PCR. Detection of Promoter Hypermethylation of WIF-1 Gene by Nested Methylation Specific Polymerase Chain Reaction in Lung Cancer Patients

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1 13 5 Vol13 No ife Science Research Oct 2009 PCR WIF-1 a, b, a, c, a a*, a b c : WIF-1,, PCR (nsp) WIF-1, PCR (SP), 58 nsp 20 WIF-1, SP 10, WIF-1 (P < 005) 20 WIF-1 ; SP(nSP) WIF-1, : ; ; WIF-1 ; : R7351 :A : (2009) Detection of Promoter Hypermethylation of WIF-1 Gene by Nested ethylation Specific Polymerase Chain Reaction in ung Cancer Patients I ei-dong a GONG Huan-yu b TAN Si-pin a ZHANG Zhi-wei c CHEN Guang-wen a XIAO Xian-zhong a* a Department of Pathophysiology Xiangya edical College Changsha b Third Xiangya Hospital Changsha c Second Xiangya Hospital Changsha Central South niversity Hunan China Abstract To evaluate the methylation status of the promoter region of Wnt inhibitory factor-1 WIF-1 gene in DNA extracted from plasma of lung cancer patients nested methylation-specific PCR nsp was used to detect WIF-1 promoter hypermethylation in plasma DNA from 58 cases of lung cancer patients plasma samples from 20 healthy individuals were used as controls Compared with methylation-specific PCR SP nsp was more sensitive in detecting the WIF-1 promoter hypermethylation Aberrant promoter methylation of the WIF-1 gene was found in 20 out of 58 plasma samples using nsp while only 10 cases was detected using SP Hypermethylation of WIF-1 gene was more frequent in smoking group No aberrant promoter methylation was detected in the peripheral plasma of the healthy individuals The data show that detection of WIF-1 gene promoter hypermethylation in the plasma of lung cancer patients by nsp can give useful information as well as an effective noninvasive method in screening and early diagnosis of lung cancer patients : : : Tel iud2005@126com * Tel

2 Key words lung cancer methylation WIF-1 nested methylation-specific PCR ife Science Research ~447 W nt Wnt Tris Sigma wingless type protein Sss NEB PCR Invitrogen [1,2] Wnt 1 Wnt inhibitory factor-1 WIF-1 13 DNA Wnt Wnt 2 m 150 [3] Wnt mmol / NaCl 10 mmol / Tris 10 mmol / Wnt WIF-1 sfrp-1 EDTA 057% SDS K DKK mg / 37 - WIF-1 DNA A 260 / A 280 WIF DNA PCR DNA nsp PCR SP CpG WIF-1 EZ DNA ethylation-gold Kit DNA μ 10 PCR Buffer 25 μ g mmol / dntp 20 μ A Taq DNA 03 μ 10 pmol / 08 μ ~79 DNA 20 μ μ PCR 95 SCC 7 8 min s 1 NSCC 51 TN I~II PCR BD 25 μ PCR 10 PCR Buffer 25 μ 5 m EDTA- g mmol / dntp 20 μ A Taq Na g 10 min DNA 04 μ 10 pmol / 4 08 μ PCR g 10 min 10 μ DNA 20 PCR 95 5 min m 30 s 1 30 s s 12 EZ DNA ethylation-gold Kit Zymo Research Orange CA dntp K 15 SP SP WIF-1 1 Invitrogen PCR 35 s s min min Sss DNA 10 μ RNaseA A Taq DNA Takara PCR 20% 50 bp DNA ladder Invitrogen EB

