1 案 例 1.1 简要案情 王某等4人携带一男孩要求鉴定,自述孩子(王小某)需要申报户口,因孩子父母均已意外身亡,无法提供孩子父母样本,由王某委托对爷爷(王某)、奶奶(张某)、外公(李某)、外婆(范某)与孩子(王小某)之间是否存在祖孙亲缘关系进行鉴定.

DNA methyltransferase 3A (DNMT3A) has been recognised as a key element of epigenetic regulation in normal development, and the aberrant regulation of DNMT3A is implicated in multiple types of cancers, especially haematological malignancies. However, its clinical significance and detailed functional role in solid tumours remain unknown, although abnormal expression has gained widespread attention in these cancers. Here, we show that DNMT3A isoform b (DNMT3Ab), a member of the DNMT3A isoform family, is critical for directing epithelial–mesenchymal transition (EMT)-associated metastasis in gastric cancer (GC). DNMT3Ab is positively linked to tumour-node-metastasis (TNM) stage, lymph node metastasis and poor prognosis in GC patients. Overexpression of DNMT3Ab promotes GC cell migration and invasion as well as EMT through repression of E-cadherin . Meanwhile, DNMT3Ab promotes lung metastasis of GC in vivo. Mechanistic studies indicate that DNMT3Ab mediates the epigenetic inaction of the E-cadherin gene via DNA hypermethylation and histone modifications of H3K9me2 and H3K27me3. Depletion of DNMT3Ab effectively restores the expression of E-cadherin and reverses TGF-β-induced EMT by reducing DNA methylation, H3K9me2 and H3K27me3 levels at the E-cadherin promoter. Importantly, DNMT3Ab cooperated with H3K9me2 and H3K27me3 contributes to the transcriptional regulation of E-cadherin in a Snail-dependent manner. Further, gene expression profiling analysis indicates that multiple metastasis-associated genes and oncogenic signalling pathways are regulated in response to DNMT3Ab overexpression. These results identify DNMT3Ab as a crucial regulator of metastasis-related genes in GC. Targeting the DNMT3Ab/Snail/ E-cadherin axis may provide a promising therapeutic strategy in the treatment of metastatic GC with high DNMT3Ab expression.

目的 探讨胃癌组织中DNA甲基转移酶3A(DNMT3A)两个异构体DNMT3A1和DNMT3A2的表达水平及与临床病理特征和预后的关系.方法 收集2005年1月至2012年12月手术切除的96例胃癌组织标本,采用免疫组化SP法检测胃癌组织中DNMT3A1和DNMT3A2的表达,分析两者表达与胃癌临床病理特征(Lauren分型、分化程度、TNM分期、淋巴结转移、远端转移、肿瘤大小、吸烟史和饮酒史)及总生存期(OS)的关系.结果 DNMT3A1和DNMT3A2蛋白在96例胃癌组织中的阳性表达率分别为88.5％(85/96)和100.0％(96/96).DNMT3A1表达与TNM分期、分化程度、淋巴结转移、Lauren分型、远处转移、肿瘤大小、吸烟史和饮酒史均无关(P＞0.05),DNMT3A2表达与分化程度、TNM分期和淋巴结转移有关(P＜0.05).Cox多因素分析显示,Lauren分型、DNMT3A2表达、肿瘤大小和淋巴结转移是影响胃癌患者OS的独立因素,DNMT3A1表达与OS无关.结论 DNMT3A1和DNMT3A2在胃癌组织表达升高.DNMT3A2高表达与预后不良有关,为胃癌诊治提供新的潜在靶点.

Increasing studies showed that long-noncoding RNAs (lncRNAs) play important roles in the biological processes, including cancer initiation and progression. However, little is known about the exact role and regulation mechanism of lncRNA UCA1 during the progression of gastric cancer (GC). In this study, we found that UCA1 was aberrantly elevated in gastric cancer tissues, and was significantly associated with lymph node metastasis and TNM stage. In vivo and in vitro, enforced UCA1 level promoted cell migration and invasion of GC cell. Depleted UCA1 expression level attenuated the ability of cell migration and invasion in GC. And then, we detected that expression level of ZEB2, a transcription factor related to tumor metastasis, was regulated by UCA1 in GC cells. miR-203 targets and suppresses to ZEB2 expression. Furthermore, we found that UCA1 could directly interact with miR-203 and lead to the release of miR203-targeted transcripts ZEB2. Herein, we revealed the novel mechanism of UCA1 on regulating metastasis-related gene by sponge regulatory axis during GC metastasis. Our findings indicated that UCA1 plays a critical role in metastatic GC by mediating sponge regulatory axis miR-203/ZEB2. To explore function of UCA1-miR-203-ZEB2 axis may provide an informative biomarker of malignancy and a highly selective anti-GC therapeutic target.

