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OBJECTIVES: The aim of this study was to assess the diagnostic usefulness of vascular endothelial growth factor (VEGF) and endostatin mRNA in cervical specimens of patients with cervical dysplasia and carcinoma. STUDY DESIGN: Transcription levels of VEGF and endostatin were detected by RT-PCR in cervical liquid-based preparation specimens and compared with cytological assessments. RESULTS: VEGF as well as endostatin mRNA expression was significantly associated with either cytological or histological diagnosis (p < 0.05). VEGF mRNA and endostatin mRNA were significantly more likely to be expressed in squamous cell carcinomas (SCC) than in cervical intraepithelial neoplasia (CIN) III (p < 0.01 and p < 0.05), and obviously also more likely to be expressed in CINII than in CINI and in CINIII than in CINII (p < 0.05). Eleven inflammation lesions gave positive results by cytology but negative results by RT-PCR for VEGF and endostatin mRNA. Twenty-four SCC lesions gave false-negative or precancerous lesion results by cytology but positive results by RT-PCR for VEGF and/or endostatin mRNA expression. CONCLUSION: Transcription levels of VEGF and endostatin by RT-PCR may be an adjunct to cytology screening for early detection of cervical carcinomas and may determine the progressive potentiality of individual lesions, especially in high-risk patients.
Assuntos
Citodiagnóstico , Endostatinas/isolamento & purificação , RNA Mensageiro/biossíntese , Displasia do Colo do Útero/diagnóstico , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Detecção Precoce de Câncer , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Transcrição Gênica , Displasia do Colo do Útero/patologiaRESUMO
BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the diagnostic utility of lung-specific X protein (LUNX) mRNA expression in bronchial brushing specimens from patients with lung cancer. METHODS: LUNX mRNA levels were assessed by performing RT-PCR on liquid-based cytology bronchial brushing specimens from patients with lung cancer (n=104) or benign lung disease (n=91). RESULTS: LUNX mRNA expression was significantly more frequent in patients with all carcinomas, squamous cell carcinomas, adenocarcinomas, as well as patients with central, peripheral and diffuse carcinomas (P<0.01), and non-small cell lung carcinomas (P<0.05) compared with patients with benign disease. The diagnostic performance of RT-PCR analysis of LUNX mRNA was significantly better than that of cytology in terms of sensitivity (93.3±4.8% vs 64.4±9.2%), negative predictive value (91.6±6.0% vs 71.1±7.9%) and accuracy (88.7±4.4% vs 81.0±5.5%), whereas specificity (83.5±7.6%) and positive predictive value (86.6±6.3%) were lower than those of cytology (100%). CONCLUSIONS: Liquid-based cytology and RT-PCR can be performed to detect LUNX mRNA expression in bronchial brushing specimens, and this technique may be a useful adjunct to cytological diagnosis of lung cancer. The sensitivity of the technique was greater than that of cytology but its lower specificity should be taken into account.
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Biomarcadores Tumorais/metabolismo , Broncoscopia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Glicoproteínas/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Background and objective: E6 and E7 proteins in human papillomavirus (HPV) 16 are major oncogenes in several types of tumors, including lung cancer. Previous studies have demonstrated that both E6 and E7 oncoproteins can upregulate GLUT1 protein and mRNA expression levels in lung cancer cells. Thus, the present study aimed to investigate the main differences in the molecular mechanisms of GLUT1 expression regulated by E6 and E7. Methods: The double directional genetic manipulation and immunofluorescence were performed to explore the molecular mechanism of E6 or E7 upregulating the expression of GLUT1 in H1299 and A549 cell lines. Results: The overexpression of E6 in well-established lung cancer cell lines upregulated thioredoxin (Trx) protein expression. Notably, plasmid transfection or small interfering RNA transfection with E7 had no regulatory effect on Trx expression. As an important disulfide reductase of the intracellular antioxidant system, Trx plays important role in maintaining oxidative stress balance and protecting cells from oxidative damage. The overexpression of Trx increased the activation of NF-κB by upregulating p65 expression and promoting p65 nuclear translocation, and further upregulated GLUT1 protein and mRNA expression levels. The results of the present study demonstrated that E6, but not E7, upregulated GLUT1 expression in lung cancer cells by activating NF-κB due to the participation of Trx. Conclusion: These results suggest that Trx plays an important role in the pathogenesis of HPV-associated lung cancer, and propose a novel therapeutic target for HPV-associated lung cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Papillomavirus Humano 16 , Neoplasias Pulmonares/etiologia , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Proteínas Repressoras/metabolismo , Tiorredoxinas/genética , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Transportador de Glucose Tipo 1/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/fisiologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologiaRESUMO
BACKGROUND: The E6 and E7 proteins in human papillomavirus 16 (HPV 16) are the main oncogenes in the occurrence of lung cancer. In recent studies, we found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is still unclear how E6 and E7 regulate LKB1 in lung cancer cells. METHODS: Double directional genetic manipulation and nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well-established lung cancer cell lines. RESULTS: E6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p-LKB1 in the cytoplasm only. This suggested that HPV 16 E6 but not E7 downregulates the antitumor activity of LKB1 by downregulating the expression of p-LKB1 in the cytoplasm only. CONCLUSIONS: Here, we demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7. Our findings provide new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggests new therapeutic targets.
