and TT cells in treatment with5-aza-dc group(treatment group) and treatment without5-aza-dc group (untreatment group).
2. The expressions of TFPI-2, TMPRSS4and MMP1were investigated by Western blotting assay in TPC-1cells and TT cells in treatment and untreatment group. The relationships between the methylation status of TFPI-2gene promoter region and TFPI-2, TMPRSS4and MMP1expression in TPC-1cells and TT cells in two groups were analyzed.
3. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. The measurement data were presented as x±s.Independent-Samples T Test was used to analyze the measurement data. Statistical significance was determined at P＜0.05level.
Results
1. The results from MSP displayed that the promoter region of TFPI-2gene in thyroid carcinoma cell TPC-1and TT cells was hyper-methylation state, whereas it was demethylation after treated with5-aza-dc.
2. The results from Western blotting showed that the expression of TFPI-2was significantly higher in thyroid carcinoma TPC-1cells and TT cells in treatment group than in untreatment. t value was32.704,18.432, P value was0.000,0.000, respectively, the expressions of TMPRSS4and MMP1were significantly lower in thyroid carcinoma TPC-1cells and TT cells in treatment group than untreatment group (P＜0.01).
Conclusions:
1. Hypermethylation of TFPI-2gene promoter region might be the important mechanism of inactivation of TFPI-2gene in papillary thyroid carcinoma.
2. Methylation status of TFPI-2gene promoter region and the expression of TFPI-2might be used as important molecular markers in the differential diagnosis of papillary thyroid carcinoma, nodular goiter and thyroid adenoma.
3. There is close relationship between the methylation status of TFPI-2gene promoter region, subsequent the expression of TFPI-2, TMPRSS4, MMP-1and the lymphatic metastasis of papillary thyroid carcinoma.
4. The role of TFPI-2in the lymphatic metastasis of papillary thyroid carcinoma might be associated lower expression of TFPI-2and higher expression of MMP-1 and TMPRSS4caused by hypermethylation of TFPI-2gene promoter region.
5. TFPI-2hypermethylation and low expression might be an indicative factor of malignant change of benign nodule.
The innovation in present study:
Incidence rate of thyroid carcinoma has increased and part of benign nodules occurs malignant change. Mechanisms of invasion in thyroid cancer remain poorly understood. But the above problem haven't been concerned. In present study, took tissue from nodular goiter, thyroid adenoma, papillary thyroid carcinoma and thyroid carcinoma cell lines as the materials, aimed to investigate the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma, so as to provide an epigenetics molecular for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma. Provide a clue to predicting malignant change of benign nodule.
