靶向p21基因saRNA抑制肝癌细胞生长实验研究

目的 探讨RNA激活p21对肝癌HepG2、Hep3b和SMMC-7721细胞生长和侵袭力的影响.方法 化学合成靶向p21的saRNA、阴性对照dsRNA,将其转染肝癌细胞系HepG2、Hep3b和SMMC-7721.每个细胞系分为3组,分别为p21-322组、阴性对照组和空白对照组,每组复3孔;p21-322组和阴性对照组分别采用p21-322 saRNA和阴性对照dsRNA进行转染,空白对照组不干预.转染后72 h,采用RT-PCR和Western blot检测各组细胞中的p21 mRNA和P21蛋白表达水平,采用MTT法检测细胞生长情况及划痕实验观察细胞侵袭能力的变化.结果 HepG2、Hep3b和SMMC-7721细胞p21-322组p21 mRNA相对表达水平分别为23.43±2.29、16.87±1.61、31.77±5.06,P21蛋白相对表达水平分别为55.93±12.66、32.91±5.17、24.96±6.81;空白对照组分别为3.53±0.07、2.39±0.02、5.70±0.89,3.21±0.03、2.91±0.14、4.15±0.12;阴性对照组分别为3.87±0.97、2.57±0.71、5.87±1.73,3.11±0.70、3.01±0.97、5.13±2.14;p21-322组p21 mRNA和蛋白相对表达水平明显高于空白对照组和阴性对照组(P＜0.01),空白对照组与阴性对照组比较差异无统计学意义(P＞0.05);细胞转染后第6天时HepG2、Hep3b、SMMC-7721细胞p21-322转染组细胞平均生长抑制率分别为41％、48％和52％;HepG2细胞p21-322组24 h后空白区域占原划痕区域面积的百分比((76±11)％)明显高于空白对照组((13±6)％)和阴性对照组((17±8)％)(P＜0.01),阴性对照组与空白对照组比较差异无统计学意义(P＞0.05).结论 靶向p21的RNAa能抑制肝癌细胞的生长和侵袭力,p21可作为一个具有肝癌治疗应用价值的靶基因.     

CADM1在大肠癌细胞系中的表达及与启动子甲基化的关系

目的：探讨CADM1在大肠癌细胞系中的表达及与启动子甲基化的关系。方法用RT－PCR法检测大肠癌细胞系中 CADM1基因 mRNA 表达水平,Western blot 法检测细胞系 CADM1蛋白表达情况,BSP 法和 MSP法检测 CADM1基因启动子甲基化状态。结果CADM1 mRNA 在24％大肠癌细胞系表达缺失,在50％细胞系中CADM1 mRNA 表达减少。75％大肠癌细胞系检测到 CADM1基因启动子甲基化阳性,且表达缺失。结论CADM1基因启动子甲基化与大肠癌的发生发展有密切关系,该基因的甲基化检测可作为癌症早期筛选、监测预后的肿瘤标志物。     

PTEN hypermethylation profiles of Chinese Kazakh patients with esophageal squamous cell carcinoma

Aberrant DNA methylation of promoter region CpG islands may serve as an alternative mechanism to genetic defects in the inactivation of tumor suppressor genes (TSGs) in human malignancies. The aim of this study was to examine the promoter methylation status of the PTEN TSG and its association with esophageal squamous cell carcinoma (ESCC) carcinogenesis in a Chinese Kazakh population, which is known to have a relatively high ESCC incidence and mortality. The methylation status of the PTEN promoter region was determined in patients with ESCC (n = 95) and healthy individuals (n = 65) using highly sensitive Sequenom Epityper assays. The methylation level of the PTEN gene was significantly higher in patients with ESCC than in healthy controls. The median methylation level was 10.0% (interquartile range [IQR]: 7.0–11.0%) in patients with ESCC and 6.0% in controls (IQR: 4.0–9.0%; P = 0.001). PTEN methylation levels were higher in male patients with ESCC than in male controls, whereas a trend toward significance was observed between female patients with ESCC and female controls (P = 0.005 and P = 0.086, respectively). The PTEN methylation level was associated with histopathological grade and lymph node metastasis in patients with ESCC (P = 0.002 and P = 0.009, respectively). To our knowledge, this is the first report to show the presence of PTEN promoter CpG hypermethylation in ESCC and its association with tumor metastasis.     

Selective organ specific inflammation in offspring harbouring microchimerism from strongly alloreactive mothers

The origins of autoimmunity are not yet understood despite significant advances in immunology. The trafficking of maternal cells to the offspring represents the very first immunological event in foetal life and is reinforced during lactation. The persistence of maternal cells in offspring's tissues and circulation has been associated with several autoimmune disorders. However a direct causal effect has never been demonstrated. Maternal T cells specifically targeting foetal insulin producing cells have been shown to generate islet inflammation without directly participating in this process. Our objective was to evaluate if alloreactive maternal cells could directly trigger a graft-versus host like reaction or indirectly influence the development of the offspring's regulatory T cells favouring autoimmunity. We adopted a breeding strategy comparing genetically identical offspring from either strongly alloreactive transgenic mothers compared to immunodeficient mothers. We detected maternal alloreactive T cells in the offspring and early signs of inflammation in small intestine of 6 weeks old offspring. Interestingly, CD4⁺ Foxp3⁺ regulatory T cell frequency was diminished in mesenteric lymph nodes from eight months old offspring born of alloreactive mothers compared to offspring of immunodeficient mothers. Our study favours a hypothesis where highly alloreactive maternal cell microchimerism indirectly predisposes offspring to autoimmunity.     

