3′-Me-DAB诱导大鼠肝癌发生过程中p53及相关基因mRNA的定量分析

背景与目的:有关肝癌中 p53基因突变及 p53蛋白表达异常已有报道 , 但其在 mRNA水平上的变化尚不清楚 . 为了解在肝癌发生、发展和预后过程中 p53、谷胱甘肽转硫酶 P( glutathione S-transferase P,GST-P) 、甲胎蛋白 ( α -fetoprotein,AFP) 和白蛋白 mRNA水平的变化 , 本研究定量分析癌前病变和癌灶中 GST-P、 AFP和白蛋白 mRNA的量 . 方法:在 3′ -甲基 -4-二甲胺偶氮苯 (3′ -methyl-4-dimethylaminoazobenzene,3′ -Me-DAB)诱发 F344大鼠肝癌过程中 , 用激光捕获显微取样仪 (LCM)准确获得大鼠肝脏中微小癌灶或癌前病变组织后 , 采用 LightCyclerTM V3 System 实时 (real-time)RT-PCR定量分析这些病灶组织中 mRNA水平 . 结果:在实验的第 6、 12和24周,癌前病变组织中p53mRNA量均显著高于正常组织(P≤0.001)。从第6周到第24周,癌前病变组织中p53mRNA逐渐降低(P<0.01)。癌组织中p53mRNA含量高于正常组织,低于同期癌前病变组织(P=0.028和0.0136)。第24周的癌前病变组织或癌组织中细胞核呈p53强染色。各实验期,癌前病变中GST-PmRNA量明显高于正常组织和癌组织(P<0.001)。癌组织中AFPmRNA的表达量显著高于癌前病变组织和正常组织(P<0.001),白蛋白mRNA的表达量显著低于这两种组织(P<0.01)。GST-P和AFP分别在癌前病变组织和癌组织中呈灶状强表达。结论：GST-P和AFPmRNA分别在癌前病灶组织和癌组织中强表达,是这些病变组织的重要标志物。p53mRNA在早期肝癌前病变和癌灶中显示高水平的表达,而较晚期p53蛋白升高。     

Lack of mutations of the p53 tumor suppressor gene in hepatocellular carcinomas induced in rats by a peroxisome proliferator

Immunohistochemical, immunoblotting, and DNA-sequencing analyses were performed on hepatocellular carcinomas induced in rats chronically fed BR931, a peroxisome proliferator, to determine whether the tumors carried mutations or other alterations of the p53 gene. None were detected. Inactivation of this tumor suppressor gene does not appear, therefore, to be involved in the carcinogenicity of BR931, a nongenotoxic chemical hepatocarcinogen.     

miR-34b表达调控及其在肿瘤发生中作用的研究进展

miRNA是一种内源性的非编码小RNA,在转录后水平调控基因表达,具有高度保守性和组织特异性.其广泛参与调控细胞的生长、发育等许多复杂生命过程,在多种肿瘤的发病中起重要作用.miR-34b为新近发现并备受关注的miRNA之一,受p53及其启动子甲基化等因素的调节,并通过调控多种靶分子参与调节细胞的增殖与凋亡,对肿瘤的发生与调控起作用,在肿瘤的治疗与预后中有广阔的应用前景.文章就miR-34b及其与肿瘤关系的研究进展进行综述.     

SNHG15, a p53-regulated lncRNA,suppresses cisplatin-induced apoptosis and ROS accumulation through the miR-335-3p/ZNF32 axis

Small nucleolar RNA host gene 15 (SNHG15) is upregulated in many malignancies and mediates the development of multiple cancers, including osteosarcoma (OS). However, data on the regulatory mechanisms and role of SNHG15 in the chemoresistance of OS remain scarce. Here, we show that p53 binds to the SNHG15 promoter, leading to decreased SNHG15 expression. Decreased SNHG15 expression promotes cisplatin-induced apoptosis and reactive oxygen species (ROS) accumulation in OS cells. Furthermore, SNHG15 sponges and inhibits the activity of endogenous miR-335-3p, leading to the upregulation of zinc finger protein 32 (ZNF32). Taken together, these findings reveal that p53 downregulates SNHG15 expression in OS. In addition, SNHG15 suppresses cisplatin-induced apoptosis and ROS accumulation through the miR-335-3p/ZNF32 pathway.     

Upregulation of p18Ink4c expression by oncogenic HPV E6 via p53-miR-34a pathway

Binding of p53 to miR-34a promoter activates the expression of tumor-suppressive miR-34a. Oncogenic human papillomavirus (HPV) infection downregulates miR-34a expression through viral E6 degradation of p53. In our report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18(+) HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint because of viral E7 degradation of pRB. In summary, our study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a and p18Ink4c and identifies p18Ink4c as a possible biomarker for cervical cancer.     

