MMR蛋白在雌激素致结肠癌细胞凋亡中的作用及SEPT9检测结直肠癌的研究
发布时间：2018-12-08 10:35
【摘要】：背景与目的:近30年来,我国结直肠癌的发生率和死亡率呈明显上升趋势,结直肠癌已成为我国的重大公众卫生问题。随着我国肿瘤防治战略的整体前移,通过预防和早诊早治来降低结直肠癌的发生率和死亡率越来越受重视。化学预防是一种较有前景的结直肠癌一级预防方法。流行病学研究表明,雌激素具有预防结直肠癌发生的作用,但雌激素预防结直肠癌的具体机制尚不明确,错配修复(mismatch repair,MMR)蛋白可能是其中较关键的因素之一。本文第一部分研究了MMR蛋白在雌激素引起的结肠癌细胞凋亡中的作用。已有充足的证据表明,筛查和早期诊断能降低结直肠癌的死亡率。粪便隐血试验是目前最常用的结直肠癌筛查方法,但是其敏感性欠佳。外周血septin9基因甲基化检测(SEPT9)是一种新的结直肠癌诊断标志物,但尚未在国内开展使用。本文第二部分研究了新一代SEPT9在中国人群中检测结直肠肿瘤的性能,并与免疫粪隐血试验(fecal immunochemical test,FIT)进行了比较。方法:我们使用了两种MMR蛋白缺陷的人结肠癌细胞株:hMLH1缺陷的HCT116细胞,和hMSH2缺陷的LoVo细胞。通过转染hMLH1和hMSH2野生型cDNA,又构建了相应的MMR蛋白正常的细胞株。在给予雌二醇(17β-estradiol,E2)处理后,观察不同MMR状态的细胞活性和凋亡率的改变,以及凋亡相关蛋白Bax,caspase-9和caspase-3表达的变化。同时,也研究了过表达雌激素受体β(estrogen receptorβ,ERβ)蛋白对结肠癌细胞凋亡的影响。选取经结肠镜检查和病理确诊的135例结直肠癌患者,169例腺瘤性息肉患者(包括84例进展期腺瘤和85例非进展期腺瘤),81例增生性息肉患者,和91例健康对照者(结肠镜检查未见异常)。所有476名患者均在结肠镜检查前抽取外周血,以Epi proColon 2.0试剂盒行septin9基因甲基化检测。其中177名患者同时接受了SEPT9和FIT检查。以结肠镜检查为金标准,计算SEPT9和FIT检测结直肠肿瘤的敏感性和特异性,并以McNemar检验比较SEPT9和FIT对结直肠癌和进展期腺瘤的检出率。结果:E2仅在hMLH1蛋白正常表达的结肠癌细胞中引起凋亡;而在hMLH1蛋白表达缺失的结肠癌细胞中,E2并不能引起明显的凋亡反应。过表达hMSH2或ERβ也能诱导结肠癌细胞凋亡,但这种效应与E2处理无关。即使在hMSH2缺陷的LoVo细胞中,过表达hMLH1蛋白也增强了E2诱导的细胞凋亡反应。SEPT9对结直肠癌的敏感性为74.8%(95%CI:67.0%-81.6%),特异性为87.4%(与非结直肠癌对比,95%CI:83.5%-90.6%)。SEPT9总的假阳性率为4.7%(95%CI:2.2%-8.6%)。SEPT9对I期结直肠癌的检出率为66.7%,II期为82.6%,III期为84.1%,IV期为100%。SEPT9对进展期腺瘤的敏感性为27.4%(95%CI:18.7%-37.6%)。FIT对结直肠癌的敏感性为58.0%(95%CI:46.1%-69.2%),特异性为82.4%(95%CI:74.4%-88.7%)。69例同时接受SEPT9和FIT检查的结直肠癌患者中,SEPT9检测出了25例FIT为阴性的结直肠癌,而FIT检测出了12例SEPT9为阴性的结直肠癌。SEPT9对结直肠癌的检测性能优于FIT,但是对进展期腺瘤的检出率与FIT相似。结论:E2导致的结肠癌凋亡是依赖于错配修复蛋白hMLH1的,hMLH1蛋白能增强E2诱导的结肠癌细胞凋亡,但这一过程可能独立于错配修复系统的错配识别和修复能力。该结果提示,在接受雌激素替代治疗前、或使用雌激素类似物预防结直肠癌前,应该评估患者错配修复蛋白状态,以使效益/风险比最大化。SEPT9检测结直肠癌的敏感性和特异性均高于FIT,可用于结直肠癌的筛查和早期诊断。但其对进展期腺瘤的敏感性较差。SEPT9和FIT两种方法在检测结直肠癌上具有互补性,联合使用这两种方法检测结直肠癌可以提高检出率。
[Abstract]:BACKGROUND & OBJECTIVE: In the past 30 years, the incidence and mortality of colorectal cancer in China have increased significantly, and colorectal cancer has become a major public health problem in our country. With the overall advancement of our country's tumor control strategy, it is more and more important to reduce the incidence and mortality of colorectal cancer by prevention and early diagnosis. Chemical prevention is a promising method for colorectal cancer. Epidemiological studies have shown that estrogen has a role in the prevention of colorectal cancer, but the specific mechanism of estrogen to prevent colorectal cancer is not clear, and mismatch repair (MMR) proteins may be one of the most critical factors. The first part of this paper studies the role of MMR proteins in the apoptosis of colon cancer cells induced by estrogen. There is ample evidence that screening and early diagnosis can reduce the mortality of colorectal cancer. The fecal occult blood test is the most commonly used screening method for colorectal cancer, but its sensitivity is not good. Peripheral blood septin9 gene methylation detection (SEPT9) is a new diagnostic marker for colorectal cancer, but has not been used in the country. The second part of this paper studies the performance of the new generation of SEPT9 in the detection of colorectal tumors in Chinese population, and compared with the faecal occult blood test (FIT). Methods: We used two types of MMR protein-deficient human colon cancer cell lines: hMLH1-deficient HCT116 cells and hMSH2-deficient LoVo cells. By transfecting hMLH1 and hMSH2 wild-type cDNA, the normal cell line of MMR protein was constructed. The changes of cell activity and apoptosis rate in different MMR states, as well as the changes of the expression of Bax, caspase-9 and caspase-3, were observed after the treatment with estradiol (17)-estadiol, E2). The effect of estrogen receptor antagonist (ER) on the apoptosis of colon cancer cells was also studied. Of the 135 patients with colorectal cancer who were diagnosed by colonoscopy and pathology, 169 patients with adenomatous polyposis (including 84 progressive and 85 non-progressive adenomas), 81 hyperplastic polyps, and 91 healthy controls (no abnormalities in colonoscopy) were selected. Peripheral blood was extracted from all 476 