子宫内膜癌中抑癌基因FHIT第5和第8外显子纯合性缺失与蛋白表达的研究
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摘要
目的 建立检测子宫内膜癌组织中FHIT基因第5和第8外显子纯合性缺失的方法，研究子宫内膜癌组织中FHIT基因5、8外显子纯合性缺失及其蛋白表达，以及与子宫内膜癌临床病理参数的关系，探讨FHIT基因外显子5、8纯合性缺失与蛋白表达的关系及其在子宫内膜癌发病机制中的作用。
方法 采用PCR法检测54例子宫内膜癌及22例癌旁正常组织、46例正常增生期子宫内膜的FHIT外显子5、8纯合性缺失情况；采用免疫组化SP法检测上述122例病例FHIT蛋白表达的情况；对FHIT基因外显子5、8纯合性缺失和蛋白表达与子宫内膜癌临床病理参数的关系进行分析。
结果 54例子宫内膜癌中外显子5的纯合性缺失率为20％(9／54)，外显子8的纯合性缺失率为9％(5／54)，有2例外显子5，8均缺失；FHIT外显子5、8纯合性缺失率为26％(14／54)，在癌旁正常组织和正常增生期子宫内膜中未发现外显子5、8纯合性缺失，三组相比有显著性差异(P＜0.05)。子宫内膜癌中FHIT蛋白表达降低和／或缺失率为35％(19／54)，在癌旁正常组织和正常增生期子宫内膜中未发现FHIT蛋白降低和／或缺失，三组相比有显著性差异(P＜0.05)，FHIT基因外显子5、8纯合性缺失和蛋白表达降低和／或缺失有关。FHIT基因外显子5、8纯合性缺失和蛋白表达降低和／或缺失仅见于子宫内膜样癌，在临床少见的浆液性癌和透明细胞癌中均未发现。FHIT基因外显子缺失和蛋白表达改变与年龄、是否绝经、肥胖、高血压、未产等雌激素相关特征及组织分化程度、FIGO分期、肌层浸润、淋巴结转移、宫颈／附件／腹膜浸润无明显关系。
结论 FHIT外显子5、8纯合性缺失是FHIT基因失活的重要方式之一，由于FHIT外显子5、8纯合性缺失导致FHIT蛋白表达降低和／或缺失。FHIT外显子5、8纯合性缺失及FHIT蛋白表达降低和／或缺失可能在子宫内膜癌的发生、发展中起重要作用，但与子宫内膜癌的临床病理参数无明显关系。FHIT外显子5、8纯合性缺失及FHIT蛋白表达降低和／或缺失仅见于子宫内膜样癌，但与子宫内膜样癌相关特征无明显关系。FHIT基因突变可能是通过非激素途
天津医科大学硕士研究生学位论文
径介导子宫内膜癌的发病机制。
Objective To investigate the homozygous deletions of exon5 and exon5 and protein expression of the tumor-suppressor gene FHIT in endometrial carcinomas and its relations with histopathological features.To explore the potential role of FHIT in the development and progress of endometrial carcinomas. Methods The homozygous deletions of exon5 and exon5 of FHIT gene was detected in 54 cancer samples of EC, 22 corresponding contiguous normal tissues and 46 normal endometrias by PCR. The protein expression of FHIT was detected in the three series by immunohistochemistry.
Results The homozygous deletions of FHIT exon5,8 was observed in 14 out of 54 rumors, with a HDs rate of 26%. The rate of exon5 is 20%(9/54), the rate of exon5 is 9%(5/54); Both HDs of exon5 and exon5 was observed in 2 tumors. The HDs rate of FHIT gene in endometrial carcinomas (26%) was significantly higher than that in corresponding contiguous normal tissues (0/22)and normal endometrias(0/46) (P<0.05). Abnormal FHIT expression (reduced or absent) was noted in 35%( 19/54) of tumor cases, no corresponding contiguous normal tissues and normal endometrias showed abnormal FHIT expression. The relationship of HDS and expression was statistically significant(P=0.000 < 0.05).The homozygous deletions of FHIT exon5,8 and abnormal FHIT expression only were observed in EEC, not in NEEC, but there was no statistically significant differences in them(P>0.05). There was no relationship between FHIT gene HDS or expression and histopathological features.