甲状腺癌是一种较常见的恶性肿瘤,乳头状甲状腺癌约占甲状腺癌的60%。乳头状甲状腺癌和甲状腺腺瘤、结节性甲状腺肿均表现为甲状腺内结节性肿块,早期临床表现相似。甲状腺腺瘤、结节性甲状腺肿为良性甲状腺病变,10～18%的良性甲状腺病变会转化成恶性病变。浸润和转移是导致甲状腺癌治疗失败和患者死亡的主要原因。因此,寻找乳头状甲状腺癌浸润和转移相关的分子标志非常重要。抑癌基因的失活被认为是肿瘤发生与演进过程中的重要机制。基因启动子区域CpG岛的甲基化是导致基因失活最常见的表观遗传机制。寻找可用于肿瘤诊断、浸润和转移判断及预测良性结节恶性变的表观遗传学分子标志已成为肿瘤研究的热点,以期为临床诊治提供理论依据。
基质金属蛋白酶(matrix metalloproteinases, MMPs)和跨膜丝氨酸蛋白酶(transmembrane proteaseserine4, TMPRSS4)可以降解细胞外基质(extracellular matrix， ECM)中成分,促进肿瘤细胞的侵袭的和转移,而肿瘤的浸润和转移与患者愈后密切相关。组织因子途径抑制物-2(tissue factor pathway inhibitor-2, TFPI-2)可抑制TMPRSS和MMPs对ECM的破坏作用,从而抑制肿瘤的浸润和转移。TFPI-2基因具有典型的管家基因特征,在大多数人类组织中表达,但在起源于这些组织的肿瘤中,常因启动子区CpG岛高甲基化而表达降低或不表达,因此,TFPI-2基因被认为可能是肿瘤抑制因子。本研究拟应用结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌组织标本及人乳头状甲状腺癌TPC-1细胞和人髓样甲状腺癌TT细胞为研究对象,探讨TFPI-2基因在结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌组织中的表达差异,探讨该基因启动子区甲基化状态对其表达的影响及在乳头状甲状腺癌转移中的作用。以期为甲状腺结节性疾病的鉴别诊断和乳头状甲状腺癌转移的判断提供表观遗传学标志,为甲状腺疾病良性结节恶性变的早期发现提供线索。
本论文分三部分：
第一部分甲状腺结节性疾病中TFPI-2的表达及其在乳头状甲状腺癌中与TMPRSS4和MMP-1表达的关系
方法
1.采用免疫组织化学技术检测30例结节性甲状腺肿、20例甲状腺腺瘤和40例乳头状甲状腺癌组织(淋巴结转移患者18例,无淋巴结转移者22例)中TFPI-2蛋白的表达情况及在40例乳头状甲状腺癌组织中TFPI-2、 TMPRSS4和MMP-1蛋白的表达情况,分析TFPI-2在三种不同甲状腺结节性疾病中蛋白表达差异及其在乳头状甲状腺癌中与TMPRSS4和MMP-1蛋白表达的相关性,分析乳头状甲状腺癌中TFPI-2、 TMPRSS4和MMP-1蛋白表达与淋巴结转移的关系。
2.统计学分析：所有数据均经SPSS14.0软件进行统计分析,计数资料采用列联表分析(crosstabs)，相关性分析采用Spearman相关分析,检验水准为a=0.05。
结果
1.免疫组化结果显示：结节性甲状腺肿组织中TFPI-2蛋白的阳性表达率为83.3%,甲状腺腺瘤组织中TFPI-2蛋白的阳性表达率为65.0%,甲状腺乳头状癌组织中TFPI-2蛋白的阳性表达率为52.5%,组间相比差异有显著性,X2=7.221,P=0.027；TFPI-2蛋白在伴转移乳头状甲状腺癌组织中的表达低于无转移甲状腺乳头状癌组织中TFPI-2蛋白的表达,差异有显著性X2=4.821,P=0.028；TMPRSS4和MMP-1蛋白在伴转移乳头状甲状腺癌组织中的表达均高于无转移甲状腺乳头状癌组织中的表达,差异有显著性,X2值分别为4.835和6.852；P值分别为0.028和0.009。
2.相关分析显示：乳头状甲状腺癌中TFPI-2蛋白与TMPRSS、MMP-1蛋白的表达均呈负相关关系,相关系数分别为-0.383和-0.339,相关系数检验P值分别为0.015和0.032。
第二部分甲状腺结节性疾病中TFPI-2基因启动子区CpG岛甲基化状态及意义
方法
1.采用甲基化特异性聚合酶链反应(methylation specific-polymerase chain reaction， MSP)方法检测30例结节性甲状腺肿、20例甲状腺腺瘤和40例乳头状甲状腺癌中TTFPI-2基因启动子区甲基化状态,分析结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌中TFPI-2基因启动子区CpG岛甲基化状态与TFPI-2蛋白表达的关系,分析乳头状甲状腺癌中TFPI-2基因启动子区甲基化状态与淋巴结转移的关系。
2.统计学分析：所有数据均经SPSS14.0软件进行统计分析,计数资料采用列联表分析(crosstabs),相关性分析采用Spearman相关分析,检验水准为a=0.05。
结果
1. MSP结果显示：结节性甲状腺肿组织中TFPI-2基因启动子区甲基化率为16.7%,甲状腺腺瘤组织中TFPI-2基因启动子区甲基化率为25.0%,乳头状甲状腺癌组织中TFPI-2基因启动子区甲基化率为50.0%。组间相比差异有显著性,X2=9.722,P=0.008。乳头状甲状腺癌(伴转移)组织中TFPI-2基因启动子区甲基化率为88.9%,乳头状甲状腺癌(无转移)组织中TFPI-2基因启动子区甲基化率为18.2%,组间相比差异有显著性,X2=19.798,P=0.000。
2.相关分析显示：结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌中TFPI-2基因启动子区CpG岛甲基化状态与TFPI-2蛋白表达均呈负相关关系,r分别为：-0.760、-0.545、-0.651,P值分别为：0.000、0.020、0.000。
第三部分甲状腺癌细胞系中TFPI-2基因启动子区CpG岛甲基化状态及其对TFPI-2、TMPRSS4和MMPl表达的影响
方法
1.以人乳头状甲状腺癌TPC-1细胞和人髓样甲状腺癌TT细胞为研究对象,采用MSP检测5-氮杂胞苷(5-aza-deoxycytidine，5-aza-dc)处理与未处理组TPC-1和TT细胞中TFPI-2基因启动子区甲基化状态。
2.采用Western blotting方法检测5-aza-dc处理与未处理组TPC-1和TT细胞中TFPI-2、TMPRSS4和MMP1的蛋白表达。分析5-aza-dc对TPC-1和TT细胞中TFPI-2基因启动子区甲基化状态的影响及甲基化状态与TFPI-2、 TMPRSS4和MMP1蛋白表达的关系。