目的:检测长链非编码RNA BANCR在胃癌组织中的表达,并探讨其对胃癌细胞迁移、侵袭生物学行为的调控作用与相关机制。方法:采用荧光定量PCR（QPCR）和TCGA-STDA数据库分析胃癌组织和癌旁组织中BANCR的表达水平,采用小干扰RNA（siRNA）技术敲低胃癌细胞MKN28 BANCR的表达,采用Transwell、QPCR检测MKN28细胞的迁移、侵袭情况以及肿瘤细胞转移相关标志物的表达水平变化。结果:胃癌组织中BANCR的表达水平呈明显上调。敲低BANCR表达后,MKN28细胞的迁移、侵袭能力被抑制,转移相关分子CDH1的表达量显著增加,而Vimentin的表达量显著降低。此外,敲低BANCR下调ZEB1表达,同时干扰miR-203能部分恢复ZEB1的表达水平。结论:BANCR在胃癌组织中呈高表达,其可能通过调节miR-203-ZEB1-CDH1轴促进胃癌细胞迁移和侵袭。

Hepatitis B virus (HBV) is mainly suspected to promote hepatocellular carcinoma (HCC) development by epigenetic alteration. The HBV X protein (HBx) plays a key role in the molecular pathogenesis of HBV-related HCC. However, the mechanism of HBx-mediated hepatocarcinogenesis remains to be elucidated. RIZ1 gene, a candidate HCC suppressor gene, is frequently found to be hypermethylated and downregulated in HCC. In the present study, we show that the expression of RIZ1 was downregulated in 65% HCC tissues. Decreased expression of RIZ1 was restored by 5'-Aza in MHCC-97H HCC cell lines. HBx recombinant transfection increased DNMT1 expression level and suppressed RIZ1 expression. Moreover, knockdown of DNMT1 by siRNA restored RIZ1 expression in HCC cell SMMC-7721 and reduced methylated CpG sites of RIZ1. ChIP results showed that DNMT1 protein could bind to RIZ1 promoter, and this interaction was further enhanced with the transfected HBX recombinant. Moreover, miR-152 was decreased and involved in upregulation of DNMT1 in HBx transfected cells, at least partly, contributed to the epigenetic inactivation of RIZ1. Taken together, our data found that HBx repressed RIZ1 expression via DNMT1, which offered a new mechanism of RIZ1 inactivation in HCC, except for the widely known DNA methylation. These results enriched the epigenetic mechanism by which HBx contributes to pathogenesis of HBV-HCC.

目的:分析全新短串联重复序列(short tandem repeat,STR)基因座在江苏人群中的基因多态性和基因频率.方法:2015年江苏省598例无血缘关系的汉族个体,利用STRtyper-10G Direct Kit试剂盒对9个STR位点进行扩增,自动基因分析仪进行片段分析并进行基因分型.结果:累积STR等位基因共发现111个,其中罕见等位基因13个.基因型频率大于0.08的基因型组合13个,其中D13S325基因座的等位基因型19/20的频率最高达到0.130 4,杂合度达到了0.851±0.068,随机个体相同表型偶合率为0.054±0.022,个体识别能力为0.946±0.022,多态信息总量为0.808±0.052,非父排除率达到了0.697±0.130.结论:通过大量的样本实验和等位基因频率总结,得到了江苏人群更多基因多样性的客观数据,以便在出现微变异等情况时满足进一步实际应用的需要.