Conclusions Loss of FHIT function by HDs or other mechanisms is an event in endometrial tumorigenesis. HDs of FHIT exon5,8 may cause abnormal FHIT expression. HDs of FHIT exon5,8 and abnormal FHIT expression only were observed in EEC, but not related to estrogenic risk factors. FHIT gene alerations may contribute in carcinogenesis in EC, especially in EEC, through an
5
estrogen-independent pathway.
方法 采用PCR法检测54例子宫内膜癌及22例癌旁正常组织、46例正常增生期子宫内膜的FHIT外显子5、8纯合性缺失情况；采用免疫组化SP法检测上述122例病例FHIT蛋白表达的情况；对FHIT基因外显子5、8纯合性缺失和蛋白表达与子宫内膜癌临床病理参数的关系进行分析。
结果 54例子宫内膜癌中外显子5的纯合性缺失率为20％(9／54)，外显子8的纯合性缺失率为9％(5／54)，有2例外显子5，8均缺失；FHIT外显子5、8纯合性缺失率为26％(14／54)，在癌旁正常组织和正常增生期子宫内膜中未发现外显子5、8纯合性缺失，三组相比有显著性差异(P＜0.05)。子宫内膜癌中FHIT蛋白表达降低和／或缺失率为35％(19／54)，在癌旁正常组织和正常增生期子宫内膜中未发现FHIT蛋白降低和／或缺失，三组相比有显著性差异(P＜0.05)，FHIT基因外显子5、8纯合性缺失和蛋白表达降低和／或缺失有关。FHIT基因外显子5、8纯合性缺失和蛋白表达降低和／或缺失仅见于子宫内膜样癌，在临床少见的浆液性癌和透明细胞癌中均未发现。FHIT基因外显子缺失和蛋白表达改变与年龄、是否绝经、肥胖、高血压、未产等雌激素相关特征及组织分化程度、FIGO分期、肌层浸润、淋巴结转移、宫颈／附件／腹膜浸润无明显关系。
结论 FHIT外显子5、8纯合性缺失是FHIT基因失活的重要方式之一，由于FHIT外显子5、8纯合性缺失导致FHIT蛋白表达降低和／或缺失。FHIT外显子5、8纯合性缺失及FHIT蛋白表达降低和／或缺失可能在子宫内膜癌的发生、发展中起重要作用，但与子宫内膜癌的临床病理参数无明显关系。FHIT外显子5、8纯合性缺失及FHIT蛋白表达降低和／或缺失仅见于子宫内膜样癌，但与子宫内膜样癌相关特征无明显关系。FHIT基因突变可能是通过非激素途
天津医科大学硕士研究生学位论文
径介导子宫内膜癌的发病机制。
Objective To investigate the homozygous deletions of exon5 and exon5 and protein expression of the tumor-suppressor gene FHIT in endometrial carcinomas and its relations with histopathological features.To explore the potential role of FHIT in the development and progress of endometrial carcinomas. Methods The homozygous deletions of exon5 and exon5 of FHIT gene was detected in 54 cancer samples of EC, 22 corresponding contiguous normal tissues and 46 normal endometrias by PCR. The protein expression of FHIT was detected in the three series by immunohistochemistry.
Results The homozygous deletions of FHIT exon5,8 was observed in 14 out of 54 rumors, with a HDs rate of 26%. The rate of exon5 is 20%(9/54), the rate of exon5 is 9%(5/54); Both HDs of exon5 and exon5 was observed in 2 tumors. The HDs rate of FHIT gene in endometrial carcinomas (26%) was significantly higher than that in corresponding contiguous normal tissues (0/22)and normal endometrias(0/46) (P<0.05). Abnormal FHIT expression (reduced or absent) was noted in 35%( 19/54) of tumor cases, no corresponding contiguous normal tissues and normal endometrias showed abnormal FHIT expression. The relationship of HDS and expression was statistically significant(P=0.000 < 0.05).The homozygous deletions of FHIT exon5,8 and abnormal FHIT expression only were observed in EEC, not in NEEC, but there was no statistically significant differences in them(P>0.05). There was no relationship between FHIT gene HDS or expression and histopathological features.
