N-myc downstream regulated gene 1 inhibition of tumor progression in Caco2 cells.

BACKGROUND: Invasion and migration are the irreversible stages of colorectal cancer (CRC). The key is to find a sensitive, reliable molecular marker that can predict the migration of CRC at an early stage. N-myc downstream regulated gene 1 (NDRG1) is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration, however the current molecular role of NDRG1 in CRC remains unknown. AIM: To explore the role of NDRG1 in the development of CRC. METHODS: NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9. Furthermore, the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot. The cell proliferation rate was measured by the cell counting kit-8 method; cell cycle and apoptosis were detected by flow cytometry; invasion and migration ability were detected by the 24-transwell method. RESULTS: NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed, while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out. This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase. Our data also demonstrated that NDRG1 promotes early cell apoptosis. Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed. CONCLUSION: NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.

Impact of fenofibrate on choroidal neovascularization formation and VEGF-C plus VEGFR-3 in Brown Norway rats.

OBJECTIVE: This study aims to explore the possible role of fenofibrate in inhibiting choroidal neovascularization (CNV) in Brown Norway (BN) rats. METHODS: BN rats underwent binocular retinal laser photocoagulation to induce CNV. On day one, fenofibrate was injected into the vitreous cavity of rats in the control and experimental groups. Fundus fluorescein angiography (FFA), isolectin B4-FITC staining, immunofluorescence staining, qRT-PCR and western blot were performed at 1, 2, 3 and 4 weeks to observe the morphological changes of CNV and the expression of the vascular endothelial growth factor C (VEGF-C) and the vascular endothelial growth factor receptor-3 (VEGFR-3). RESULTS: CNV with the spontaneous gradual regression and scarring phenomenon appeared in BN rats. In neovascularization, VEGF-C was mainly distributed in the ganglion cell layer, while VEGFR-3 was mainly expressed in the choroid. In the control group, choroidal VEGF-C initially increased, and subsequently decreased, while VEGFR-3 level maintained a constant level after the decrease. Both had a decreasing expression in the retina. The early formation of CNV was significantly weakened in the experimental group, but there was no difference in the later period. VEGF-C and VEGFR-3 expression in the choroid and retina were lower than in the control group. Furthermore, VEGFR-3 protein was not expressed in the retina. However, this gradually increased in the early period and declined in the terminal stage in the choroid. CONCLUSION: VEGF-C and VEGFR-3 participated in the laser-induced CNV formation in BN rats. Fenofibrate could inhibit CNV formation.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging.

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews (Tupaia belangeri chinensis). The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

Differential expression and subcellular localization of Prohibitin 1 are related to tumorigenesis and progression of non-small cell lung cancer.

Lung cancer remains the leading cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer. With a variety of biological functions, Prohibitin1 (PHB1) has been proved tumor-associated. But there are conflicting data regarding the involvement of PHB1 in tumorigenesis and few studies regarding the role of PHB1 in lung cancer. The studies reported herein used a combination of clinical observations and molecular methods to investigate the possible role of PHB1 in NSCLC tissues and cell lines. PHB1 expression was evaluated by RT-PCR, RT-qPCR, Western blotting and immunohistochemistry analysis. Flow cytometric analysis was used to determine the surface expression profiles of PHB1 in lung cell lines. The results showed that PHB1 expression were generally increased in lung cancer tissues when compared with matched noncancerous tissues and closely related with tumor differentiation and lymph node invasion. PHB1 expression levels was also increased in three lung cancer cell lines (SK-MES-1, NCI-H157 and NCI-H292) as compared with BEAS-2B cells. Moreover, there were various subcellular localization of PHB1 in different lung cancer cells and the presence of PHB1 on the surface of lung cancer cells was significantly reduced. In conclusion, PHB1 expression is increased in NSCLC and the up-regulation of PHB1 is associated with clinically aggressive phenotype. The different subcellular localization of PHB1 in NSCLC cells and the loss of the membrane-associated PHB1 probably related to the tumorigenesis and progression of NSCLC and suggests that PHB1 may play different roles in various types of NSCLC.

Down-regulation of protease-activated receptor 4 in lung adenocarcinoma is associated with a more aggressive phenotype.

The role of protease-activated receptors (PARs) in lung tumors is controversial. Although PAR4 is preferentially expressed in human lung tissues, its possible significance in lung cancer has not been defined. The studies reported herein used a combination of clinical observations and molecular methods. Surgically resected lung adenocarcinomas and associated adjacent normal lung tissues were collected and BEAS-2B and NCI-H157 cell lines were grown in tissue culture. PAR4 expression was evaluated by RT-PCR, RT-qPCR, Western blotting and immunohistochemistry analysis. The results showed that PAR4 mRNA expression was generally decreased in lung adenocarcinoma tissues as compared with matched noncancerous tissues (67.7%) and was associated with poor differentiation (p=0.017) and metastasis (p=0.04). Western blotting and immunohistochemical analysis also showed that PAR4 protein levels were mostly decreased in lung adenocarcinoma tissues (61.3%), and were also associated with poor differentiation (p=0.035) and clinical stage (p=0.027). Moreover, PAR4 expression was decreased in NCI-H157 cells as compared with BEAS-2B cells. In conclusion, PAR4 expression is significantly decreased in lung adenocarcinoma, and down-regulation of PAR4 is associated with a more clinically aggressive phenotype. PAR4 may acts as a tumor suppressor in lung adenocarcinoma.