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in developmental process and diseases including cancer. To elucidate the functional preferred site of DNMTs, we analyzed the feature of distinct methylated sequences and established the defined relationship between DNMTs and preference genomic DNA sequences. Small interfering RNA (siRNA) construct of DNTM1, DNMT3A, and DNMT3B was transfected into the human hepatocellular carcinoma cell line SMMC-7721, respectively. Distinguishing methylated fragments pool was enriched by SHH method in cells which is knocked down DNMT1, DNMT3A, DNMT3B, separately. The defined binding transcription factors (TFs) containing of 5′CpG islands were obtained with bioinformatics software and website. In SMMC-7721 hepatocellular carcinoma (HCC) cell line, DNMT1, DNMT3A, and DNMT3B were specific suppressed by their corresponding siRNA construct, separately. A 46, 42, 67 distinctive methylated fragments from three different DNMTs were evaluated according to genomic DNA database. Those separated fragments were distributed among genomic DNA regions of all chromosome complements, including coding genes, repeat sequences, and genes with unknown function. The majority of coding genes contain CpG islands in their promoter region. Cluster analysis demonstrated all of preference sequences identified by three DNMTs shares their own conserved sequences. In depleting of different DNMTs cells, 80 % of 103 upregulation genes induced by DNMT1 knock-down contain CpG sites; 76 % of 25 upregulation genes induced by DNMT3A knock-down contain CpG sites; 63 % of 126 upregulation genes induced by DNMT3B knock-down contain CpG sites. Our findings suggested that distinctive DNMTs targeted DNA methylation site to their preference sequences, and this targeting might be associated with diverse roles of DNMTs in tumorigenesis. Meanwhile, the analysis of preference sequences provides an alternative way to find out the individual function of DNMTs.

目的：分析16个X‐S T R基因座在江苏汉族人群中的遗传多态性。方法收集江苏地区116例亲子鉴定无血缘关系的血样，检测16个X‐STR基因座的等位基因频率（pi）、多态性信息总量（PIC）、男性个体识别能力（DPm ）及女性个体识别能力（DPf ）。结果在16个 X‐STR基因座中，HPRTB基因座检测7种等位基因（11～17），pi分布为0.0067～0.4331，其中等位基因13较为常见，pi为0.4331；HPRTB基因座的PIC为0.6785，DPm和DPf分别为0.7189和0.8807。结论16个X‐STR基因座在江苏汉族人群中具有高度的多态性，为江苏地区个体识别和亲权鉴定提供客观依据。

目的:利用3130遗传基因测序仪的电泳图谱分析江苏人群D11S4463、D2S1776、D14S1434、D4S2408、D10S1043、D20S482、D12SATA63、D19S433等12个非CODIS STR基因座多态性分布,计算该12个基因座的基因频率(P)、个体鉴别能力(DP)、无偏倚期望杂合度(H)、多态性信息总量(PIG)、二联体非父排除率(PEduo)和三联体非父排除率(PEtrio),并评估其法医学应用价值.方法:利用PCR扩增,产物通过3130测序仪毛细管电泳.读出数据后用分析软件进行分析.结果:12个非CODISSTR基因座的频率分布在本组人群中均符合Hardy-Weinberg平衡.杂合度分布为0.707 6～0.804 6,个体鉴别能力为0.846 8～0.948 6,二联体非父排除率为0.278 6～0.482 6,三联体非父排除率为0.446 7～0.663 2,多态性信息总量为0.643 8～0.807 4.结论:通过大量的样本实验和等位基因频率的统计,得出等位基因多态性数据,用于比对验证,具有很好的辅助作用,在一些CPI值不足10 000及突变案例中具有重要的补充作用.

Little is known about the roles of DNA methyltransferase 3A (DNMT3A) in gastric carcinogenesis. Here, we reported that the exogenous expression of DNMT3A promoted gastric cancer (GC) cell proliferation by accelerating the G1/S transition. Subsequently, p18INK4C was identified as a downstream target of DNMT3A. The elevated expression of DNMT3A suppressed p18INK4C at least at the transcriptional level. Depletion of p18INK4C expression in GC cells induced cell cycle progression, whereas its re-expression alleviated the effect of DNMT3A overexpression on G1/S transition. Furthermore, we found that DNMT3A modulated p18INK4C by directly binding to and silencing the p18INK4C gene via promoter hypermethylation. In clinical GC tissue specimens analyzed, the level of methylation of p18INK4C detected in tumor tissues was significantly higher than that in paired non-tumor tissues. Moreover, elevated level of DNMT3A expression was associated with the differentiation of GC tissues and was negatively correlated with the p18INK4C expression level. Taken together, our results found that DNMT3A contributes to the dysregulation of the cell cycle by repressing p18INK4C in a DNA methylation-dependent manner, suggesting that DNMT3A-p18INK4C axis involved in GC. These findings provide new insights into gastric carcinogenesis and a potential therapeutic target for GC that may be further investigated in the future.