Assuntos
Papillomavirus Humano 16/metabolismo , Cinesinas/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Masculino , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , TransfecçãoRESUMO
BACKGROUND: There is an immediate need for research on the mechanism underlying telomerase activation and overexpression. MATERIALS & METHODS: A total of 174 patients with lung cancer (n = 106) and benign lung disease (n = 68) were recruited for the current study. The mRNA expression levels of E6, E7, LKB1, Sp1, and hTERC in brushing cells were detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and hTERC amplification was also detected by fluorescence in situ hybridization (FISH). To investigate the potential mechanism, bidirectional genetic manipulation was performed in well-established lung cancer cell lines. RESULTS: Our results indicated that the mRNA expression levels of E6, E7, Sp1, and hTERC and the amplification level of hTERC were significantly increased in the malignant group compared with those of the benign group (p < 0.01). Conversely, the mRNA expression level of LKB1 was significantly decreased in the malignant group (p < 0.01). The correlation between E6, E7, Sp1, and hTERC expression was positive but was negative with LKB1 (p < 0.01). Our results also showed that HPV16 E6/E7 downregulated the expression of LKB1 at both the protein and mRNA levels. The loss of LKB1 upregulated Sp1 expression, and also promoted Sp1 activity. Sp1 further upregulated hTERC at the mRNA and gene amplification levels. Thus, we proposed a HPV-LKB1-Sp1-hTERC axis of E6/E7 upregulation of hTERC expression. CONCLUSION: We demonstrated for the first time that E6 and E7 promoted hTERC mRNA expression and the amplification of hTERC by relieving the effect of LKB1 on the phosphorylation of Sp1. Sp1 further activated hTERC by directly binding to the promoter regions of hTERC.
RESUMO
Liver kinase B1 (LKB1) is a critical tumor suppressor that is frequently mutated in human cancers. LKB1 has serine/threonine protein kinase activity, which regulates gene expression by phosphorylation of Yes-Associated protein (YAP). The phosphorylation-dependent YAP shuttling is critically important intracellular mechanism in the Hippo pathway. In our previous study, we found that the amplification of hTERC was significant higher in the bronchial brushing cells of patients with lung cancer, however, the underlying molecular mechanism is not clear. In this study, we showed that LKB1 overexpression could phosphorylate YAP and promoted its nuclear rejection. Silencing LKB1 could dephosphorylate YAP and promoted its entry into the nucleus. Here, we found that LKB1 inhibited the mRNA expression and the amplification of hTERC. YAP further up-regulated hTERC at mRNA and gene amplification levels. Therefore, we suggest that LKB1 may inhibit the expression and amplification of hTERC through the axis of LKB1-pYAP(YAP)-hTERC.