Conclusions Loss of FHIT function by HDs or other mechanisms is an event in endometrial tumorigenesis. HDs of FHIT exon5,8 may cause abnormal FHIT expression. HDs of FHIT exon5,8 and abnormal FHIT expression only were observed in EEC, but not related to estrogenic risk factors. FHIT gene alerations may contribute in carcinogenesis in EC, especially in EEC, through an
5
estrogen-independent pathway.
引文
1．糜若然主编．妇产科疾病诊断治疗学．北京：中国医药科技出版社，2000，741
2. Ohta M, Inoue H,Grazia M, et al.The FHIT Gene, Spanning the Chromosome 3p14.2 Fragile Site and Renal Carcinoma-Associated t(3;8) Breakpoint, Is Abnormal in Digestive Tract Cancers. Cell, 1996, 84:587-597
3. Xiao GH , Jin F, Klein-Szanto AJ,et al. The FHIT gene product is highly expressed in the cytoplasm of renal tubular epithelium and is down-regulated in kidney cancers.Am J Pathol, 1997; 151:1541-1547
4. Barnes L, Garrison P, Siprashvili Z, et al. Fhit,a Putative Tumor Suppressor in Humans, Is a Dinucleoside 5' , 5'" -P~1, p~3-Triphosphate Hydrolase.Biochemistry, 1996,35:11529-11535
5. Siprashvili Z, Sozzi G, Barnes L, et al. Replacement of Fhit in cancer cells suppresses tumorigencity. Proc Natl Acad Sci USA, 1997,94:13711-13776
6. Pace HC, Garrison PN, Robinson AK, et al. Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit. Proc Natl Acad Sci USA,1998,95:5484-5489
7. Sard L, Accornero P, Tomielli S, et al. The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci USA, 1999, 96:8489-8492
8. Roz L, Gramegna M, Ishii H, et al. Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppress tumorigenicity in lung and cervical cancer cell lines. Proc Natl Acad Sci USA, 2002, 99:3615-3620
9. Ji L, Fang B, Yen N, Roz L, Gramegna M, Ishii H,et al. Induction of Apoptosis and Inhibition of Tumorigencity and Tumor Growth by Adenovirus Vector-mediated Fragile Histidine Triad(FHIT). Cancer
Research, 1999,59:3333-3339
10. Otterson G, Xiao G, Geradts J, et al. Protein Expression and Functional Analysis of the FHIT Gene in Human Tumor cells. J Natl Cancer hast, 1998,90:426-432
11. SHI Y, Zou M, Farid NR, et al. Association of FHIT (fragile histidine triad ), a candidate tumor suppressor gene, with the ubiquitin-conjugating enzyme hUBC9. Biochem J, 2000, 352:443-448
12. Chaudhuri AR, Khan IA, Prasad V, et al. The Tumor Suppressor Protein Fhit. J Biol Chem, 1999, 274:24378-24382
13. Wistuba I, Montellano F, Milchgrub S, et al. Deletion of Chromosome 3p Are Frequent and Early Events in the Pathogenesis of Uterine Cervical Carcinoma. Cancer Research, 1997,57:3154-3158
14. Yoshino K, Enomoto T, Nakamura T, et al.Aberrant FHIT Transcripts in Squamous Cell Carcinoma of the Uterine Cervix.Int J Cancer, 1998,76:176-181
15. Helland A, Kraggerud SM, Kristensen GB, et al. Primary cervical carcinomas show 2 common regions of deletion at 3p,1 within the FHIT gene: evaluation of allelic imbalance at FHIT, RB1 and TP53 in relation to survival, Int J Cancer, 2000, 88:217
16. Greenspan D, Connolly D, Wu R, et al. Loss of FHIT Expression in cervical Carcinoma Cell lines and Primary Tumors. Cancer Research, 1997,57:4692-4698