Expression of p73, a novel protein related to the p53 tumour suppressor p53, and apoptosis in cholangiocellular carcinoma of the liver.

p73, the first homologue of the tumour suppressor protein p53, was recently discovered on chromosome 1p36 and has been shown to induce apoptosis in a p53-like manner. The present study was performed with the aim of investigating the expression of p53, its new homologue p73 and the occurrence of apoptosis in cholangiocellular carcinoma. Protein levels of p73 were examined in 41 patients with curatively (R0-) resected cholangiocellular carcinomas with an antiserum, raised against a peptide in the N-terminal domain of p73. The incidence of mutations in the p53 gene was analysed by direct sequencing and also immunohistochemically. Apoptotic cell death was assessed using in-situ end-labelling (ISEL) technique in combination with morphological criteria. The results obtained were correlated with patient survival. Immunostaining of p73 protein was detected in 17/41 carcinomas examined (41%). The immunoreactivity was confined to the cell nucleus. In 15/41 patients (37%), mutations of the p53 gene were observed. Eleven out of these 15 patients stained also positive for p73. In contrast, out of 26 patients without any detectable p53 mutation, only six exhibited p73 immunostaining. We failed to observe a correlation between p73 expression or p53 and apoptosis within a given tumour. Survival analysis including the parameters stage and grade of disease, p73 and p53, and also apoptosis, showed that tumour stage and grade as well as p53 and p73 were significantly related to prognosis. In Cox regression survival analysis, however, only extent of primary tumour and lymph node status had an independent prognostic impact. Our results with a high prevalence of p73 within tumours harbouring mutated p53 gene suggest that p73 could compensate for p53 function. We failed to establish p73 or p53 as independent prognostic factors in cholangiocellular carcinoma of the liver.     

γ-射线诱导人白血病T细胞株Jurkat细胞凋亡及其机制

目的　观察γ 射线诱导p5 3基因突变的白血病细胞株Jurkat细胞凋亡的时间和剂量效应 ,以探求辐射诱发肿瘤细胞凋亡通路的分子机制。方法　分别以 5、10、2 0Gyγ 射线照射Jurkat细胞 ,于照射后继续培养 6、12、2 4、3 6、48h ,分别收获细胞。流式细胞术 (FCM )定量观察细胞凋亡的变化。采用WesternBlot法检测Jurkat细胞株p5 3基因蛋白的表达。结果　细胞受照射后 ,在一定的时间范围内 ,凋亡细胞的比例随作用时间和照射剂量的增加而增加 ,显示出较好的量效和时效关系。本实验所用的Jurkat细胞株是p5 3基因突变的细胞株 ,p5 3基因蛋白不表达。 结论　确立了γ 射线诱导Jurkat细胞凋亡时间和剂量效应关系 ,而导致凋亡的机制独立于肿瘤抑制基因 p5 3 ,这为探讨新的凋亡通路机制奠定了基础。     

Co-carcinogenic effect of cyclohexanol on the development of preneoplastic lesions in a rat hepatocarcinogenesis model

Cyclohexanol is a basic industrial chemical widely used because of its versatility as an industrial solvent. No studies have been conducted to evaluate the carcinogenic/co-carcinogenic hazards associated with cyclohexanol exposure. In male Fisher 344 rats liver preneoplastic lesions were induced by N-nitrosodiethylamine (150 mg/Kg) i.p., followed by the tumor promoter 2-acetylaminofluorene (2-AAF: 20 mg/kg) orally administered on three consecutive days before partial hepatectomy. The cyclohexanol administration in this hepatocarcinogenesis assay revealed that it has a strong tumor co-promoter potential. There is clear evidence that oxidative stress and the CYP2E1 are components of carcinogenesis. Although no changes in the lipid peroxidation levels were observed between treated and untreated animals, a significant increase in CYP2E1 expression was observed when cyclohexanol was administered 24 h after the last 2-AAF dose. On the other hand, levels of the proliferation markers PCNA and Ki-67 were not increased after treatment with cyclohexanol, but a marked downregulation of the Bax proapoptotic protein was found exclusively in mitochondrial extracts of animals treated with cyclohexanol. This study represents the first report of the ability of cyclohexanol-induced lesions, when administered simultaneously with 2-AAF, to potentiate the development of preneoplastic liver.     

Isozyme pattern of fructose diphosphate aldolase during hepatocarcinogenesis induced by 2-acetylaminofluorene in rat liver

The biosynthesis of aldolase A and B subunits has been studied in rat liver during the administration of carcinogen AAF

1 The abbreviations used in this paper are: FDP — fructose-1,6-diphosphate; F-1-P — fructose-1-phosphate; AAF — 2-acetylaminofluorenea

. Transition from a predominance of aldolase B to A was observed during carcinogenesis in rat liver. Changes in isozymic pattern and FDP to F-1-P cleavage activity ratio were observed before histological alterations typical of hepatoma could be detected. Our data support the hypothesis of dedifferentiation during hepatocarcinogenesis which in an early stage results in switching on of the gene for aldolase A with simultaneous continuation of biosynthesis of aldolase B within single cells.     

Alterations in the structural gene and the expression of p53 in rat liver tumors induced by aflatoxin B1.