3.统计学分析：应用SPSS14.0统计软件进行统计学处理,计量资料采用(x±s)表示；计量资料采用两样本均数t检验。检验水准为α=0.05。
结果
1.MSP结果显示：甲状腺癌TPC-1和TT细胞中TFPI-2基因启动子区处于甲基化高状态,而5-aza-dc处理后TPC-1和TT细胞中TFPI-2基因启动子区呈去甲基化状态。
2.Western blotting结果显示,5-aza-dc处理组TPC-1和TT细胞中TFPI-2蛋白表达织高于5-aza-dc未处理组,差异有统计学意义,t值分别为：32.704和18.432,P值分别为：0.000和0.000；而5-aza-dc处理组TPC-1和TT细胞中TMPRSS4和MMP1蛋白低于5-aza-dc未处理组,P值均小于0.01。
结论
1.TFPI-2基因启动子区CpG岛高甲基化可能是乳头状甲状腺癌中TFPI-2失活的重要机制
2.TFPI-2基因启动子区CpG岛甲基化状态和TFPI-2蛋白表达可能是结节性甲状腺肿、甲状腺腺瘤和乳头状甲状腺癌的鉴别诊断的重要分子标志。
3.TFPI-2基因启动子区甲基化状态、TFPI-2、 TMPRSS4和MMP-1蛋白表达可能与乳头状甲状腺癌的转移存在密切的关系。
4.TFPI-2在乳头状甲状腺癌转移中的作用可能与TFPI-2因基因启动子区高甲基化而表达降低,从而使MMP-1和TMPRSS4表达增高有关。
5.TFPI-2可能成为检测良性结节恶性变的指示因子。
本研究创新点：
甲状腺癌的发病率不断上升,且部分良性结节会发生恶性变,但少有人关注。以结节性甲状腺肿、甲状腺腺瘤、乳头状甲状腺癌和甲状腺癌细胞系为研究对象,从组织和细胞培养等不同角度探讨了TFPI-2基因启动子区甲基化状态在乳头状甲状腺癌转移中的作用及其在乳头状甲状腺癌、结节性甲状腺肿和甲状腺腺瘤的鉴别诊断中的意义。为甲状腺结节性疾病的鉴别诊断和乳头状甲状腺癌转移的判断提供了新的表观遗传学标志物,为甲状腺疾病良性结节恶性变提供了早期检测的线索。
Objectives
Thyroid carcinoma is a common malignant tumor. Papillary thyroid carcinoma accounts for60%of all thyroid carcinoma, and invasion and metastasis are the main reasons of treatment failure and leading to death of patient with thyroid carcinoma. Accordingly, it becomes increasingly concerned to looking for the related specific molecular markers as the invasion and metastasis of papillary thyroid cancer. Papillary thyroid carcinoma, nodular goiter and thyroid adenoma represent an enlarged thyroid gland containing circumscribed nodules within its substance. It is difficult to discriminate these which the clinical manifestation is similar.10-18%out of benign nodules have the possibility of malignant change. Deactivation of cancer suppressor genes is taken for important mechanism in the process of carcinogenesis, and CpG island methylation in the cancer suppressor gene promoter region is the most common change of deactivation. So, it has been the hot topic to search for the epigenetic molecular markers which used to diagnosis, estimating of invasion and metastasis and predicting malignant change, may be it can be used to guiding the treatment. Matrix metalloproteinase can degrade the proteins in extracellular matrix (ECM) and destroyed the barrier for protecting normal tissue. This is the most important reason of invasion and metastasis related to recovery. More and more studies have shown that tissue factor pathway inhibitor-2(TFPI-2) gene promoter was characterized by housekeeping gene, which expressed in most human tissues. But it was down-expression or no-expression in the tumors origining from above organizations, which often dued to promoter CpG island methylation. There for, TFPI-2gene was estimated as a cancer suppressor gene. The studies also showed that TFPI-2could inhibit the combination of transmembrane proteaseserine4(TMPRSS4), matrix metalloproteinases (MMPs) and the extracellular matrix (ECM), reduced the damaging effects of the TMPSS4and MMPs to the ECM, which inhibited tumor invasion and metastasis. In our study, the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma were investigated, so as to provide molecular genetics target for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma, and in order to get the information about detecting the indicator of predicting malignant change earlier..