High Incidence of Activating TERT Promoter Mutations in Meningiomas Undergoing Malignant Progression

Meningiomas are common central nervous system tumors. The World Health Organization (WHO) defines three grades, predictive of the risk of recurrence. These tumors can relapse frequently and sometimes undergo malignant transformation. Maintenance of telomere length is a key process in malignant progression, and mutations in TERT promoter have recently been identified in various types of cancer. We sequenced the TERT promoter in 85 meningiomas from 73 patients. We found a high incidence of TERT promoter mutations in patients with meningiomas undergoing malignant histological progression (28%, n = 5/18 patients). In this subset of patients with histological progression, TERT promoter mutations were found in both the lowest and the highest grade tumors, and in both NF2‐mutated and nonmutated samples. In contrast, one mutation was identified in 35 meningiomas without recurrence or progression, belonging to various histological grades. This sample was an aggressive meningioma in a patient who died shortly after surgery. Interestingly, tumors showing relapse without histological progression were not mutated for TERT promoter (n = 20). Finally, TERT promoter mutations were associated with a marked increase in TERT expression. Thus, TERT promoter mutations are pivotal genetic alterations involved in malignant progression of meningiomas and could be used as a biomarker to identify meningiomas at risk of malignant transformation.     

A C?As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C⋅As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe²⁺-dependent MAs(III) demethylation. In addition, ArsI cleaves the C⋅As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C⋅As lyase.The metalloid arsenic is the most common environmental toxic substance, entering the biosphere primarily from geochemical sources, but also through anthropogenic activities (1). Arsenic is a group 1 human carcinogen that ranks first on the Agency for Toxic Substances and Disease Registry Priority List of Hazardous Substances (www.atsdr.cdc.gov/SPL/index.html). Microbial arsenic transformations create a global arsenic biogeocycle (1). These biotransformations include redox cycles between the relatively innocuous pentavalent arsenate and the considerably more toxic and carcinogenic trivalent arsenite (2, 3). In addition, many microbes, both prokaryotic and eukaryotic, have arsM genes for inorganic arsenite [As(III)] S-adenosylmethionine methyltransferases that methylate inorganic As(III) to mono-, di-, and tri-methylated species (4, 5). The genes encoding arsenic transforming enzymes are widely distributed, and these arsenic biotransformations have been proposed to play significant roles in the arsenic biogeocycle and in remodeling the terrain in volcanic areas such as Yellowstone National Park and regions of the world with high amounts of arsenic in soil and water such as West Bengal and Bangladesh (3, 6).Arsenicals, both inorganic and organic, have been used in agriculture in the United States for more than a century (7). Historically, the use of inorganic arsenical pesticides/herbicides has been largely replaced by methylated arsenicals such as monosodium methylarsonic acid (MSMA), which is still in use as an herbicide for turf maintenance on golf courses, sod farms, and highway rights of way, and for weed control on cotton fields (7). More complex pentavalent aromatic arsenicals such as roxarsone [4-hydroxy-3-nitrophenylarsonic acid, Rox(V)] have been largely used since the middle of the 1940s as antimicrobial growth promoters for poultry and swine to control Coccidioides infections and improve weight gain, feed efficiency, and meat pigmentation (8, 9). These aromatic arsenicals are largely excreted unchanged and introduced into the environment when chicken litter is applied to farmland as fertilizer (8). Pentavalent organoarsenicals are relatively benign and less toxic than inorganic arsenicals; however, aromatic (8–10) and methyl (11, 12) arsenicals are degraded into more toxic inorganic forms in the environment, which may contaminate the foods and water supplies. Although microbial degradation of environmental organoarsenicals has been documented (8, 9, 11, 13), no molecular details of the reaction have been reported. We recently demonstrated that a microbial community in Florida golf course soil carries out a two-step pathway of MSMA reduction and demethylation (14). Here we report the isolation of an environmental methylarsonous acid [MAs(III)]-demethylating bacterium Bacillus sp. MD1 (for “MAs(III) demethylating”) from Florida golf course soil and the cloning of the gene, termed arsenic inducible gene (arsI), responsible for MAs(III) demethylation. The gene product, ArsI, is nonheme iron-dependent dioxygenase with C⋅As lyase activity. ArsI cleaves the C⋅As bond in a wide range of trivalent organoarsenicals, including the trivalent roxarsone [Rox(III)], into As(III), which strongly suggests that the environmental pentavalent phenylarsenicals such as Rox(V) also undergo a two-step pathway of sequential reduction and ArsI-catalyzed dearylation, in analogy with the demethylation of MSMA by a microbial community. Thus, ArsI-catalyzed C⋅As bond cleavage is a newly identified mechanism for degradation of organoarsenical herbicides and antimicrobial growth promoters.     

OSCC中ER-α、RAR-β、MGMT。及P16INK4a基因启动子甲基化的临床研究

目的：探讨肿瘤相关基因ER-α、RAR-β、MGMT及P16INK4a启动子在口腔鳞状细胞癌（OSCC）组织中的甲基化状态。方法：20例病理确诊为OSCC的组织切片,经酶消化法提取组织DNA后双硫法检测ER-αL、RAR-β、MGMT及P16INK4a基因启动子的甲基化状态,比较分析4种基因启动子甲基化状态和临床病理参数的相关性。结果：20例中,P16INK4a、MGMT启动子甲基化发生率均为10％,RAR-B启动子甲基化发生率为40％,ER-d启动子甲基化发生率为55％,两株OSCC细胞系中,ER-α、RAR-β启动子均出现甲基化,而MGMT及P16INK4a启动子均未见甲基化。结论：RAR-β、ER-α基因启动子的甲基化较P16INK4a、MGMT＇g更为常见,提示前两者可能在OSCC的发生中具有更重要的作用。