3 5 : PCR WIF PCR WIF-1 Table 1 The primer sequences used to analyze promoter hypermethylation of WIF-1 for nested-sp Primer sequences 5-3 Anealing / ength / bp Outer Sense Inner ethylated Sense nmethylated Sense TAG GGG TTT TTG AGT GTT T ACC TAA ATA CCA AAA AAC CTA C CGT TTT ATT GGG CGT ATC GT ACT AAC GCG AAC GAA ATA CGA GGG TGT TTT ATT GGG TGT ATT GT AAA AAA ACT AAC ACA AAC AAA ATA CAA AC SPSS P< SP nsp 58 WIF-1 SP nsp 1724% 10 / nsp WIF % 20 / SP nsp DNA WIF bp 2 20 WIF-1 DNA DNA nsp WIF-1 2 DNA WIF-1 ar C C N control P1 P2 water 150 bp 1 WIF-1 nsp ar: 50 bp DNA ladder marker; : ; : ; C: ; N: ; P1: ; P2: ; Water: Fig1 Representative results showing promoter methylation amplified by nsp of WIF-1 gene in plasma C Tumor plasma N Normal plasma ar 50 bp DNA ladder marker anes and amplified products with primers recognizing methylated and unmethylated sequences respectively P1 and P2 lymphocyte DNA unmethylated or methylated in vitro by excessive CpG Sss methylase was used as unmethylation and methylation positive control respectively Water: Water blanks ar C5 C9 C5 C9 150 bp 2 nsp SP nsp SP ar: 50 bp DNA ladder marker; : ; : ; C5, nsp, SP ; C9, nsp SP Fig2 Representative results showing promoter methylation amplified by nsp and SP and represents amplified products by nsp and SP ar 50 bp DNA ladder marker and amplified products with primers recognizing methylated and unmethylated sequences respectively For sample C5 nsp can produce a weak band while SP can t amplify anything as for sample C9 both SP and nsp can amplify a strong band

4 Table 2 2 WIF-1 Clinical and pathological correlation of WIF-1 gene promoter methylation Clinical data N ethylation / % P Gender ale Female Age Years 60 >60 Smoking Yes No Hisological type SCC NSCC TN stage I~ # 0468 # 0017 * 0726 # 0849 # Notes: # P > 005 * P < WIF-1 PCR 2 / [10] TN DNA WIF-1 nsp SP 58 P > 005 WIF-1 WIF-1 nsp 3448% SP P < % WIF-1 Wnt Wnt WIF-1 CpG 12ql43 WIF-1 CG WIF-1 CpG [4,5] [1,2,11~13] Wnt CpG WIF-1 CpG WIF-1 83% [6,7] WIF-1 [14] / WIF-1 [8,9] [3] SP CpG WIF-1 CpG 58 SP 20 WIF-1 20 WIF-1 [3] PCR nsp WIF-1 PCR

5 5 : PCR WIF WIF-1 WIF-1 [J] J Cancer Res Clin Oncol (References): [1] WISSANN C WID P J KAISER S et al WIF-1 a component of the Wnt pathway is down-regulation in prostate breast lung and bladder cancer[j] J Pathol [2] BEHRENS J STIG B The Wnt connection to tumorigenesis[j] Int Dev Biol [3] [J] X Xin-juan Wnt 5958 region hypermethylation as an adjuvant diagnostic marker for non-small cell lung cancer-related malignant pleural effusions [7] YANAGAWA N TARA G OIZI H et al Promoter hypermethylation of RASSF1A and RNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers[j] ung Cancer 2007 [8] ESTEER SANCHEZ-CESPEDES ROSE R et al Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients[j] Cancer Res [9] ZO H Z Y B WANG Z W et al Detection of aberrant p16 methylation in the serum of colorectal cancer patients[j] Oncogene [10] PAISANO W A DIVINE K K SACCOANNO G et al Predicting lung cancer by detecting aberrant promoter methylation in sputum[j] Cancer Res [11] BATRA S SHI Y KCHENBECKER K et al Wnt inhibitory factor-1 a Wnt antagonist is silenced by promoter hypermethylation in malignant pleural mesothelioma[j] Bio- DING Wen-bai HAN Yu et al Study of abnormal methylation of the canonical Wnt signaling pathway-related genes in lung carcinoma carcinogenesis[j] Guangdong edical chem Biophys Res Commun Journal [12] TANIGCHI H YAAOTO H HIRATA T et a1 [4] ESTEER Dormant hypermethylated tumour suppressor Frequent epigenetic inactivation of Wnt inhibitory factor-1 in genes questions and answers[j] J Pathol human gastrointestinal cancers[j] Oncogene [5] FENG Q HAWES S E STERN J E et al DNA methylation in tumor and matched normal tissues from non-small cell lung cancer patients[j] Cancer Epidemiol Biomarkers Prev [13] [14] AZIERES J HE B YO et al Wnt signaling in lung cancer[j] Cancer ett l-10 AZIERES J HE B YO et al Wnt inhibitory factor-1 is silenced by promoter hyperthylation in human lung cancer [6] YANG T E S W I J et al WIF-1 promoter [J] Cancer Res

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