Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer

AIM： To identify the methylation of secreted frizzled-related protein 1 （SFRP1） in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients.
METHODS： We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific （MSP） PCR and RT-PCR respectively. Fisher＇s exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1.
RESULTS： In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2′-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 （44%） and 8 （15%） specimens respectively （x^2= 10.34, P 〈 0.01）. Loss of SFRP1 expression was detected in 17（33%） and 6 （12%） specimens respectively （x^2= 6.75, P 〈 0.01）. There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type.
CONCLUSION： SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.     

乙型肝炎病毒相关性肝细胞癌中分泌型卷曲相关蛋白基因甲基化分析

目的 研究HBV相关性肝细胞癌(HCC)中分泌型卷曲相关蛋白(SFRP)1、SFRP2基冈甲基化状态及其与肝细胞癌发牛发展的关系.方法 利用甲基化特异性聚合酶链反应(MSP)法检测45例肝细胞癌患者术中取得癌组织、癌旁组织及6例胆囊结石或肝脏血管瘤患者正常肝组织中SFRP1、SFRP2基因的甲基化状态.数据行X2检验、Fisher's 确切概率法统计分析.结果 在45例HCC患者中,癌组织和癌旁组织中SFRP1基因的甲基化率分别占62.2%和35.6%(X2=6.403,P＜0.05);SFRP2基因的甲基化率分别占51.1%和28.9%(X2=4.630,P＜0.05);6例正常肝组织均未检测到甲基化.癌组织中SFRPI与SFRP2基因甲基化在性别、年龄、HBV血清标志物、癌旁组织类型、有无转移和病理分级等因素之间差异均无统计学意义(P＞0.05),癌组织中SFRP1与SFRP2基因异常甲基化具有线性相关性(r=0.381,P=0.01).结论 SFRP1和SFRP2基因甲基化在HBV相关性HCC中是个频发事件,将来有可能作为一种预测HCC形成的分子生物学标志物.     

分泌卷曲相关蛋白在甲状腺相关眼病发病中的作用研究

目的:研究分泌卷曲相关蛋白(SFRP)在甲状腺相关眼病(TAO)发病中的作用。方法:收集9例TAO并行鼻内镜下眶减压术患者(病变组)眶脂肪组织及7例眼科由于外伤行眼球摘除术或眼外肌矫形术患者(对照组)术中切除的正常眶脂肪组织,应用实时荧光定量PCR技术检测病变组与对照组眶脂肪组织中SFRP1~5以及过氧化物酶体增殖物激活受体γ(PPAR-γ)mRNA的表达,应用蛋白质印迹技术检测病变组与对照组眶脂肪组织中SFRP2和PPAR-γ的蛋白表达情况,分析其差异。结果:病变组眶脂肪组织中SFRP2的mRNA表达水平高于对照组(P<0.05),SFRP1、SFRP3、SFRP4、SFRP5的mRNA表达水平2组间差异无统计学意义。病变组眶脂肪组织中PPAR-γ的mRNA表达水平高于对照组(P<0.01)。病变组眶脂肪组织中SFRP2及PPAR-γ的蛋白水平均较对照组明显升高(均P<0.01)。结论:SFRP2可能在TAO患者发病中起一定作用,并可能是通过PPAR-γ的升高引起眶脂肪增生。     

Silencing of secreted frizzled-related protein genes in MSI colorectal carcinogenesis

BACKGROUND/AIMS: The epigenetic pathway of colorectal carcinogenesis has recently become the focus of attention. The most common epigenetic change is the promoter hypermethylation of tumor suppressor genes. Secreted frizzled-related proteins have been identified and are reported to act as inhibitors of the Wnt signaling pathway. It has also been reported that microsatellite unstable cancers show more frequent hypermethylation in tumor suppressor genes and less frequent APC mutation. METHODOLOGY: Fifty-one sporadic colorectal cancers were investigated, of which 22 were MSI-H and 29 were MSI-L/MSS. Methylation-specific polymerase chain reaction was performed to detect the methylation status of SFRP1, 2 and 5 genes. RESULTS: All of the samples showed hypermethylation in the promoter region of SFRP1. MSI-H cancers showed more frequent hypermethylation in SFRP2 than MSI-L/MSS cancers, though there was no statistical significance. SFRP5 promoter hypermethylation was significantly more frequent in MSI-H cancers than in MSI-L/MSS cancers. CONCLUSIONS: SFRPs may act as an important inhibitor of the Wnt signaling pathway in MSI-H cancers.     

Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor

INTRODUCTION Secreted frizzled-related proteins (sFRPs) comprise a family of five secreted glycoproteins that antagonize Wnt canonical and noncanonical signaling by different mechanisms directly or indirectly[1]. Wnt signaling regulates cell growth, motil…     

Association of single nucleotide polymorphisms in secreted frizzled-related protein 1 gene with bone mineral density in Japanese women

Aim:   Recent studies have demonstrated that the Wnt signaling pathway plays an important role in bone metabolism. The purpose of this study was to examine whether the gene of secreted frizzled-related protein 1 ( SFRP1 ), a Wnt antagonist, is involved in the etiology of osteoporosis using association study.
Methods:   Seven single nucleotide polymorphisms (SNP) in the SFRP1 gene were genotyped and analyzed for association with bone mineral density (BMD) in 931 Japanese women (63.5 ± 6.7 years old, mean ± standard deviation).
Results:   One SNP (rs16890444) located in intron and another (rs3242) located in the 3'-untranslated region of the sFRP1 gene were significantly associated with the lumbar spine BMD value, and BMD values for both the femoral neck and the total hip, respectively. Women with the T/T genotype of the former SNP had a lower BMD value of the lumbar spine (L2–L4) compared with those with C/C or C/T (BMD value adjusted for age, duration after menopause, and body mass index: 0.781 vs 0.830, P  = 0.037), while women with the T/T genotype of the latter SNP had higher BMD values of femoral neck and total hip compared with those with C/C or C/T (adjusted BMD value: femoral neck, 0.721 vs 0.633, P  = 0.025; total hip, 0.834 vs 0.737, P  = 0.027).
Conclusion:   These results suggest that the SFRP1 may be a candidate gene for a BMD determinant, but further studies need to consolidate the present findings.     

富含半胱氨酸的酸性分泌蛋白与原发性肝癌发生发展的关系

原发性肝癌是临床常见的恶性肿瘤,富含半胱氨酸的酸性分泌蛋白在肝癌的发展与转移过程中发挥着非常重要的作用。介绍了富含半胱氨酸的酸性分泌蛋白的结构与生物学功能,分析了其在恶性肿瘤中的作用机制及其与肝癌发展、转移的关系,并对其在肝癌诊治中的应用价值进行了分析与展望。     

分泌型卷曲相关蛋白基因启动子甲基化状态检测在急性白血病中的意义

目的 探讨分泌型卷曲相关蛋白(SFRP)家族基因启动子异常甲基化状态与急性白血病(AL)发生发展的关系.方法 采用甲基化特异性PCR(MSP)检测AL患者、AL细胞系和正常人外周血单个核细胞中SFBP(1、2、4、5)基因启动子区的甲基化状态.结果 正常人单个核细胞中不存在SFRP基因的甲基化.AL患者中,SFRP1、2、4、5基因甲基化总发生率分别为35.6%(31/87)、25.3%(22/87)、12.6%(11/87)、17.2%(15/87),其中在急性髓系白血病(AML)患者中的甲基化率分别为33.9%(20/59)、23.7%(14/59)、6.8%(4/59)、10.2%(6/59),在急性淋巴细胞白血病(ALL)患者中的甲基化率分别为39.3%(11/28)、28.6%(8/28)、25.0%(7/28)、32.1%(9/28).SFRP1、2、5基因在HL60、NB4、Molt-4和Jurkat细胞系中均呈完全甲基化状态,SFRP4在NB4、Molt-4和Jurkat细胞系中呈完全甲基化状态,在HL60细胞系中则呈部分甲基化状态.结论 在AL患者及细胞系中,SFRP(1、2、4、5)基因已出现高频率甲基化.推测SFRP基因的异常甲基化模式与AL的发生密切相关,其可能成为诊断和监测AL的基因标志物.     

Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes beta-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.     

Epigenetic and genetic alterations of PTEN in hepatocellular carcinoma

Aim:  To investigate the roles of epigenetic and genetic alterations of the phosphatase and tensin homologue on chromosome 10 gene (PTEN) in carcinogenesis and the development of hepatocellular carcinomas (HCC).
Methods:  A total of 56 cases of HCC tissues and six liver cell lines were studied for the expression of PTEN by immunohistochemistry and Western blot analysis. The PTEN gene mutations in exon5 and exon8 were detected by a combination of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Methylation-specific PCR (MSP) was used to identify PTEN promoter methylation.
Results:  Of the 56 cases of HCC, 24 (42.9%) expressed the PTEN protein. All surrounding liver tissues of the hepatoma (32 cases) were positive for PTEN. Of the six cell lines, three liver cancer cell lines showed a low expression of PTEN. Five mutations of 56 HCC samples were detected. All of them were located at intron4. No mutation was found in exon5 and exon8. After MSP analysis, we found nine cases of PTEN promoter methylation in 56 specimens (16.1%). However, no CpG island of PTEN was found to be methylated in all six liver cell lines.
Conclusion:  The level of PTEN protein was altered in part of the HCC. The downregulation of PTEN expression may not be mainly associated with the PTEN mutations, but partly due to PTEN promoter methylation and other epigenetic regulation.     

Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females

Osteoarthritis (OA) is a leading cause of disability in Western society with multiple risk factors, including a complex genetic pattern. Identifying loci involved in the heredity of OA might lead to insights into the molecular pathogenesis of this common disorder. A previous genome scan mapped a primary hip OA susceptibility locus to chromosome 2q with a maximum multipoint logarithm of odds score of 1.6 in 378 affected sibling pair families. Here, microsatellite targeting of eight candidate genes in this region from 2q23-2q32 demonstrated significant associations with the tumor necrosis factor alpha-induced protein 6 gene in all probands and the integrin alpha 6 and frizzled motif associated with bone development (FRZB) genes in female probands. However, genotyping showed lack of association for a nonsynonymous single-nucleotide polymorphism in tumor necrosis factor alpha-induced protein 6, whereas a single-nucleotide polymorphism in FRZB resulting in an Arg324Gly substitution at the carboxyl terminus was associated with hip OA in the female probands (P = 0.04). This association was confirmed in an independent cohort of female hip cases (n = 338; P = 0.04). In addition, a haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in FRZB was a strong risk factor for primary hip OA, with an odds ratio of 4.1 (P = 0.004). FRZB encodes secreted frizzled-related protein 3, which is a soluble antagonist of wingless (wnt) signaling. Variant secreted frizzled-related protein 3 with the Arg324Gly substitution had diminished ability to antagonize wnt signaling in vitro. Hence, functional polymorphisms within FRZB confer susceptibility for hip OA in females and implicate the wnt signaling pathway in the pathogenesis of this disease.     

结直肠癌组织及细胞株中分泌型卷曲相关蛋白家族的甲基化研究

目的 研究分泌型卷曲相关蛋白(SFRP)基因启动子区CpG岛甲基化与结直肠癌的关系.方法 采用甲基化特异性PCR(MSP)技术检测20对结直肠癌及相应癌旁组织中SFRP基因启动子区CpG岛甲基化状况,T-A克隆测序验证MSP产物.5-氮杂-2'-脱氧胞苷对结直肠癌细胞株HCT116、SW480进行去甲基化处理,MSP和Western印迹法分别检测细胞株中SFRP基因甲基化和蛋白表达.结果 SFRP 1、2、4、5在结直肠癌中甲基化率分别为19/20、17/20、3/20、13/20,癌旁组织中分别为12/20、8/12、1/20、7/20.结直肠癌中SFRP 1、2、5甲基化率均高于癌旁组织,差异有统计学意义(P<0.05).HCT116细胞株SFRP 1、2、4、5基因均发生甲基化,而SW480细胞株中仅SFRP1和SFRP2出现启动子甲基化.SFRP蛋白表达与启动子甲基化呈明显负相关,经5-氮杂-2'-脱氧胞苷处理后能有效恢复SFRP蛋白表达.结论 SFRP 1、2、5甲基化可能与结直肠癌的发生有关,SFRP基因甲基化与其表达失活密切相关.     

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

Purpose

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status.

Methods

Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway.

Results

Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells.

Conclusions

WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.     

Epigenetic regulation of insulin-like growth factor axis in hepatocellular carcinoma

The insulin-like growth factor (IGF) signaling pathway is an important pathway in the process of hepatocarcinogenesis, and the IGF network is clearly dysregulated in many cancers and developmental abnormalities. In hepatocellular carcinoma (HCC), only a minority of patients are eligible for curative treatments, such as tumor resection or liver transplant. Unfortunately, there is a high recurrence of HCC after surgical tumor removal. Recent research efforts have focused on targeting IGF axis members in an attempt to find therapeutic options for many health problems. In this review, we shed lights on the regulation of members of the IGF axis, mainly by microRNAs in HCC. MicroRNAs in HCC attempt to halt the aberrant expression of the IGF network, and a single microRNA can have multiple downstream targets in one or more signaling pathways. Targeting microRNAs is a relatively new approach for identifying an efficient radical cure for HCC.