摘要
目的
检测KLK9和KLK14基因mRNA及其蛋白在正常卵巢、卵巢交界性瘤和卵巢癌组织中的表达差异,并分析KLK9和KLK14基因与卵巢癌临床病理特征的关系及其临床意义。观察雌、孕和雄激素对卵巢癌细胞系中KLK9和KLK14基因mRNA及蛋白表达的影响,以及诱导卵巢癌细胞增殖和凋亡作用。探讨KLK9、KLK14基因和雌、孕及雄激素在卵巢癌发生和侵袭中作用及相关分子机理,为卵巢癌的基因诊断和药物治疗提供科学的实验依据。
方法
一.应用半定量RT-PCR方法,检测55例卵巢癌及其正常对照组织中KLK9和KLK14基因mRNA表达,探讨并分析其与卵巢癌临床病理特征的关系。
二.采用SP免疫组化和/或Western Blot印迹分析法,检测正常卵巢(n=55)、卵巢交界性肿瘤(n=16)、卵巢癌(n=55)组织中KLK9和KLK14蛋白的表达。分析KLK9和KLK14蛋白表达与卵巢癌临床病理特征的关系。
三.雌、孕和雄激素作用SKV卵巢癌细胞系24、48、72小时后,应用Bacton Dickonson FACScan流式细胞仪,测定其细胞周期和细
ObjectiveTo test the expression differences of KLK9 and KLK14 gene mRNA and proteins in normal,borderline and malignant ovarian tissues.To analyze the relationship between KLK9 、 KLK14 gene and clinicopathological features of ovarian cancer. To observe the effect of estrogen,progestogen and androgen on the expression of KLK9 and KLK14 gene mRNA in ovarian cancer cell lines; and the effect of induction to the cancer cell proliferation and apoptosis. To explore their roles in tumorgenesis and metastasis of ovarian cancers,to supply an experimental basis for the gene diagnosis and medical treatment of the ovarian cancers.Methods1 、 KLK9 and KLK14 gene mRNA were examined in ovarian cancers (n=55)and the control group (n=55 ) by semiquantitative RT-PCR.2、 KLK9 and KLK14 protein expression were examined in normal ovaries(n=55),borderline ovarian diseases(n=16) and ovarian cancers (n=55) by means of immunohistochemical SP method and Western Blot.3、 We use Bacton Dickonson FACS to test the cell cycles and apoptosis of the SKV cell lines after the use of estrogen、 progestogen and
androgen for 24、 48、 72 hours.4、 We examine the different expression of KLK9 and KLK14gene mRNA and protein in SKV cell lines after the use of estrogen 、 proestogen and androgen by RT-PCR and Western Blot.Results1、 The expression of KLK9 gene mRNA and protein was significantly lower in ovarian cancers than that in normal ovarians (p<0.05 ) .The expression of KLK9 gene mRNA and protein in early stage of ovarian cancer was significantly higher than that in late stage .(p<0.01), but the expression has no relationship with the pathological stages and histological differentiation of ovarian cancers.2、 The expression of KLK14 gene mRNA and protein increased gradually in normal ovaries,borderline tumors and ovarian cancers;and it is higher in borderline and carcinomatous tissues than that in normal ovary tissues (P<0.05). The expression of KLK14 gene mRNA was closely related with the clinicopathological features of ovarian cancers. The experiments showed that the expression of KLK14 gene mRNA and protein was higher in late stage than that in early stage of ovarian cancers, and higher in low differentiated ones than that of in high differentiated cancers. And the expression levels have no relationship with the different histological type.
3、 Compared with the control group ,after the induction of estrogen, progestogen and androgen to the ovarian cancer call lines, in estrogen group cells in S phase increased obviously while cells in G2/M phase decreased.(P<0.0l)4、 compared with the control group, the cell lines showed increased expression of KLK9 gene mRNA and protein after the use of estrogen and increased expression of KLK14 gene mRNA and protein after the use of progestogen and increased expression of KLK14gene mRNA and protein after the use of androgen.Conclusions1 、 The down-regulated expression of KLK9 gene mRNA and protein existed in the ovarian cancers. It was correlated with the clinicopathological stage of the ovarian cancer and can be a monitoring marker for the prognosis of ovarian cancer2、 The up-regulated expression of KLK14 gene mRNA and protein existed in the ovarian cancers.It was closely related with the clinicopathological stage and differentiation degree of the cancer,and can be a biomarker to predict the biological behavior.3、 Estrogen,progestogen and androgen are closely related with the progression of the ovarian cancers.Progestogen can enhance the apoptosis of the ovarian cancer cells and inhibit the growth of the cancer cells.The induction of these three hormones to the cancer cells can be a guidance to
引文
1. Diamandis EP, Yousef GM, Luo LY, et al.The new human kallikrein gene family: implications in carcinogenesis[J]. Trends Endocrinol Metab. 2000,11(2):54-60.
2. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease[J].Endocr Rev. 2001,22(2): 184-204.
3. Yousef GM, Diamandis EP. The expanded human kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9)[J].Genomics. 2000, 65(2):184-94.
4. Magklara A, Scorilas A, Katsaros DJ, et al. The human KLK8(neuropson/ovasin) gene: identification of two novel splice variants and its prognostic value in ovarian cancer[J].Cancer Res. 2001, 7:806-811.
5.武春龙,赵玉兰,吴华星等.C-erbB-3在肺癌中的表达及临床意义.中华癌症姑息医学杂志[J].2003,2(2),94-6.
6. Meyer T, Rustin GJ.Role of tumour markers in monitoring epithelial ovarian cancer[J].Br J Cancer. 2000,82(9):1535-8.
7. George M, Yousef, Lianna G, et al. Quantitative expression of the human kallikrein gene (KLK9) in ovarian cancer: A new independent and favorable prognostic marker[J].Cancer Res.2001,61(21):7811-18.
8. Fortier AH, Nelson BJ, Grella DK, et al. Antiangiogenic activity of prostate-specific antigen[J].J Natl Cancer Inst. 1999,91(19): 1635-40.
9. Diamandis EP. Prostate-specific antigen or human kallikrein 3. Recent developments[J] .Tumour Biol. 1998;19(2):65-7; discussion 67-8.
10.Goyal J, Smith KM, Cowan JM,et al. The role for NES1 serine protease as a novel tumor suppressor [J]. Cancer Res. 1998,58(21):4782-6.
11.Tanimoto H, Underwood LJ, Shigemasa K,et al. The stratum corneum chymotryptic enzyme that mediates shedding and desquamation of skin cells is highly overexpressed in ovarian tumor cells [J]. Cancer. 1999,86(10):2074-82.
12. Kim H, Scorilas A, Katsaros D, et al. Human kallikrein gene 5 (KLK5) expression is an indicator of poor prognosis in ovarian cancer[J].Br J Cancer. 2001,84(5):643-50.
13. Yousef GM, Kyriakopoulou LG, Scorilas A, et al. Quantitative expression of the human kallikrein gene 9 (KLK9) in ovarian cancer: a new independent and favorable prognostic marker[J]. Cancer Res. 2001,61(21):7811-8.
14. Underwood LJ, Tanimoto H, Wang Y, et al. Cloning of tumor-associated differentially expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma [J]. Cancer Res. 1999,59(17):4435-9.
1. Yousef GM, Magklara A, Chang A. Cloning of a new member of the human kallikrein gene family , KLK14 , which is down-regulated in different malignancies. Canc Res, 2001, 61: 3425-3431
2. Hooper JD , Bui LT, Rae FK, et al. Identification and charicterization of KLK14, a novel Kallikrein serine protease gene located on human chromosome19q13.4 and expressed in prostate and skeletal muscle. Genomics. 2001, 73: 117-122.
3. Diamandis EP, Yousef GM. Human tissue kallikrein gene family : A rich source of novel disease biomarkers. Expert Rev Mol Diagnl.2001, 1: 182-190.
4. Yousef GM, Polymeris MK, Yacoub GM , et al. Parallel Overexpression of Seven Kallikrein Genes in Ovarian Cancer. Cancer Research 2003, 63: 2223-2227.
5. Dong Y, Kaushal A, Brattsand M, et al. Differential Splicing of KLK5 and KLK7 in Epithelial Ovarian Cancer Produces Novel Variants with Potential as Cancer Biomarkers. Clin Cancer Res, 2003, 9(5) : 1710-1720.
6. Yousef GM, Diamandis EP. Kallikreins, steroid hormones and ovarian cancer: is there a link? Minerva Endocrinol. 2002, 27: 157-166.
7. Dong Y, Kaushal A, Bui L, et al. Human kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas. Clin. Cancer Res. 2001, 7: 2363-2371.
