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IGFBP7 and Its Fuctions 浙江大学 来茂德等 IGFBP7 的 抗肿瘤效应 IGFBP7 的 抗肿瘤效应.

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Presentation on theme: "IGFBP7 and Its Fuctions 浙江大学 来茂德等 IGFBP7 的 抗肿瘤效应 IGFBP7 的 抗肿瘤效应."— Presentation transcript:

1 IGFBP7 and Its Fuctions 浙江大学 来茂德等 IGFBP7 的 抗肿瘤效应 IGFBP7 的 抗肿瘤效应

2 结直肠癌发生与发展 5q APC 12p Ki-ras 18q Smad4DCC Increase of genetic variation Accumulation of 10 4 - 10 12 mutations Chromosomal gains or losses 10 -2 per chromosome per generation EarlyadenomaLateadenomaInvasivecarcinomaMetastaticcarcinomaNormalepithelium 17p p53Othermutations 5q APC 12p Ki-ras 18q Smad4DCC MMR Deficiency 错配修复缺陷 Vogelstein 模型 1990

3 In 1999, by SSH, We constructed three libraries normal adenomacancer APC, etc.P53, etc. T NA

4 The derivation of IGFBP7 The derivation of IGFBP7 T N B2 TTTT B2 IGFBP7 97% tester driver World J Gastroenterol 2001;7(5):726-31 A B C D E M 5’-RACE

5 Gene card of IGFBP7 http://www.genecards.org/cgi-bin/carddisp.pl?gene=IGFBP7 Gene description: Gene description: insulin-like growth factor binding protein 7, belongs to the IGFBP family insulin-like growth factor binding protein 7, belongs to the IGFBP family Gene aliases: FSTL2, IGFBP-rP1, MAC25, PSF, AGM,TAF Gene aliases: FSTL2, IGFBP-rP1, MAC25, PSF, AGM,TAF Location:4q12 Location:4q12 282 AA 3272107156 160 264 IGFBP likeKazal like Ig like Signal peptide

6 I: IGFBP7 in colon cancer tissue I: IGFBP7 in colon cancer tissue IGFBP7 在癌组织中的表达

7 F=6.29 , p=0.000; spearman’s rho=0.40,p=0.000 P25 N B A T 0 0.5 1 1.5 2 IGFBP7 AU N B A T p75 p25 Expression of IGFBP7 at mRNA level in colorectal carcinomas (T), adenomas(A), tissue adjacent to tumor(B),and normal tissues(N) 从正常腺瘤到癌表达渐升高

8 T vs. N ,  2=12.05 ( df=1 ), p<0.01 Immunohistochemistry result 11 65 32 46 30 85.5% 60% 60.5%

9 IHC of IGFBP7 in B and T in the same sample

10 Clin Cancer Res. 2007;13(17):5082-8 Overexpression of IGFBP7 correlated with favourable prognosis in CRC patients IGFBP7 高表达预后好

11 LoVo HCT8 SW1116 RKO HT29 COLO205 Hce8693 CW2 SW620 SW480 Caco2 Control Marker GAPDH IGFBP7 IGFBP7 has a low expression in CRC cell lines

12 II: The regulation mechanism of II: The regulation mechanism of IGFBP7 in colon cancer IGFBP7 in colon cancer

13 Prediction of CpG islands of IGFBP7 http://www.ebi.ac.uk/emboss/cpgblot/#andNewcpgseekhttp://www.ebi.ac.uk/emboss/cpgblot/#andNewcpgseek; http://www.urogene.org/methprimer/index1.html http://www.urogene.org/methprimer/index1.html -355 +803 CpG island: Length >200bp; GC% >50%; Obs./Exp. CpG >0.6 134 the putative promoter region, exon 1 and partial intron 1

14 Analyzing the methylation status of putative promoter region, exon1, intron1 in 10 cell lines using Bisulfite Sequencing PCR (BSP) F1 R1 F2 R2 F3 R3 F4 R4 Putative promoter region exon1 intron1

15 Methylation pattern of putative promoter region HCT8 SW1116 HCE8693 COLO205 HT29 RKO CW2 SW620 SW480 CACO2 HCT8 SW1116 HCE8693 COLO205 HT29 RKO CW2 SW620 SW480 CACO2 -335 -329 -302 -298 -291 -286 -271 -251 -237 -223 -218 -214 -206 -202 -191 -185 -182 -174 -163 -160 -147 -146 -144 -142 -133 -131 -125 -117 -112 -108 -99 -88 -83 -69 -67 -58 -54 -49 -46 -35 -32 -28 -23 -18 -9 -7 methylation unmethylation