This study includes the following three parts.
Part1The expression of TFPI-2in thyroid nodular diseases and the relationships between TFPI-2andTMPRSS4, MMP-1in papillary thyroid carcinoma
Methods
1. The expressions of TFPI-2, TMPRSS4and MMP-1protein were detected by immunohistochemistry (IHC) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid cancer. The differences of the expression of TFPI-2in three thyriod nodular disease were analyzed, and the correlations between TFPI-2and TMPRSS4, MMP-1expressions in papillary thyroid carcinoma were analyzed. In papillary thyroid carcinoma, the relationships between TFPI-2, TMPRSS4, MMP-1expression and lymphatic metastasis in papillary thyroid carcinoma were analyzed.
2. Statistical analysis:All the data were statistically analyzed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P＜0.05level.
Results
1. The results from IHC revealed that:the positive expression rate of TFPI-2protein was83.3%in nodular goiter,65.0%in thyroid adenoma and52.5%in thyroid carcinoma, respectively. There was significant difference among the groups, χ2=7.221, P=0.027; The expression of TFPI-2protein was significantly lower in papillary thyroid carcinoma with metastasis than that in papillary thyroid carcinoma without metastasis χ2=4.821, P=0.028; The expressions of TMPRSS4and MMP-1protein were significantly higher in papillary thyroid carcinoma with lymphatic metastasis than that in papillary thyroid carcinoma without lymphatic metastasis. The χ2value is4.835and6.852and the P value is0.028and0.009, respectively.
2. The data from correlation analysis showed that there were significant negative correlation of expression between TFPI-2and TMPRSS4, MMP-1in papillary thyroid carcinoma. The correlation coefficient r is-0.383and-0.339, respectively, and the corresponding P value is0.015and0.032, respectively.
Part2The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2in thyroid nodular diseases
Methods
1. The methylation status of TFPI-2gene promoter region was detected by methylation-specific PCR (MSP) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid carcinoma. The relationship between the methylation status of TFPI-2gene promoter region and the expression of TFPI-2in thyroid nodular diseases, and the effects caused by the methylation status of TFPI-2gene promoter region on TFPI-2and the relationship between the methylation status and lymphatic metastasis in papillary thyroid carcinoma was analyzed.
2. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P<0.05level.
Results
1. The results from MSP indicated that①the methylation rate of TFPI-2gene promoter region was16.7%in nodular goiter,25.0%in thyroid adenoma,50.0%in papillary thyroid carcinoma, respectively. There was significant difference among the groups.χ2=9.722, P=0.008.②The methylation rate of TFPI-2gene promoter region was88.9%in papillary thyroid carcinoma with lymphatic metastasis,18.2%in papillary thyroid carcinoma without lymphatic metastasis, respectively. There was significant difference between the groups.χ2=19.798, P=0.000.
2. The data from correlation analysis suggested that there were significant negative correlation between the methylation status of TFPI-2gene promoter region and TFPI-2expression in nodular goiter, thyroid adenoma, and papillary thyroid carcinoma (P＜0.05or0.01). r value is-0.760,-0.545,-0.651,and P value is0.000、0.020、0.000, respectively.
Part3The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2, TMPRSS4and MMP-1in thyroid carcinoma cells
Methods
1. Taken human papillary thyroid carcinoma TPC-1cells and human medullary thyroid carcinoma TT cells as study targets, the methylation status of TFPI-2gene promoter region was detected by MSP in TPC-1cells and TT cells in treatment with5-aza-dc group(treatment group) and treatment without5-aza-dc group (untreatment group).
2. The expressions of TFPI-2, TMPRSS4and MMP1were investigated by Western blotting assay in TPC-1cells and TT cells in treatment and untreatment group. The relationships between the methylation status of TFPI-2gene promoter region and TFPI-2, TMPRSS4and MMP1expression in TPC-1cells and TT cells in two groups were analyzed.
3. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. The measurement data were presented as x±s.Independent-Samples T Test was used to analyze the measurement data. Statistical significance was determined at P＜0.05level.
Results
1. The results from MSP displayed that the promoter region of TFPI-2gene in thyroid carcinoma cell TPC-1and TT cells was hyper-methylation state, whereas it was demethylation after treated with5-aza-dc.
2. The results from Western blotting showed that the expression of TFPI-2was significantly higher in thyroid carcinoma TPC-1cells and TT cells in treatment group than in untreatment. t value was32.704,18.432, P value was0.000,0.000, respectively, the expressions of TMPRSS4and MMP1were significantly lower in thyroid carcinoma TPC-1cells and TT cells in treatment group than untreatment group (P＜0.01).
Conclusions:
1. Hypermethylation of TFPI-2gene promoter region might be the important mechanism of inactivation of TFPI-2gene in papillary thyroid carcinoma.
2. Methylation status of TFPI-2gene promoter region and the expression of TFPI-2might be used as important molecular markers in the differential diagnosis of papillary thyroid carcinoma, nodular goiter and thyroid adenoma.
3. There is close relationship between the methylation status of TFPI-2gene promoter region, subsequent the expression of TFPI-2, TMPRSS4, MMP-1and the lymphatic metastasis of papillary thyroid carcinoma.
4. The role of TFPI-2in the lymphatic metastasis of papillary thyroid carcinoma might be associated lower expression of TFPI-2and higher expression of MMP-1 and TMPRSS4caused by hypermethylation of TFPI-2gene promoter region.
5. TFPI-2hypermethylation and low expression might be an indicative factor of malignant change of benign nodule.
The innovation in present study:
Incidence rate of thyroid carcinoma has increased and part of benign nodules occurs malignant change. Mechanisms of invasion in thyroid cancer remain poorly understood. But the above problem haven't been concerned. In present study, took tissue from nodular goiter, thyroid adenoma, papillary thyroid carcinoma and thyroid carcinoma cell lines as the materials, aimed to investigate the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma, so as to provide an epigenetics molecular for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma. Provide a clue to predicting malignant change of benign nodule.
引文
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