奶牛乳铁蛋白基因5’侧翼区遗传多态性及其与乳腺炎关联性分析

牛乳铁蛋白(Lactoferrin,LF)是保护乳腺组织的防御因子之一,是具有多种功能的糖蛋白,关于牛LF基因的多态性研究的报道较多,但其多态性与奶牛乳腺炎相关性的研究较少,文章采用PCR-RFLP、CRS-PCR对268头中国荷斯坦牛LF基因启动子区的-926(G/A)、-915(T/G)、-478(/G)、+72(T/C)突变进行基因型分型,应用最小二乘线性模型分析LF基因多态性与体细胞评分(Somatic cell score,SCS)的相关性,结果表明,新发现+72(T/C)和-478(/G)对SCS有显著影响,而其他两个位点对SCS影响不显著(P>0.05),+72(T/C)的AB基因型是优良基因型,其个体的SCS值均显著低于AA型(P<0.01),BB型个体(P<0.05).-478(/G)位点的C等位基因是优良的等位基因,CC基因型个体的SCS值极显著低于CD、DD基因型个体(P<0.01).因此,LF基因+72(T/C)的AB基因型和-478(/G)位点的CC基因型均是奶牛乳腺炎抗性的优良基因型,可作为分子标记应用于奶牛乳腺炎抗性筛选.     

Methylation of CpG loci in 5'-flanking region alters steady-state expression of adenomatous polyposis coli gene in colon cancer cell lines

The APC genetic locus has been linked to the tumorigenesis and progression of colorectal cancer, although the precise mechanism of its involvement in this disease remains unknown. We used high sensitivity mapping of the methylated cytosine, Northern blot analysis and immunocytochemical staining in six colorectal cancer cell lines (DLD-1, SW480, Colo320, HT29, WiDr, and Colo201) to examine the relationship between the methylation status of the CpG loci in the 5'-flanking region of the APC gene and its expression. APC mRNA expression levels determined by Northern blot analysis correlated well with APC protein levels visualized by immunocytochemistry. In these colorectal cancer cell lines, no major genetic alterations of the APC gene, such as amplification or deletion, were detected. Analysis of the epigenetic control of APC gene expression in these lines revealed that methylation of the CpG loci in the 5'-untranslated region of APC mRNA repressed steady-state expression of the gene. Furthermore, epigenetic alteration of the APC gene was independent of the APC protein truncation and CpG methylation of the hMLH1 promoter. Although less eminent than protein truncation by point mutation within the coding region of the APC gene, epigenetic alteration suppressing APC gene expression may significantly contribute to oncogenesis and the progression of colorectal cancer.     

Increased frequency of CpG island methylator phenotype and CDH1 methylation in a gastric cancer high-risk region of china

This study aimed to profile the methylation statuses of CDH1/E-cadherin and five CpG island methylator phenotype (CIMP)-associated genes (p16, hMLH1, MINT1, MINT2, and MINT31) in gastric specimens of 47 Dalian long-term residents with and 31 without gastric cancers (GCs). CIMP patterns were classified as CIMP-H with over three methylated genes, CIMP-L with one to two methylated genes, and CIMP-N without methylation. Of 47 GC cases, 24 (51.1%) were CIMP-H, 18 (38.3%) were CIMP-L, and 5 (10.6%) were CIMP-N, whereas 5 of 21 (23.8%) premalignant lesions were CIMP-H and 15 (71.4%) were CIMP-L. CIMP-L was found in 75% (12/16) of GC-adjacent mucosa and in 38.7% (12/31) of mucosa from GC-free patients. CDH1 methylation occurred in 48.9% (23/47) of cancer, in 23.8% (5/21) of premalignant, and in 25% (4/16) of noncancerous tissues and was correlated with patients' age (P = .01), lymph node metastasis, and CIMP severity (P = .000-.028). Our results demonstrated that the frequencies of CIMP-H in Dalian GCs, CIMP-L, and p16 methylation in GC-adjacent tissues and in GC-free mucosa were much higher than those reported previously, indicating the elevated methylation pressure in this GC high-risk region. The close correlation between CDH1 methylation and CIMP severity suggests the necessity of their combination in GC prevention and earlier diagnosis.     

中国人胃癌染色体17q21区域基因遗传不稳定性研究

To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.     