16 Intron 1 HCT8 SW1116 HCE8693 COLO205 HT29 RKO CW2 SW620 SW480 CACO2 +505 +509 +529 +532+536+544 +557+560+565 +569 +571+574+580 +585 +587 +599+608 +610+652+654+668 +675+678 +724 +728 +746+772 +801 +812 methylation unmethylation

17 Exon 1 HCT8 SW1116 HCE8693 COLO205 HT29 RKO CW2 SW620 SW480 CACO2 HCT8 SW1116 HCE8693 COLO205 HT29 RKO CW2 SW620 SW480 CACO2 +3 +12 +19 +29 +33 +36 +41 +43 +55 +58 +6 +108 +118 +127 +132+144 +156 +175+182 +184+186+188 +193 +211 +215 +217 +220 +226 +233 +235 +244+247 +250+265 +267+270 +283 +293+302+316 +328+331 +336+346 +349+355+368 +375 +382+388 +391 +405+409+422 +424+427 +442+449 +451+490 methylation unmethylation

18 CW2 Hce8693 SW1116 HCT8 HT29 RKO COLO205 SW620 SW480 Caco2 Control Marker M U M U M U M U M U M U M U M U M U M U M, methylated product; U, unmethylated product; M-P, positive control of methylation; U-P, positive control of unmethylation; C, blank control of PCR system unmethylation Confirmation using MSP IGFBP7(+)

19 Cluster analysis depending on the methylation status of exon 1 IGFBP7+

20 Restoration of IGFBP7 after 5-aza-dC treatment RT-PCR HT29 SW620 COLO205 LoVo Control TreatedControlTreated Immunohistochemistry

21 Methy-and deMethy sites of IGFBP-rP1gene 5’-region - , control ; + , treated cells ; Black :methy-per centage J Pathol. 2007;212(1):83-90. Methylation of IGFBP7 exon 1:the key regulation mechanism silencing IGFBP7 expression in CRC cells 外显子 1 甲基化抑制基因表达

22 Aberrant methylation of IGFBP7 exon 1 in colorectal cancer tissues 在结直肠癌中的异常表达

23 casesmedianP 25 ~ P 75 P N460.210.07 ~ 0.49<0.001 T460.610.30 ~ 0.85 Wilcoxon signed ranks test: Z=-5.131, P<0.001 N: matched colorectal normal tissue; T: colorectal cancer tissues RT-PCR of IGFBP7 in colorectal cancer and matched normal tissues

24 T N Expression of IGFBP7 protein in colorectal cancer and matched normal tissues normal tissues

25 0123P N1126810.007 T623134 Wilcoxon signed ranks test: Z=-2.674, P=0.007 N: matched colorectal normal tissue; T: colorectal cancer tissues; 0, lack of staining; 1, mild staining; 2, intermediate staining; 3, strong staining Immunohistochemical staining Expression of IGFBP7 protein in colorectal cancer and matched normal tissues

26 MSP in colorectal cancer tissues N, colorectal normal mucosa; T, colorectal cancer tissues; M, methylated products; U, unmethylated products; M-P, positive control of methylation; U-P, positive control of unmethylation; C, blank control of PCR system

27 Methylation status of IGFBP7 in colorectal cancer tissue methylationunmethylationP N3790.023 T2818 Wilcoxon signed ranks test: Z=-2.268, P=0.023 N: matched colorectal normal mucosa; T: colorectal cancer tissues

28 mRNAExpressionMethylation status (%) P N0.2180.40.044 T0.6160.9 Relationship between IGFBP7 expression and the exon 1 methylation status r=-0.210, P=0.044 IHCunmethylationmethylationP Low (0, 1)15510.004 High (2, 3)1214 r=-0.299, P=0.004

29 III: The biological behaviour of IGFBP7 in CRC cells 生物学行为 CRC cells 生物学行为

30 IGFBP7 suppressed the growth of RKO cells and SW620 cells 抑制癌细胞生长 RKO SW620 *, P=0.0066 IGFBP7-RKO transfectants versus control-vector transfectants, **, P<0.0001 IGFBP7-SW620 transfectants versus SW620 control-vector transfectants. ***P=0.0108 SW480 versus SW620 cells

31 IGFBP7 reduced the soft agar colony formation ability of CRC cells 减少集落形成 Control-RKOBP7-RKO Control-SW620BP7-SW620 *, P=0.0004 for IGFBP7-RKO transfectants versus control **, P=0.0026 for IGFBP7-SW620 transfectants versus control

