长链非编码RNAIFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100 μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IF...     

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.     

p16INK4A and p19ARF act in overlapping pathways in cellular immortalization

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.     

Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer

Although the considerable progress against gastric cancer, it remains a complex lethal disease defined by peculiar histological and molecular features. The purpose of the present study was to investigate pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expressions, and analyze their possible correlations with clinicopathological factors. The expression patterns were examined by immunohistochemistry in 47 patients, 27 evaluated of intestinal-type, and 20 of diffuse-type, with a mean follow up of 56 months and by Western blot in AGS, N87, KATO-III, and YCC-2, -3, -16 gastric cell lines. Overall, stomach cancer showed EZH2 correlated with high levels of p53, Ki-67, and cytoplasmic pRb2/p130 (P < 0.05, and P < 0.01, respectively). Increased expression of EZH2 was found in the intestinal-type and correlated with the risk of distant metastasis (P < 0.05 and P < 0.01, respectively), demonstrating that this protein may have a prognostic value in this type of cancer. Interestingly, a strong inverse correlation was observed between p27(KIP1) expression levels and the risk of advanced disease and metastasis (P < 0.05), and a positive correlation between the expression levels of p21(WAF1) and low-grade (G1) gastric tumors (P < 0.05), confirming the traditionally accepted role for these tumor-suppressor genes in gastric cancer. Finally, a direct correlation was found between the expression levels of nuclear pRb2/p130 and low-grade (G1) gastric tumors that was statistically significant (P < 0.05). Altogether, these data may help shed some additional light on the pathogenetic mechanisms related to the two main gastric cancer histotypes and their invasive potentials.     

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.     

野生型和突变型p16在H460细胞株的表达

应用ＰＣＲ体外定点突为技术,构建了ｐ１６－Ｐ４８Ｌ和ｐ１６－Ｄ７４ｎ突变体。野生型和突变型ｐ１６ｃＤＮＡ克隆地ｐｃＤＮＡ３真核表达载体,导入纯合缺失ｐ１６基因的人肺癌细胞株Ｈ４６０,经ＲＮＡ点杂交初筛吴Ｇ４１８抗性的细胞株,再用Ｎｏｒｔｈｅｒｎ印迹证实外源ｐ１６表达。     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Cloning of the novel gene TM6SF1 reveals conservation of clusters of paralogous genes between human chromosomes 15q24-->q26 and 19p13.3-->p12

As the result of the EUROIMAGE Consortium sequencing project, we have isolated and characterized a novel gene on chromosome 15, TM6SF1. It encodes a 370 amino acid product with enhanced expression in spleen, testis and peripheral blood leukocytes. We have identified another gene, paralogous to TM6SF1 on chromosome 19p12, TM6SF2, with an overall similarity of 68% and 52% identity at the protein level. This conservation has led us to uncover a series of eleven genes in 19p13.3-->p12 with close homology to genes in 15q24--> q26. The percentage of sequence similarity between each paralogous pair of genes at the protein level ranges between 43 and 89%. A partial conservation of synteny with mouse chromosomes 7, 8 and 9 is also observed. The corresponding orthologous genes in mouse of human TM6SF1 and TM6SF2 show a high degree of amino acid sequence conservation.     

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.     

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.     

P48L和D74N突变对p16基因功能的影响

应用DNA聚合酶链式反应(PCR)技术,对p16抑癌基因(CDKN2)进行体外定点突变,在p16cDNA中引入第48位密码子CCG(Pro)→CTG(Leu)和第74位密码子GAC(Asp)→AAC(Asn)突变,构建了p16-P48L和p16-D74N突变体。野生型和突变型p16 cDNA克隆于pcDNA3构建pCMV-p16、pCMV-p16P48L和pCMV-p16D74N真核表达载体,导入纯合缺失p16基因的人肺癌细胞株H460,经RNA点杂交、RNA印迹和细胞免疫化学染色,检测到P16表达。通过比较表达野生型和突变型P16的H460细胞在~3H-TdR掺入及细胞所在周期的差异,证实P16表达抑制细胞进入S期,而P48L和D74N突变体对细胞进入S期没有什么影响,提示P48L和D74N突变导致P16蛋白功能丧失。     

Status of p53, p21, mdm2, pRb proteins, and DNA methylation in gonocytes of control and gamma-irradiated rats during testicular development

In fetal and newborn rat testes, gonocytes, which stop cycling for about 8 days, become highly radiosensitive. The presence of p53, p21, mdm2, and pRb, which are involved in cell cycle, apoptosis control, or both, were studied by immunohistochemistry to determine if their expression is related to this radiosensitivity. A strong cytoplasmic expression of p53 and p21 was detected. Cytoplasmic expression of p53 occurred only in arrested gonocytes, whereas that of p21 was observed before and after the block. P21 was found to colocalize with mitochondria. No expression of mdm2 was detected and pRb was present only when the gonocytes started cycling again. In animals exposed to 1.5 Gy of gamma-irradiation at Day 19 postcoitum, p53 expression was prolonged in time, whereas no change was observed in p21 amounts and localization, compared with controls. Using antibodies against 5-methyl cytosine, it was shown that gonocyte DNA passed from a hypomethylated to a methylated status 1 day after gonocytes stopped cycling. A prolonged survival of gonocytes after exposure to radiation was followed by their progressive apoptosis, which finally involved the entire gonocyte population between Days 6 and 12 postpartum. The elevated but delayed sensitivity of gonocytes to genotoxic stress may be related to the unusual expression of p53 and p21, which may itself be related to the large DNA methylation changes.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

脑胶质瘤组织中p16基因启动子区CpG岛甲基化检测及其临床意义研究

目的：研究脑胶质瘤中p16基因启动子区甲基化情况及其临床意义。方法：用甲基化特异性PCR技术检测42例脑胶质瘤组织和癌旁正常脑组织中p16基因启动子甲基化,并分析该基因启动子甲基化与临床病理特征之间的关系。结果：脑胶质瘤组织中p16基因异常甲基化率（38.27%）显著高于癌旁正常脑组织中p16基因的异常甲基化率（8.8%,P=0.000）。发生甲基化的肿瘤组织或者正常脑组织中p16基因mRNA和蛋白表达显著降低。此外,p16基因异常甲基化和肿瘤病理分级有相关性（P=0.007）,而与患者性别、年龄及肿瘤类型等临床特征无关（P=0.669,0.869和0.944）。结论：p16基因启动子区CpG岛高甲基化与p16表达下调相关,推测p16启动子区CpG岛高甲基化是导致p16基因在脑胶质瘤中表达下调的重要因素,有望成为脑胶质瘤早期辅助诊断的分子标志物之一。