17. Yoshino K, Enomoto T, Nakamura T, et al. FHIT alterations in cancerous and non-cancerous cervical epithelium. Int J Cancer, 2000, 85:6-13
18. Wu Q, Shi H, Suo Z, et al. 5'-CpG Island Methylation of the FHIT Gene is Associated with Reduced Protein Expression and Higher Clinical Stage in Cervical Carcinomas. Ultrastruct Pathol. 2003, 27(6):417-22
19. Sozzi G, Pastorino U, Moiraghi L, et al. Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res, 1998,58:5032-5037
20. Negrini M, Monaco C, Vorechovsky I, et al. The FHIT gene at 3p14.2 is abnormal in breast carcinomas. Cancer Res, 1996,56:3173-3179
21. Ohta M, Inoue H, Cotticelli,et al. The FHIT gene,spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancer. Ce11,1996,84:587-597
22. Mao L, Fan, Y-H, et al. Frequent abnormalities of FHIT, a candidate tumor suppressor gene, in head and neck cancer cell lines. Cancer Res, 1996,56:5128-5131
23. Larson A, Kern S, Curtiss S, et al. High resolution analysis of chromosome 3p alterations in cervical carcinoma. Cancer Res, 1997,57:4082-4090
24. Buttitta F, Marchetti A, Radi O, et al. Evaluation of FHIT gene alterations in ovarian cancer. British J Cancer,1998,77:1048-1051
25. Ozaki K, Enomoto T, Yoshino K, et al. FHIT alterations in endometrial carcinoma and hyperplasia. Int J Cancer,2000,85:306-312
26. Ishii H, Dumon KR., Vecchione A, et al. Potential cancer therapy with the fragile histidine triad gene .JAMA,2001, 286:2441-2449
27. Bulter D, Sc M, Collins C, et al. Loss of Fhit as a potential marker of malignant progression in preinvasive cervical cancer. Gynecologe Oncologe,2002,86:144-149
28. Kdvak T, McBroom J, Seidman J,et al. Abnormal Fragile Histidine Triad(FHIT) Expression in Advanced Cervical Carcinoma:A Poor Prognostic Factor. Cancer Res,2001,61:4382-4385
29. Terry G, Ho L, Londesborough P, et al. Abnormal FHIT expression profiles in cervical intraepithelial neoplastic(CIN) lesions. Bri J
Cancer,2002,86:376-381
30. Su T, Wang J, Tseng H, et al. Analysis of FHIT transcripts in cervical and endometrial cancers. Int J Cancer, 1998,76:216-222
31. Ozaki K, Enomoto T, Yoshino S, et al. FHIT alterations in endometrial carcinoma and hyperplasia. Int J Cancer,2000,85:306-312
32. Hendricks D, Taylor R, Reed M,et al. FHIT Gene Expression in Human Ovarian, Endometrial, and Cervical Cancer Cell Lines. Cancer Res,1997,57:2112-2115
33. Druck T, Hadaczek P, Fu T, et al. Structure and Expression of the Human FHIT Gene in Normal and Tumor Cells. Cancer Res, 1997,57:504-512
34．孙立峰，周青，汪渊等抑癌基因FHIT第8和第5外显子在原发性肝癌中纯合性缺失与点突变的研究 实用肿瘤杂志 2002，17：308-310
35．周清华 陈军 覃扬等 人非小细胞肺癌中FHIT等位基因缺失和突变的研究 中国肺癌杂志 2001，4：10-14
36. Yura Y, Mandai M, Konishi I, et al. Loss of Fhit protein expression in high-grade and advanced stage endometrial. Anticancer Res,2003,23(3C):2837-43
37. Hadaczek P, Gronwald J, Chosia M, et al. Fhit protein expression in endometrial cancers: no correlation with histological grade. Pol J Pathol, 2001,52(4): 199-203.