Specific site methylation in the 5'-flanking region of CYP1A2 interindividual differences in human livers

Human cytochrome P4501A2 (CYP1A2) is involved in the metabolism of a large number of common drugs and is responsible for the metabolic activation of numerous promutagens and procarcinogens. Large interindividual differences exist in the expression of this enzyme. This variability has important implications for drug efficacy and cancer susceptibility. In this sudy, the methylation status of the CCGG site (bp -2759) located adjacent to an AP-1 site in the 5'-flanking region of the CYP1A2 gene was assessed in liver samples from a pool of 55 human donors. DNA methylation is an important epigenetic mechanism controlling gene expression and may be one of the molecular mechanisms underlying the interindividual variation. Analysis was conducted using Hpa II digestion and PCR. Results showed that individual samples varied in the methylation status at this site. The site was found to be hypermethylated in approximately 60% of the samples. To compare methylation status with level of CYP1A2 expression, results were analyzed by median test. In 44% of the samples with expression levels above the median the CCGG site was hypermethylated. However, for those samples with levels below the median hypermethylation of the site was found in 73% of the samples. The difference was statistically significant (p<0.05). These findings indicate that CpG methylation may be involved in controlling the expression of CYP1A2, with hypermethylation reducing expression. Moreover, this approach can be useful in assessing the role of site-specific DNA methylation in interindividual variation of CYP1A2. Analysis of other CpG sites in potentially important regulatory elements of the CYP1A2 gene will continue.     

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.     

中国人结肠癌nm23H1基因遗传不稳定性的研究

Techniques such as DNA extraction from paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain, Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to study microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 at the 17th chromosome of Chinese patients and their influence on the expression of gene nm23H1, and to clarify the relationship between the genetic instability of gene nm23H1 and the development of colon cancer, which may provide experimental basis for clinical treatment. In our experiments, the frequency of MSI, LOH and nm23H1 protein reacted positive of 30 cases of colon cancer were 26.67%, 20.00% and 53.33% respectively. In tumor node metastasis (TNM) staging, the positive frequency of MSI (43.75%) and nm23H1 protein (81.25%) in stage I + II were more than those (MSI 7.14%, p < 0.05 and nm23H1 21.43%, p < 0.01) in stage III + IV, while the frequency of LOH (35.71%), which had a rising trend along with the Duke's staging increasing, was higher than that of LOH (6.25%, p < 0.05) in stage I + II. The positive frequency of nm23H1 protein in the group of tubular adenocarcinoma (60.00%) was distinctively higher than that in the group of mucoid adenocarcinoma (20.00%, p < 0.01), showing a rising trend along with the increase of the differentiation degree of tubular adenocarcinoma. Furthermore, the positive frequency of nm23H1 protein in MSI positive group was also higher than MSI negative group (p < 0.05). And there was no difference in nm23H1 protein expression analyzed by computer imaging techniques. The results of experiments indicated that both MSI and LOH controlled the development of sporadic colon cancer independently in different paths. LOH occurred mostly in the late period of sporadic colon cancer and endowed with it a high aggressive and poor prognosis. In contrast, MSI was an early period molecule marker of sporadic colon cancer. Increasing the amount of nm23H1 protein expression could effectively restrain colon cancer metastasis and improved prognosis of sporadic colon cancer patients.     

Genetic polymorphisms of XRCC1 gene and susceptibility to gastric cancer in Chinese Han population

Abstract

This study aims to evaluate whether the c.1471G?>?A and c.1686C?>?G genetic polymorphisms of XRCC1 gene influencing gastric cancer susceptibility. A total of 813 subjects with Chinese Han ethnicity were enrolled. Our data suggest that the allele and genotype frequencies are significantly different from gastric cancer patients with cancer-free controls. We find that c.1471G?>?A and c.1686C?>?G genetic polymorphisms statistically increase the risk of gastric cancer. Our findings indicate these two genetic polymorphisms are related with the susceptibility to gastric cancer, and could be used as molecular markers for detecting gastric cancer in Chinese Han ethnicity.     

hMLH1基因启动子CpG岛甲基化致MSI与肿瘤的关系

通过人类错配修复基因( hMLHl)启动子CpG岛甲基化与微卫星不稳定性(MSI)的分析,探讨癌症发病的机制.错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制,而hMLH1的表达失活则可导致MSI的产生,促进癌症的发生.根据一系列研究得出结论,在肿瘤组织中hMLH1基因启动子CpG岛甲基化和微卫星不稳定(MSI)有显著相关性,并在癌症早期发生、发展过程中起重要作用.因此临床检测hMLH1基因启动子CpG岛甲基化及微卫星不稳定可能成为癌症鉴别诊断、评价预后、指导化疗的分子标志物之一.     

Structural organization of the bovine thyroglobulin gene and of its 5'-flanking region

The structural organization of the bovine thyroglobulin gene has been investigated by a combination of Southern genomic blotting and direct analysis of cloned gene fragments isolated from a chromosomal DNA library. The entire locus is spread over more than 200,000 base pairs which makes it one of the largest eukaryotic genes studies to date. The coding information is scattered into at least 42 exons, 34 of which have been precisely identified. A different evolutionary origin of the 5' and 3' regions of the gene is supported by the highly different proportion of exonic material they contain (12% and 3%, respectively) and by the existence of sequence homology between the 3' region of thyroglobulin and acetylcholinesterase. Detailed sequence analysis of the 5' region of the gene and its flanking segment demonstrated that a significant homology exists between bovine and human thyroglobulin sequences, except for the presence within the ruminant promoter region of a 220-base-pair sequence belonging to the bovine monomer repeated family.