patients prior to the colonoscopy and the septin9 gene methylation was detected with the Epi proColon 2.0 kit. Of these, 177 patients received both the SEPT9 and FIT tests. The sensitivity and specificity of SEPT9 and FIT in the detection of colorectal tumors were calculated using colonoscopy as the gold standard, and the detection rates of SEPT9 and FIT for colorectal cancer and advanced adenoma were compared with McNemar test. Results: E2 induced apoptosis only in colon cancer cells that were normally expressed in hMLH1 protein. In colon cancer cells with hMLH1 protein expression, E2 could not cause a significant apoptosis reaction. Overexpression of hMSH2 or ER antigen can also induce apoptosis of colon cancer cells, but this effect is not related to E2 treatment. Even in the hMSH2-deficient LoVo cells, the overexpression of hMLH1 protein also enhances the E2-induced apoptosis reaction. The sensitivity of SEPT9 to colorectal cancer was 74.8% (95% CI: 67. 0%-81.6%), and the specificity was 87.4% (compared with non-colorectal cancer, 95% CI: 83.5%-90.6%). The total false positive rate of SEPT9 was 4.7% (95% CI: 2.2%-80.6%). The detection rate of SEPT9 in stage I colorectal cancer was 65.7%, 82.6% in phase II, 84.1% in phase III and 100% in stage IV. The sensitivity of SEPT9 to the progression of colorectal cancer was 27. 4% (95% CI: 18. 7%-37. 6%). The sensitivity of FIT to colorectal cancer was 58. 0% (95% CI: 46. 1%-69.2%), and the specificity was 82.4% (95% CI: 74. 4%-85.7%). 69 of 69 patients with colorectal cancer who received both SEPT9 and FIT examination showed 25 FIT-negative colorectal cancer, and FIT detected 12 SEPT9-negative colorectal cancer. The detection performance of SEPT9 for colorectal cancer is superior to that of FIT, but the detection rate of the progression adenoma is similar to that of FIT. Conclusion: The apoptosis of colon cancer cells induced by E2 is dependent on the mismatch repair protein hMLH1, and the hMLH1 protein can enhance the apoptosis of colon cancer cells induced by E2, but this process may be independent of mismatch identification and repair capacity of the mismatch repair system. The results suggest that the patient's mismatch repair protein status should be assessed before the estrogen replacement therapy or the use of an estrogen analog to prevent colorectal cancer to maximize the benefit/ risk ratio. The sensitivity and specificity of SEPT9 in the detection of colorectal cancer are higher than that of FIT, which can be used for screening and early diagnosis of colorectal cancer. but its sensitivity to the progression of the adenoma is poor. The two methods of SEPT9 and FIT are complementary in the detection of colorectal cancer, and the detection of colorectal cancer by combination of these two methods can improve the detection rate.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.3
本文编号：2368205
[Abstract]:BACKGROUND & OBJECTIVE: In the past 30 years, the incidence and mortality of colorectal cancer in China have increased significantly, and colorectal cancer has become a major public health problem in our country. With the overall advancement of our country's tumor control strategy, it is more and more important to reduce the incidence and mortality of colorectal cancer by prevention and early diagnosis. Chemical prevention is a promising method for colorectal cancer. Epidemiological studies have shown that estrogen has a role in the prevention of colorectal cancer, but the specific mechanism of estrogen to prevent colorectal cancer is not clear, and mismatch repair (MMR) proteins may be one of the most critical factors. The first part of this paper studies the role of MMR proteins in the apoptosis of colon cancer cells induced by estrogen. There is ample evidence that screening and early diagnosis can reduce the mortality of colorectal cancer. The fecal occult blood test is the most commonly used screening method for colorectal cancer, but its sensitivity is not good. Peripheral blood septin9 gene methylation detection (SEPT9) is a new diagnostic marker for colorectal cancer, but has not been