8. Obiezu CV, Scorilas A, Katsaros D, et al. Higher human kallikrein gene 4 (klk4) expression indicates poor prognosis of ovarian cancer patients. Clin. Cancer Res. 2001, 7: 2380-2386.
9. Kim H., Scorilas A., Katsaros D. , Yousef G. M, et al. Human kallikrein gene 5 (KLK5) expression is an indicator of poor prognosis in ovarian cancer. Br. J. Cancer. 2001 , 84: 643-650.
10. Luo L. Y, Katsaros D, Scorilas A, et al. Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma. Clin. Cancer Res. 2001, 7: 2372-2379.
11. Luo L, Bunting P, Scorilas A, Diamandis E. P. Human kallikrein 10: a novel tumor marker for ovarian carcinoma?. Clin Chim Acta. 2001, 306: 111-118.
12. Diamandis EP, Okui A, Mitsui S, et al. Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma. Cancer Res. 2002, 62: 295-300.
13. Anisowicz A, Sotiropoulou G, Stenman G, et al. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol. Med. 1996, 2: 624-636.
14. Tanimoto H, Underwood LJ, Shigemasa K, et al. Increased expression of protease M in ovarian tumors. Tumour Biol. 2001, 22: 11-18.
15. Underwood LJ, Tanimoto H, Wang Y, et al. Cloning of tumor-associated differentially expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma. Cancer Res. 1999, 59: 4435-4439.
16. Tanimoto H, Underwood LJ, Shigemasa K, et al. The stratum corneum chymotryptic enzyme that mediates shedding and desquamation of skin cells is highly overexpressed in ovarian tumor cells. Cancer (Phila. ). 1999, 86: 2074-2082.
17. Yousef GM, Borgono CA, et al. Quantitative analysis of human kallikrein gene 14 expression in breast tumors indicates association with poor prognosis. British Journal of Cancer.2002, 87 (11): 1287-1293.
18. Rittenhouse HG, Finlay JA, Mikolajczyk SD, et al. Human kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate. Crit. Rev. Clin. Lab. Sci. 1998, 35: 275-368.
19. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr Rev. 2001, 22: 184-204.
20. Dong Y, Kaushal A, Bui L, et al. Human kallikrein 4 (KLK4) is highly expressed in serous ovarian carcinomas. Clin Cancer Res. 2001, 7: 2363-2371.
21. Obiezu CV, Scorilas A, Katsaros D, et al. Higher human kallikrein gene 4 (klk4) expression indicates poor prognosis of ovarian cancer patients. Clin Cancer Res. 2001, 7: 2380-2386.
22. Diamandis EP, Yousef GM, Soosaipillai AR., et al. Human kallikrein 6 (zyme/protease M/neurosin): a new serum biomarker of ovarian carcinoma. Clin. Biochem. 2000, 33: 579-583.
23. Luo L, Bunting P, Scorilas A, et al. Human kallikrein 10: a novel tumor marker for ovarian carcinoma?. Clin Chim Acta. 2001, 306: 111-118,.
24. Diamandis EP, Okui A, Mitsui S, et al. Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma. Cancer Res. 2002, 62: 295-300.
1. Guzeloglu KO, Kayisli UA, Al-Rejjal R, et al. Regulation of PTEN (phophatase and tensin homolog deleted on chromosome 10 ) expression by estradiol and progesterong in human endometrium[J]. J Clin Endocrinol Metab, 2003, 88: 5017-5026.
2. Franke HR, Kole S, Ciftei Z, et al. In vitro effects of estradiol, dytrogesterong, tamoxifen and cyclophosphamide on proliferation vs. death in human breast cancer cells[J]. Cancer Lett, 2003, 190: 113-118.
3. Godwin AK, Testa JR, Hamilton TC. The biology of ovarian cancer development. Cancer (Phila) 1993,71;530-536.
4. Holschneider CH, Berek JS. Ovarian cancer: epidemiology, biology, and prognostic factors. Semin. Surg Oncol.2000, 19: 3-10.
5. Hackenberg R, Luttchens S, Hofmann J, et al.Androgen sensitivity of the new human breast cancer cell line MFM-223.Cancer Res. 1991, 51, 5722-5727.
6. Thongngram T , Jenkins JK , Mmurray RW. Estrogen and peogesterong differentially modulate cell cycling , apoptosis, and response to lipopolysaccharide in monocyte cell [J].Allergy Clin Immunol, 2002, 109 (1) S233.
7. Mabuchi S, Ohmichi M, et al. Estrogen and paclitaxel-induced