38. Segawa T, Sasagawa T, Saijoh K, et al. Clinicopathological significance of fragile histidine triad transcriptio protein expression in endometrial carcinomas. Clin Cancer Res. 2000,6(6):2341-8
39. Oehler MK, Brand A, Wain GV. Molecular genetics and endometrial cancer. J Br Menopause Soc. 2003,9(1):27-31
40. Mark E. Sherman M.D. Theories of Endometrial Carcinogenesis: A Multidisciplinary Approach. Mod Pathol .2000,13(3):295-308
41. Maxwell GL, Risinger JI, Alvarez AA, et al. Favorable survival associated with microsatellite instability in endometrioid endometrial cancers. Obstet Gynecol. 2001 , 97(3):417-22
2. Ohta M, Inoue H,Grazia M, et al.The FHIT Gene, Spanning the Chromosome 3p14.2 Fragile Site and Renal Carcinoma-Associated t(3;8) Breakpoint, Is Abnormal in Digestive Tract Cancers. Cell, 1996, 84:587-597
3. Xiao GH , Jin F, Klein-Szanto AJ,et al. The FHIT gene product is highly expressed in the cytoplasm of renal tubular epithelium and is down-regulated in kidney cancers.Am J Pathol, 1997; 151:1541-1547
4. Barnes L, Garrison P, Siprashvili Z, et al. Fhit,a Putative Tumor Suppressor in Humans, Is a Dinucleoside 5' , 5'" -P~1, p~3-Triphosphate Hydrolase.Biochemistry, 1996,35:11529-11535
5. Siprashvili Z, Sozzi G, Barnes L, et al. Replacement of Fhit in cancer cells suppresses tumorigencity. Proc Natl Acad Sci USA, 1997,94:13711-13776
6. Pace HC, Garrison PN, Robinson AK, et al. Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit. Proc Natl Acad Sci USA,1998,95:5484-5489
7. Sard L, Accornero P, Tomielli S, et al. The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci USA, 1999, 96:8489-8492
8. Roz L, Gramegna M, Ishii H, et al. Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppress tumorigenicity in lung and cervical cancer cell lines. Proc Natl Acad Sci USA, 2002, 99:3615-3620
9. Ji L, Fang B, Yen N, Roz L, Gramegna M, Ishii H,et al. Induction of Apoptosis and Inhibition of Tumorigencity and Tumor Growth by Adenovirus Vector-mediated Fragile Histidine Triad(FHIT). Cancer
Research, 1999,59:3333-3339
10. Otterson G, Xiao G, Geradts J, et al. Protein Expression and Functional Analysis of the FHIT Gene in Human Tumor cells. J Natl Cancer hast, 1998,90:426-432
11. SHI Y, Zou M, Farid NR, et al. Association of FHIT (fragile histidine triad ), a candidate tumor suppressor gene, with the ubiquitin-conjugating enzyme hUBC9. Biochem J, 2000, 352:443-448
12. Chaudhuri AR, Khan IA, Prasad V, et al. The Tumor Suppressor Protein Fhit. J Biol Chem, 1999, 274:24378-24382
13. Wistuba I, Montellano F, Milchgrub S, et al. Deletion of Chromosome 3p Are Frequent and Early Events in the Pathogenesis of Uterine Cervical Carcinoma. Cancer Research, 1997,57:3154-3158
14. Yoshino K, Enomoto T, Nakamura T, et al.Aberrant FHIT Transcripts in Squamous Cell Carcinoma of the Uterine Cervix.Int J Cancer, 1998,76:176-181
15. Helland A, Kraggerud SM, Kristensen GB, et al. Primary cervical carcinomas show 2 common regions of deletion at 3p,1 within the FHIT gene: evaluation of allelic imbalance at FHIT, RB1 and TP53 in relation to survival, Int J Cancer, 2000, 88:217
16. Greenspan D, Connolly D, Wu R, et al. Loss of FHIT Expression in cervical Carcinoma Cell lines and Primary Tumors. Cancer Research, 1997,57:4692-4698
17. Yoshino K, Enomoto T, Nakamura T, et al. FHIT alterations in cancerous and non-cancerous cervical epithelium. Int J Cancer, 2000, 85:6-13