Rat hepatocellular carcinomas (HCCs) induced by aflatoxin B1 (AFB) treatment were examined for changes in the p53 tumor suppressor gene and in p53 suppressor gene expression. A high proportion of HCCs (nine of 11 tumors in six of eight animals) exhibited new p53 restriction fragments, indicating genomic alterations of one of the p53 alleles. Each tumor with an altered p53 restriction-fragment pattern exhibited a new fragment in one of two size classes (3 kb or 7 kb with EcoRI digestion) that were missing portions of the 3' end of the p53 gene. These findings indicate that apparently similar genomic rearrangements or deletions occurred independently in AFB-induced tumors. When compared with nontumor liver tissue from the same animal, the tumors with p53 gene alterations showed dramatically reduced levels of p53 mRNA and protein and greatly increased levels of histone H2B and retinoblastoma tumor suppressor (Rb) mRNA. In two HCCs showing no evidence of p53 restriction-fragment alterations, mutant p53 protein was detected. Mutant protein was also detected in two liver samples containing an adenoma and altered foci. These data suggest that alterations of the p53 tumor suppressor gene are involved in the induction of rat HCC by AFB.     

Low p38 MAPK and JNK activation in cultured hepatocytes of DRH rats; a strain highly resistant to hepatocarcinogenesis

DRH rats are a hepatocarcinogenesis-resistant strain isolated from hepatocarcinogenesis-sensitive Donryu rats, and the liver of DRH shows less histological damage and fewer/smaller neoplastic hepatic lesions by the treatment with hepatocarcinogens. To investigate the mechanism of the resistance, the properties of hepatocytes of DRH and Donryu were compared. In primary culture, DRH hepatocytes exhibited higher proliferation and less apoptosis than Donryu hepatocytes in the presence of EGF and insulin. However, such difference was not correlated to the degree of DNA damage associated with cell culture or cell cycle checkpoint function. Although the mitogen-activated protein kinases [EGF receptor (EGFR) and extracellular signal regulating kinases (ERK1/2)] were activated to the same degree, the stress-activated protein kinases [p38 mitogen-activated protein kinase (p38) and c-jun N-terminal kinase (JNK)] were activated to a lesser degree in the DRH hepatocytes. Treatment with 2-acetylaminofluorene (2-AAF) in vivo also resulted in less JNK and p38 activation in the DRH livers. Furthermore, apoptosis signal-regulating kinase 1 (ASK1) was inhibited by the lysate from the DRH but not by the Donryu hepatocytes. The low activation of the stress-activated protein kinases may be linked to the resistance to cellular stress, which may underlie the hepatocarcinogenesis-resistance in DRH rats.     

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1-1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single-nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5'-upstream region of the H19 promoter of the parental alleles.     

Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients

Background:

Owing to late diagnosis in advanced disease stages, prognosis of patients with epithelial ovarian cancer (EOC) is poor. The quantification of deregulated levels of microRNAs could facilitate earlier diagnosis and improve prognosis of EOC.

Methods:

Seven microRNAs (miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429) were quantified in the serum of 180 EOC patients and 66 healthy women by TaqMan PCR microRNA assays. Median follow-up time was 21 months. The effects of miR-7 and miR-429 on apoptosis, cell proliferation, migration and invasion were investigated in two (EOC) cell lines.

Results:

Serum levels of miR-25 (P=0.0001) and miR-93 (P=0.0001) were downregulated, whereas those of miR-7 (P=0.001) and miR-429 (P=0.0001) were upregulated in EOC patients compared with healthy women. The four microRNAs discriminated EOC patients from healthy women with a sensitivity of 93% and a specificity of 92%. The levels of miR-429 positively correlated with CA125 values (P=0.0001) and differed between FIGO I–II and III–IV stages (P=0.001). MiR-429 was an independent predictor of overall survival (P=0.011). Overexpressed miR-429 in SKOV3 cells led to suppression of cell migration (P=0.037) and invasion (P=0.011). Increased levels of miR-7 were associated with lymph node metastases (P=0.0001) and FIGO stages III–IV (P=0.0001). Overexpressed miR-7 in SKOV3 cells resulted in increased cell migration (P=0.001) and invasion (P=0.011). Additionally, the increased levels of miR-376a correlated with FIGO stages III–IV (P=0.02).

Conclusions:

Our data indicate the diagnostic potential of miR-7, miR-25, miR-93 and miR-429 in EOC and the prognostic potential of miR-429. This microRNA panel may be promising molecules to be targeted in the treatment of EOC.     

微RNA与p53调控的关系研究进展

微RNA(miRNA)是近年发现的一类内源性非编码单链小RNA,在转录后水平负调控基因表达,参与发育、凋亡和细胞增殖等重要生命过程.p53是一重要转录因子,调控细胞周期与凋亡.两者的异常表达均与肿瘤的生成密切相关.     

微RNA与p53调控的关系研究进展

微RNA(miRNA)是近年发现的一类内源性非编码单链小RNA,在转录后水平负调控基因表达,参与发育、凋亡和细胞增殖等重要生命过程.p53是一重要转录因子,调控细胞周期与凋亡.两者的异常表达均与肿瘤的生成密切相关.