32 Control-RKOBP7-RKO Control-SW620BP7-SW620 IGFBP7 suppressed the cell growth in soft agar

33 IGFBP7 induced apoptosis in CRC cells 诱导凋亡

34 A B AZADC 抑制结肠癌细胞的生长( A )、促进凋亡( B ) Cancer Biol Ther. 2008;7(12):1896-900.

35 IGFBP-rP1 inducing senescence 诱导老化 诱导老化 Exp Mol Pathol. 2008;85(2):141-5.

36 IGFBP7 was a potential tumor suppressor gene in colon cancer IGFBP7 was a potential tumor suppressor gene in colon cancer 是一个抑癌基因 是一个抑癌基因 Cancer Biol Ther. 2007;6(3):354-9 Exp Mol Pathol. 2008;85(2):141-5

37 Explore the molecular mechanism…… Explore the molecular mechanism…… IGFBP7 transfectants Control ? What’s different inside

38 Identify the differentially expressed genes in IGFBP7-RKO transfectants versus control RP5 RP6EV6 EV5 EV7RP7 vsvs vs

39 EV5 RP5 RP7 RP6 EV6 EV7 cluster Identify the differentially expressed genes using Affymetrix® GeneChip® U133 Plus 2.0

40 Reproducible in at least 2 clones: 91 genes KEGG pathway analysis: MAPK pathway: GADD45B , FOS , FLNB , NR4A1 , RASA1 TGF-  pathway: SMAD3, CDKN2B, ID1, ID3 IGFBP7 could activate MAPK pathway and inhibit TGF-  pathway. Interesting information resides in the chip results:

41 Reproductive in 3 clones: 16 genes Verify the chip results(using realtime RT-PCR)

42 Analysis of the 16 gene expression in IGFBP7- SW620 transfectants and control.

43 PH 5 Control-RKO ( EV) BP7-RKO (RP) Comparative 2-DE of BP7-RKO and control 8

44 EV RP EV RP EV RP Spot Protein descriptionSequence coverage(%) * Swissprot ID Theoretical Mr /Pi ** 1Serum albumin5.74%P0276869367/6.42 2Serum albumin7.97%P0276869367/6.42 3Serum albumin6.86%P0276869367/6.42 4pyruvate kinase, muscle22.45%Q9UK316002/7.58 5Phenylalanyl-tRNA synthetase beta chain12.56%Q9NSD966130/6.39 6Actin, cytoplasmic 1or 233.33%P6326141793/5.31 7Actin, cytoplasmic 1or 223.20%P6326141793/5.31 860 kDa heat shock protein, mitochondrial precursor2.96%P1080961055/5.7 960 kDa heat shock protein, mitochondrial precursor28.52%P1080961055/5.7 *Sequence coverage was calculated by amino acid count **Calculated from the database entry without any processing Unpublished data

45 The association between IGFBP7 and fasting glucose IGFBP7 表达与空腹血糖相关 Expression level of IGFBP7 Endocr Relat Cancer. 2004;11(1):141-8.

46 Standard curve (ng/mL) Evaluate IGFBP7 level in serum in patients of type 2 diabetes using Elisa

47 Healthy persons Frequency of donor’s age age

48 CRC and DM VS healthy persons IGFBP-rP1(µg/L) Healthy persons CRC patients

49 3T3-L1 preadipoctytes (×100) 3T3-L1 adipocytes (stain with Oil Red-O) (×100) 3T3-L1 adipocytes (×100) IGFBP7 inhibit insulin-stimulated 2-deoxyglucose (DOG) Uptake in 3T3-L1 adipocytes

50 Metabolic syndrome is reversible 代谢综合征是二十一世纪流行病,可逆

51 12 3 4 5 6 7 ALS IGF-I insulin extracellular ? intracellular IGFBP-RIGFBP-rpR IGF-IR Insulin R MAPKPI3k

52 Mechanism of IGFBP-rP1 Burger et al. Eur J Cancer, 41: 1515-1527, 2005

53 IGFBP7 may be a molecule contribute to insulin resistance, which may play important roles in the initiation and development of type 2 diabetes. 可能参与 2 型糖尿病的发生和发展 Unpublished data

54 IGFBP7 binds insulin with high affinity: IGFBP7 binds insulin with high affinity: 500 fold higher than IGFBP1-6 does. 500 fold higher than IGFBP1-6 does. What’s the binding structure of IGFBP7 to insulin? Character of IGFBP7 J Biol Chem. 1997;272(49):30729-34.