The de-regulation of the miR-29 family and DNA methyltransferase 3A (DNMT3A) is associated with gastric cancer (GC). While increasing evidence indicates miR-29b/c could regulate DNA methylation by targeting DNMT3A, it is currently unknown if epigenetic silencing of miR-29b/c via promoter hypermethylation in GC is caused by abnormal expression of DNMT3A. Thus, we aimed to evaluate whether cross-talk regulation exists between miR-29b/c and DNMT3A and whether it is associated with a malignant phenotype in GC. First, wound healing and Transwell assays revealed that miR-29b/c suppresses tumor metastasis in GC. A luciferase reporter assay demonstrated that DNMT3A is a direct target of miR-29b/c. We used bisulfite genomic sequencing to analyze the DNA methylation status of miR-29b/c. The percentage of methylated CpGs was significantly decreased in DNMT3A-depleted cells compared to the controls. Furthermore, the involvement of DNMT3A in promoting GC cell migration was associated with the promoter methylation-mediated repression of CDH1. In 50 paired clinical GC tissue specimens, decreased miR-29b/c was significantly correlated with the degree of differentiation and invasion of the cells and was negatively correlated with DNMT3A expression. Together, our preliminary results suggest that the following process may be involved in GC tumorigenesis. miR-29b/c suppresses the downstream gene DNMT3A, and in turn, miR-29b/c is suppressed by DNMT3A in a DNA methylation-dependent manner. The de-regulation of both of miR-29b/c and DNMT3A leads to the epigenetic silencing of CDH1 and contributes to the metastasis phenotype in GC. This finding reveals that DNA methylation-associated silencing of miR-29b/c is critical for GC development and thus may be a therapeutic target.

Background: MicroRNAs (miRNAs) play an important role in a tumor-suppressive or oncogenic manner in carcinogenesis. Alteration expression patterns of miRNAs in gastric cancer (GC) are associated with cancer initiation and progression. In the present study, we evaluated miR-29a-3p expression pattern and its function in gastric carcinogenesis.Methods: The expression of miR-29a-3p in GC tissue samples and cell lines was detected by quantitative real-time PCR (qRT-PCR). After transfected with miR-29a-3p mimics or inhibitor, the cell proliferation, cell migration, and invasion ability were assessed by CCK-8 assay, wound healing assay, and Trans-well assay, respectively. The level of CDK2, CDK4, CDK6, and CyclinD1 were determined by qRT-PCR and Western blot.Results: Compared with the corresponding non-tumor tissues, miR-29a-3p showed a significant down-regulated expression in tumor tissues. In vitro functional assays demonstrated that enforced miR-29a-3p expression inhibited cell proliferation by reducing the expression of CDK2, CDK4, and CDK6. Wound healing and Transwell assays revealed that miR-29a-3p suppressed tumor metastasis in GC.Conclusions: Our preliminary results suggest that altered expression of miR-29a-3p is involved in gastric cancer process. The present study provides the first insight into the specific role of miR-29a-3p in gastric carcinogenesis.

DNA-methyltransferase (DNMT)-3A which contains DNMT3A1 and DNMT3A2 isoforms have been suggested to play a crucial role in carcinogenesis and showed aberrant expression in most cancers. Accumulated evidences also indicated that single nucleotide polymorphisms (SNP) in DNMT genes were associated with susceptibility to different tumors. We hypothesized that genetic variants in DNMT3A1 promoter region are associated with gastric cancer risk. We selected the tagSNPs from the HapMap database for the Chinese and genotyped in a case-control study to evaluate the association with gastric cancer (GC) in a Chinese population. We identified that the functional tagSNP rs7560488 T>C associated with a significantly increased risk of GC. In vitro functional analysis by luciferase reporter assay and EMSA indicated that the tagSNP rs7560488 T>C substantially altered transcriptional activity of DNMT3A1 gene via influencing the binding of some transcriptional factors, although a definite transcriptional factor remains to be established. Compared with TT homozygotes, subjects who were TC heterozygotes and CC homozygotes exhibited a reduced expression of DNMT3A1. Furthermore, stratified analysis showed that individuals who harbor TC or CC genotypes less than 60 years old were more susceptible to GC. Our results suggest that the genetic variations in the DNMT3A1 promoter contribute to the susceptibility to GC and also provide an insight that tagSNP rs7560488 T>C may be a promising biomarker for predicting GC genetic susceptibility and a valuable information in GC pathogenesis.

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.