used in the country. The second part of this paper studies the performance of the new generation of SEPT9 in the detection of colorectal tumors in Chinese population, and compared with the faecal occult blood test (FIT). Methods: We used two types of MMR protein-deficient human colon cancer cell lines: hMLH1-deficient HCT116 cells and hMSH2-deficient LoVo cells. By transfecting hMLH1 and hMSH2 wild-type cDNA, the normal cell line of MMR protein was constructed. The changes of cell activity and apoptosis rate in different MMR states, as well as the changes of the expression of Bax, caspase-9 and caspase-3, were observed after the treatment with estradiol (17)-estadiol, E2). The effect of estrogen receptor antagonist (ER) on the apoptosis of colon cancer cells was also studied. Of the 135 patients with colorectal cancer who were diagnosed by colonoscopy and pathology, 169 patients with adenomatous polyposis (including 84 progressive and 85 non-progressive adenomas), 81 hyperplastic polyps, and 91 healthy controls (no abnormalities in colonoscopy) were selected. Peripheral blood was extracted from all 476 patients prior to the colonoscopy and the septin9 gene methylation was detected with the Epi proColon 2.0 kit. Of these, 177 patients received both the SEPT9 and FIT tests. The sensitivity and specificity of SEPT9 and FIT in the detection of colorectal tumors were calculated using colonoscopy as the gold standard, and the detection rates of SEPT9 and FIT for colorectal cancer and advanced adenoma were compared with McNemar test. Results: E2 induced apoptosis only in colon cancer cells that were normally expressed in hMLH1 protein. In colon cancer cells with hMLH1 protein expression, E2 could not cause a significant apoptosis reaction. Overexpression of hMSH2 or ER antigen can also induce apoptosis of colon cancer cells, but this effect is not related to E2 treatment. Even in the hMSH2-deficient LoVo cells, the overexpression of hMLH1 protein also enhances the E2-induced apoptosis reaction. The sensitivity of SEPT9 to colorectal cancer was 74.8% (95% CI: 67. 0%-81.6%), and the specificity was 87.4% (compared with non-colorectal cancer, 95% CI: 83.5%-90.6%). The total false positive rate of SEPT9 was 4.7% (95% CI: 2.2%-80.6%). The detection rate of SEPT9 in stage I colorectal cancer was 65.7%, 82.6% in phase II, 84.1% in phase III and 100% in stage IV. The sensitivity of SEPT9 to the progression of colorectal cancer was 27. 4% (95% CI: 18. 7%-37. 6%). The sensitivity of FIT to colorectal cancer was 58. 0% (95% CI: 46. 1%-69.2%), and the specificity was 82.4% (95% CI: 74. 4%-85.7%). 69 of 69 patients with colorectal cancer who received both SEPT9 and FIT examination showed 25 FIT-negative colorectal cancer, and FIT detected 12 SEPT9-negative colorectal cancer. The detection performance of SEPT9 for colorectal cancer is superior to that of FIT, but the detection rate of the progression adenoma is similar to that of FIT. Conclusion: The apoptosis of colon cancer cells induced by E2 is dependent on the mismatch repair protein hMLH1, and the hMLH1 protein can enhance the apoptosis of colon cancer cells induced by E2, but this process may be independent of mismatch identification and repair capacity of the mismatch repair system. The results suggest that the patient's mismatch repair protein status should be assessed before the estrogen replacement therapy or the use of an estrogen analog to prevent colorectal cancer to maximize the benefit/ risk ratio. The sensitivity and specificity of SEPT9 in the detection of colorectal cancer are higher than that of FIT, which can be used for screening and early diagnosis of colorectal cancer. but its sensitivity to the progression of the adenoma is poor. The two methods of SEPT9 and FIT are complementary in the detection of colorectal cancer, and the detection of colorectal cancer by combination of these two methods can improve the detection rate.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.3
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相关期刊论文 前4条
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