55 amino sequence: 172-176 , 196-201 , 217-220 , 235-244 Predicting the binding domain of IGFBP7 to insulin: Predicting the binding domain of IGFBP7 to insulin: ATGGAGCGGCCGTCGCTGCGCGCCCTGCTCCTCGGCGCCGCTGGGCTGCTGCTCCTGCTCCTGCCCCTCTC CTCTTCCTCCTCTTCGGACACCTGCGGCCCCTGCGAGCCGGCCTCCTGCCCGCCCCTGCCCCCGCTGG GCTGCCTGCTGGGCGAGACCCGCGACGCGTGCGGCTGCTGCCCTATGTGCGCCCGCGGCGAGGGCGA GCCGTGCGGGGGTGGCGGCGCCGGCAGGGGGTACTGCGCGCCGGGCATGGAGTGCGTGAAGAGCCG CAAGAGGCGGAAGGGTAAAGCCGGGGCAGCAGCCGGCGGTCCGGGTGTAAGCGGCGTGTGCGTGTG CAAGAGCCGCTACCCGGTGTGCGGCAGCGACGGCACCACCTACCCGAGCGGCTGCCAGCTGCGCGC CGCCAGCCAGAGGGCCGAGAGCCGCGGGGAGAAGGCCATCACCCAGGTCAGCAAGGGCACCTGCG AGCAAGGTCCTTCCATAGTGACGCCCCCCAAGGACATCTGGAATGTCACTGGTGCCCAGGTGTACTTG AGCTGTGAGGTCATCGGAATCCCGACACCTGTCCTCATCTGGAACAAGGTAAAAAGGGGTCACTATG GAGTTCAAAGGACAGAACTCCTGCCTGGTGACCGGGACAACCTGGCCATTCAGACCCGGGGTGGCC CAGAAAAGCATGAAGTAACTGGCTGGGTGCTGGTATCTCCTCTAAGTAAGGAAGATGCTGGAGAATAT GAGTGCCATGCATCCAATTCCCAAGGACAGGCTTCAGCATCAGCAAAAATTACAGTGGTTGATGCCTT ACATGAAATACCAGTGAAAAAAGGTGAAGGTGCCGAGCTATAA C N P W

56 Protein Marker Whole BL21 lysate Without W Supernatant of sonicated BL21 Precipitate of sonicated BL21 Purified fusion protein W-pET28a Protein Marker Whole BL21 lysate Without I Supernatant of sonicated BL21 Precipitate of sonicated BL21 Purified fusion protein I-pET41a Protein Marker Whole BL21 lysate Without 01 Supernatant of sonicated BL21 Precipitate of sonicated BL21 Purified fusion protein 01-pET28a Protein Marker Whole BL21 lysate Without 02 Supernatant of sonicated BL21 Precipitate of sonicated BL21 Purified fusion protein 02-pET41a W-pET28a(+)P-pET41a(+) N-pET28a(+) C-pET41a(+) Express the fragments of IGFBP7 Express the fragments of IGFBP7

57 W PNC Insulin SDS-PAGE cut the gel at 6KD transmembrane Incubated with fusion protein with His-tag wash Incubated with His-tag Ab wash Incubated with secondary Ab For odssey Analyze the binding affinity of the IGFBP7 fragments to insulin Analyze the binding affinity of the IGFBP7 fragments to insulin 正在对关键氨基酸进行鉴定

58 Conclusions The expression of IGFBP7 was upregulated in CRC 1.The expression of IGFBP7 was upregulated in CRC tissue. Overexpression of IGFBP7 correlates with tissue. Overexpression of IGFBP7 correlates with favourable prognosis in CRC patients. favourable prognosis in CRC patients. Methylation of exon1 was the key regulating mechanism 2.Methylation of exon1 was the key regulating mechanism of IGFBP7. of IGFBP7. IGFBP7 played potential tumor suppressor role in 3. IGFBP7 played potential tumor suppressor role in colorectal carcinogenesis. colorectal carcinogenesis. 4.The patients with insulin resistance are associated with The serum IGFBP7 higher incidence of some cancers.The serum IGFBP7 level was higher in type 2 diabetes and colorectal cancer level was higher in type 2 diabetes and colorectal cancer patients. IGFBP7 may be a molecule contributing to patients. IGFBP7 may be a molecule contributing to insulin resistance. insulin resistance.

59 Grants China National “973” program ( 2007CB914304 ) China National “973” program ( 2007CB914304 ) the National Scientific Foundation of China the National Scientific Foundation of China (30200333, 30570840, 30770989, 30900236) (30200333, 30570840, 30770989, 30900236) Zhejiang Natural Science Foundation of China ( D2080011 ) Zhejiang Natural Science Foundation of China ( D2080011 )

60 Persons contributed to the research Department of Pathology, Zhejiang University Dr. Maode Lai Dr. Minjie Luo Dr. Lina Shao Dr. Jie Lin Dr. Wenjing Ruan Dr. Wenjing Ruan Dr. Yu Ma Dr. Fangying Xu Dr. Fangying Xu Lipei Wang Haibing Wang Haibing Wang Youzhao Li …… Department of Chemistry, Zhejiang University Dr. Tao Wu Dr. Xin Chen


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