[Pathological study of lung cancer induced by Yunnan tin mine dusts in F344 rats].

OBJECTIVE: To set up animal models of the lung cancer induced by Yunnan tin mineral dusts (no radon) in F344 rats and to explore the process of carcinogenesis and pathologic alterations in various stages of malignant transformation in the animal models. METHODS: One hundred and ninety F344 rats were randomly divided into Yunnan tin mineral dust group (100 rats), furfural physiological saline group (30 rats), physiological saline group (30 rats) and normal control group (30 rats). The intratracheal instillation with mass fraction of 6% suspension liquid mixture Yunnan tin mineral dusts, volume fraction of 2% furfural physiological saline and physiological saline 0.2 ml was performed in the rates once per week respectively except normal control group. Then the rats were sacrificed in batch periodically after one week. The last rat was exposed to the tin mine dusts for 100 weeks. The morphological process and tumor formation were dynamically observed under LM and TEM. Immunohistochemistry detection of cytokeratin of High MW and low MW was used for tumor classification. Pollak stein was used to evaluate the development of fibrosis of lung in the rats. RESULTS: Bronchoalveolar inflammation occurred in the early stage after the intratracheal instillation of Yunnan tin mineral dust was performed in F344 rates. Along with reduction of inflammation, collagen fibrils increased at alveolar interstices. Simple hyperplasia, papillary hyperplasia and metaplasia of the epithelial cells in alveolar and bronchi were observed, followed by atypical adenomatous hyperplasia and squamous dysplasia. Lung cancer was induced in the end. Among the 14 cases of lung cancer, 9 cases were adenocarcinoma, 2 squamous cell carcinoma and 3 mixed carcinoma. No lung cancer occurred in other three control groups. There was a significant difference in the malignancy rate between the experimental group and the three control groups (P < 0.01). The squamous metaplasia and squamous carcinoma were found in alveoli that expressed cytokeratin of High MW. Lung fibrosis was found in 31 cases of in the tin mineral dust group. The greater the mineral dust deposit was, the more serious the alveolar fibrosis was. CONCLUSION: Yunnan tin mineral dusts without radon induce lung cancer in rates. The adenocarcinoma and squamous carcinomas induced in F344 rat lung can occur in the alveoli. The further study on whether type II alveolar epithelial cells are the origin cells of adenocarcinoma and some peripheral squamous lung carcinomas is worthwhile.

[Loss of exons of FHIT gene and FHIT protein expression in Yunnan tin miners with lung cancer].

BACKGROUND & OBJECTIVE: Yunnan tin miners have an extremely high incidence of lung cancer. Abnormality of the fragile histidine triad (FHIT) gene has been proved to closely relate to lung cancer development. This study was to explore the loss of exons 3, 4, 5 and 8 of FHIT gene and FHIT protein expression in Yunnan Tin miners with lung cancer. METHODS: The exons 3, 4, 5, and 8 of FHIT gene in lung cancer cell line YTMCC of a Yunnan Tin miner, and 30 specimens of lung cancer from Yunnan Tin miners in Gejiu district and 22 specimens of lung cancer from non-miners in other regions were detected by polymerase chain reaction (PCR). The expression of FHIT protein in 90 specimens of human lung cancer and 43 specimens of lung cancer developed by the intratracheal instillation of Yunnan Tin mineral dust in F344 rats was detected by immunohistochemistry. RESULTS: Losses of exon 3 and exon 8 were detected in YTMCC cells. Lung cancer samples from Yunnan Tin miners and non-miners exhibited heterozygous loss of FHIT gene among exons 3, 4, 5 and 8. The percentages of FHIT gene deletion and loss of FHIT protein expression in Yunnan Tin miners with lung cancer were 68.2% and 70.6%, respectively. The percentages of FHIT gene heterozygous loss was significantly higher in lung cancer tissue than in normal lung tissue (P < 0.01). Loss of FHIT protein expression was detected in the early stage of F344 rat lung canceration after treatment of Yunnan tin Mineral dust:the percentage was 100% in 8 specimens of squamous dysplasia, carcinoma in situ, and early stage of squamous cell carcinoma. CONCLUSIONS: Deletion and abnormal expression of FHIT gene are common in Yunnan tin miners and non-miners with lung cancer. The high rate of loss of FHIT expression in precancerous lesions and lung cancer at early stage indicates that FHIT could be an early screening target of lung cancer.