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Cervical liquid-based cytology plays an important role in the diagnosis of cervical squamous intraepithelial lesion (SIL). However, cytological evaluation alone has a relatively low sensitive. To overcome this problem, HPV DNA testing or HPV DNA combined with cytology has been applied. HPV DNA testing significantly improved the sensitivity, but the specificity is low, especially in cancer and high-grade SIL (HSIL) cases. The aim of this study was to evaluate the diagnostic utility of p16 overexpression in cervical cells of patients with HSIL and cancer. The expression of p16 was detected by immunostaining in liquid-based cells from cervical brushing in 278 patients which including: Cancer ( n = 13), HSIL ( n = 112), low-grade SIL (LSIL) ( n = 45), and Benign ( n = 108). The expression levels of p16 were significantly higher in the cancer and HSIL groups when compared with the LSIL and Benign groups ( P < 0.01). The accurate diagnostic rates of cancer and HSIL were significantly increased by p16 immunostaining plus cytology than that by cytology alone ( P < 0.01). The false negative or false positive of p16 immunostaining occurred with a unicellular pattern. With sensitivity of 96.0% and accuracy of 91.7%, the diagnostic performance of p16 immunostaining was much better than that of cytology alone with sensitivity of 36.0% and accuracy of 70.9% ( P < 0.01). p16 immunostaining in cervical brushing cells may not only be used as an ancillary tool to cytological diagnosis of cervical neoplasia but also help to distinguish HSIL from LSIL and the triage of transient infection.
Assuntos
Infecções por Papillomavirus/metabolismo , Displasia do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Feminino , Humanos , Imuno-HistoquímicaRESUMO
Chronic infection of HPV16 E6/E7 is frequently associated with lung cancers, especially in non-smokers and in Asians. In our previous studies, we found that HPV16 E6/E7 up-regulated HIF-1α at protein level and further up-regulated GLUT1 at both protein and mRNA levels in well-established lung cancer cell lines. In one of our further mechanism study, the results demonstrated that HPV16 E6/E7 up-regulated the expression of GLUT1 through HPV-LKB1-HIF-1α-GLUT1 axis. However, there are multiple pathways involved in HPV16 E6/E7 regulation of HIF-1α expression. In current study, using double directional genetic manipulation in well-established lung cancer cell lines, we showed that both E6 and E7 down-regulated the expression of RRAD at both protein and mRNA levels. Like LKB1, RRAD is one of the cancer suppressor genes. The loss of RRAD further activated NF-κB by promoted cytoplasmic p65 translocated to nucleus, and up-regulated the expression level of the p-p65 in nucleus. Furthermore, p-p65 up regulated HIF-1α and GLUT1 at both protein and mRNA levels. Thus, we proposed HPV16 E6/E7 up-regulated the expression of GLUT1 through HPV-RRAD-p65- HIF-1α- GLUT1 axis. In conclusion, we demonstrated for the first time that E6 and E7 promoted the expression of HIF-1α and GLUT1 by relieving the inhibitory effect of RRAD which resulted in the activation of NF-κB by promoting cytoplasmic p65 translocated to nucleus, and up-regulated the expression of the p-p65 in nucleus in lung cancer cells. Our findings provided new evidence to support the critical role of RRAD in the pathogenesis of HPV-related lung cancer, and suggested novel therapeutic targets.
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Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Renais , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas/patologia , Neoplasias Renais/cirurgia , Neoplasias Renais/patologia , Neoplasias Pulmonares/patologia , Pulmão/patologiaRESUMO
Current human papillomavirus (HPV)16 DNA testing has high sensitivity but low specificity, while mRNA testing (qualitative) improves the specificity. However, both techniques are not able to discriminate between transient and persistent infections. To overcome the disadvantages, we quantitatively detected E6 and E7 mRNAs by quantitative real-time polymerase chain reaction (qRT-PCR) in cervical brushing cells from 87 HPV16+ and 31 HPV16- patients. Our results showed that the expression levels of E6 mRNA or E7 mRNA were significantly increased in HPV16-positive cases than that in the negative cases. Furthermore, in HPV16+ cases, the expression levels of E6 mRNA were significantly increased in invasive cancer compared with high-grade squamous intraepithelial lesion (HSIL; p < 0.01), and HSIL compared with low-grade squamous intraepithelial lesion (LSIL; p < 0.01). There were no significant changes between LSIL and benign lesions. The expression levels of E7 mRNA presented no significant difference among the above-mentioned four groups. To test whether qRT-PCR can discriminate between transient and persistent infections, 57 HPV16+ patients were followed up for 1 year, and our results demonstrated that the expression levels of both E6 mRNA and E7 mRNA in the persistent infection group were significantly increased relative to the transient infection group ( p < 0.01 or 0.05). Thus, a quantitative detection of the expression levels of E6 mRNA in cervical brushing cells may not only be used as an ancillary tool to cytological diagnosis of cervical neoplasia, but may also help to determine the severity of the lesions and the triage of transient infection.