18. Wu Q, Shi H, Suo Z, et al. 5'-CpG Island Methylation of the FHIT Gene is Associated with Reduced Protein Expression and Higher Clinical Stage in Cervical Carcinomas. Ultrastruct Pathol. 2003, 27(6):417-22
19. Sozzi G, Pastorino U, Moiraghi L, et al. Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res, 1998,58:5032-5037
20. Negrini M, Monaco C, Vorechovsky I, et al. The FHIT gene at 3p14.2 is abnormal in breast carcinomas. Cancer Res, 1996,56:3173-3179
21. Ohta M, Inoue H, Cotticelli,et al. The FHIT gene,spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancer. Ce11,1996,84:587-597
22. Mao L, Fan, Y-H, et al. Frequent abnormalities of FHIT, a candidate tumor suppressor gene, in head and neck cancer cell lines. Cancer Res, 1996,56:5128-5131
23. Larson A, Kern S, Curtiss S, et al. High resolution analysis of chromosome 3p alterations in cervical carcinoma. Cancer Res, 1997,57:4082-4090
24. Buttitta F, Marchetti A, Radi O, et al. Evaluation of FHIT gene alterations in ovarian cancer. British J Cancer,1998,77:1048-1051
25. Ozaki K, Enomoto T, Yoshino K, et al. FHIT alterations in endometrial carcinoma and hyperplasia. Int J Cancer,2000,85:306-312
26. Ishii H, Dumon KR., Vecchione A, et al. Potential cancer therapy with the fragile histidine triad gene .JAMA,2001, 286:2441-2449
27. Bulter D, Sc M, Collins C, et al. Loss of Fhit as a potential marker of malignant progression in preinvasive cervical cancer. Gynecologe Oncologe,2002,86:144-149
28. Kdvak T, McBroom J, Seidman J,et al. Abnormal Fragile Histidine Triad(FHIT) Expression in Advanced Cervical Carcinoma:A Poor Prognostic Factor. Cancer Res,2001,61:4382-4385
29. Terry G, Ho L, Londesborough P, et al. Abnormal FHIT expression profiles in cervical intraepithelial neoplastic(CIN) lesions. Bri J
Cancer,2002,86:376-381
30. Su T, Wang J, Tseng H, et al. Analysis of FHIT transcripts in cervical and endometrial cancers. Int J Cancer, 1998,76:216-222
31. Ozaki K, Enomoto T, Yoshino S, et al. FHIT alterations in endometrial carcinoma and hyperplasia. Int J Cancer,2000,85:306-312
32. Hendricks D, Taylor R, Reed M,et al. FHIT Gene Expression in Human Ovarian, Endometrial, and Cervical Cancer Cell Lines. Cancer Res,1997,57:2112-2115
33. Druck T, Hadaczek P, Fu T, et al. Structure and Expression of the Human FHIT Gene in Normal and Tumor Cells. Cancer Res, 1997,57:504-512
34．孙立峰，周青，汪渊等抑癌基因FHIT第8和第5外显子在原发性肝癌中纯合性缺失与点突变的研究 实用肿瘤杂志 2002，17：308-310
35．周清华 陈军 覃扬等 人非小细胞肺癌中FHIT等位基因缺失和突变的研究 中国肺癌杂志 2001，4：10-14
36. Yura Y, Mandai M, Konishi I, et al. Loss of Fhit protein expression in high-grade and advanced stage endometrial. Anticancer Res,2003,23(3C):2837-43
37. Hadaczek P, Gronwald J, Chosia M, et al. Fhit protein expression in endometrial cancers: no correlation with histological grade. Pol J Pathol, 2001,52(4): 199-203.
38. Segawa T, Sasagawa T, Saijoh K, et al. Clinicopathological significance of fragile histidine triad transcriptio protein expression in endometrial carcinomas. Clin Cancer Res. 2000,6(6):2341-8
39. Oehler MK, Brand A, Wain GV. Molecular genetics and endometrial cancer. J Br Menopause Soc. 2003,9(1):27-31
40. Mark E. Sherman M.D. Theories of Endometrial Carcinogenesis: A Multidisciplinary Approach. Mod Pathol .2000,13(3):295-308
41. Maxwell GL, Risinger JI, Alvarez AA, et al. Favorable survival associated with microsatellite instability in endometrioid endometrial cancers. Obstet Gynecol. 2001 , 97(3):417-22