Assuntos
Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/diagnóstico , RNA Mensageiro/genética , Proteínas Repressoras/genética , Lesões Intraepiteliais Escamosas Cervicais/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Adulto , Idoso , Feminino , Papillomavirus Humano 16/isolamento & purificação , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , RNA Mensageiro/análise , Lesões Intraepiteliais Escamosas Cervicais/virologia , Neoplasias do Colo do Útero/virologia , Adulto JovemRESUMO
Glacier alluvial deposits record persistent organic pollutants (POPs) not only derived from the atmospheric deposition but also from the release of glacial melting. The evidence for melting glacier in the Tibetan Plateau (TP) as a secondary source of pollutants is introduced through investigating the concentration of organochlorine pesticides (OCPs) in four deposited profiles collected at the edge of the Changwengluozha glacier. Two concentration peaks were observed for dichlorodiphenyltrichloroethanes (DDTs) and hexachlorocyclohexanes (HCHs) in the past century. The first peak was observed in the 1970s, corresponding with the heavy usage of HCHs and DDTs in the surrounding countries and regions. The second one was in 2000 when the production and usage of DDTs and HCHs were strictly limited, which possibly indicated a significant release from melting glacier. This result was further supported by the enantiomeric fraction values for α-HCH and o,p'-DDT. On the other hand, the dramatic increase of polycyclic aromatic hydrocarbons (PAHs) from atmospheric deposition, which was associated with the socioeconomic development in Tibet, shaded the release of PAHs from melting glacier. This study reveals not only the air deposition history of legacy POPs but also a substantial release of OCPs from glacier to the adjacent environment. Our research supports the hypothesis that the melting glacier in the TP represents a secondary source of OCPs, which is consistent with the findings in the Alps glaciers.
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Monitoramento Ambiental , Sedimentos Geológicos/química , Hidrocarbonetos Clorados/análise , Camada de Gelo/química , Praguicidas/análise , Hidrocarbonetos Policíclicos Aromáticos/análise , TibetRESUMO
The characteristic distribution patterns of hydrocarbons have been used for fingerprinting to identify their sources. The historical air depositions of hydrocarbons recorded in natural media help to understand the evolution of the air environment. Travertine is a natural acceptor of air deposition that settles on the ground layer by layer. To reconstruct the historical air environment of hydrocarbons in the North Tibetan Plateau (NTP), a unique background region, twenty-seven travertine samples were collected systematically from a travertine column according to its precipitated year. For each sample, the precipitated year was dated while n-alkanes and polycyclic aromatic hydrocarbons (PAHs) were determined. Based on source identification, the air environment of hydrocarbons in the past century was studied for the region of NTP. Before World War II, the anthropogenic sources of hydrocarbons showed little influence on the air environment. During World War II and China's War of Liberation, hydrocarbons increased significantly, mainly from the use of fossil fuels. Between 1954 and 1963, hydrocarbons in the air decreased significantly because the sources of petroleum combustion decreased. From the mid-1960s through the end of the 1990s, air hydrocarbons, which mainly originated from biomass burning, increased gradually because agriculture and animal husbandry were developing steadily in Tibet and China. From the late 1990s, hydrocarbons in the atmosphere increased rapidly due to the rapid increase of tourism activities, which might increase hydrocarbon emissions from traffic. The reconstruction of the historical air hydrocarbons in NTP clearly reflects the evolution of the region and global development.
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Poluentes Atmosféricos/análise , Poluição do Ar/estatística & dados numéricos , Monitoramento Ambiental , Hidrocarbonetos Policíclicos Aromáticos/análise , Atmosfera/química , China , Material Particulado/análise , TibetRESUMO
The fraction of trans-chlordane (TC) in chlordane was used to indicate racemic degradation while the enantiomer fractions (EFs) indicated enantioselective depletion. In 44 soils of the Central Tibetan Plateau, the fractions of TC ranged from 0.368 to 0.411. The EFs ranged from 0.174 to 0.696 for TC and from 0.483 to 0.672 for cis-chlordane (CC). (-) enantiomer excess (ee) was found to be 80.0% in the soils for TC and (+) ee was 86.5% for CC. The fraction of TC changed with the clay content while the EFs changed with the soil organic carbon. Meanwhile, the fractions of TC and the EFs were determined for the surficial sediments in Yamzhog Yumco Lake, which were compared with those in the soils at its catchment area. The composition and chiral signature of chlordane did not vary between soils and sediments. Our results will help to elucidate the transformation of chlordane in soils and in surficial transport.