A human-specific insertion promotes cell proliferation and migration by enhancing TBC1D8B expression.

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution.

[Construction and evaluation of a gradient stress model of PC12 cells induced by corticosterone].

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 µmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 µmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 µmol/L, 25 µmol/L, 50 µmol/L, 100 µmol/L, 150 µmol/L and 200 µmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.

[Effects of three Polyphenolic compounds on the intestinal flora of mice exposed simulated intermittent plateau hypoxia].

OBJECTIVE: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. METHODS: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. RESULTS: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (P＜0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(P＜0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(P＜0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(P＜ 0.05). CONCLUSION: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.

[The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism].

OBJECTIVE: To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. METHODS: Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. RESULTS: With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. CONCLUSION: Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

[Effects of soybean isoflavones on the energy metabolism of swimming mice].

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(P<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(P<0.05). CONCLUSIONS: Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus.

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10-24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10-3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10-3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10-3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10-3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10-4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10-5). KC corneas also had increased mtDNA damage (P = 3.63×10-10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.

TGF-ß and NF-κB signaling pathway crosstalk potentiates corneal epithelial senescence through an RNA stress response.

The corneal epithelium plays important roles in the maintenance of corneal transparency for good vision, and acts as a protective barrier against foreign insults. Structural and functional changes with aging in the corneal epithelium have been documented. Here we found that transforming growth factor-ß (TGF-ß) is highly expressed in the elderly donor corneal epithelium, as are senescence-associated genes, such as p16 and p21. In human corneal epithelial cell (HCEC) models, TGF-ß induces cellular senescence, characterized by increased SA-ß-gal positive cells and elevated expression of p16 and p21. Pharmacological inhibition of TGF-ß signaling alleviates TGF-ß-induced cellular senescence. In addition, we determined that senescence-associated inflammation was significantly aggravated in TGF-ß-induced cellular senescence by detecting the expression of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFα). Both genetic and pharmacological approaches revealed that blocking nuclear factor-κB (NF-κB) signaling not only inhibited the production of inflammatory factors, but also rescued the senescent phenotype induced by TGF-ß in HCECs. Mechanistically, TGF-ß induced an atypical RNA stress responses, leading to accelerated mRNA degradation of IκBα, an inhibitor of NF-κB. Together, our data indicate that TGF-ß-driven NF-κB activation contributes to corneal epithelial senescence via RNA metabolism and the inflammation blockade can attenuate TGF-ß-induced senescence.

Evaluating the Association between Keratoconus and Reported Genetic Loci in a Han Chinese Population.

BACKGROUND: Keratoconus (KC) is a complex degenerative disorder of the cornea. Genetic, environmental, and lifestyle factors may all contribute to the pathogenesis of KC. Most of the reported KC-associated SNPs have been detected in Caucasians and Australians. To investigate whether the reported associated SNPs can be found in a Chinese population, we performed a replication study of the significantly associated SNPs. MATERIALS AND METHODS: A total of 210 unrelated Chinese KC patients and 191 unrelated controls were included in the present study. SNPs rs4954218 (Near RAB3GAP1 (5')), rs4894535 (FNDC3B), rs2956540 (LOX), rs3735520 (Near HGF (5')), rs1324183 (MPDZ-NF1B), rs1536482 (RXRA-COL5A1), rs7044529 (COL5A1), rs2721051 (Near FOXO1 (3')), rs9938149 (BANP-ZNF469) and rs6050307 (VSX1) were assessed for their association with KC. The genotype of each SNP was detected using the Sequenom MassARRAY-Assay. RESULTS: SNP rs1324183 located in MPDZ-NF1B was associated with an increased risk of KC (OR=3.108, 95% CI=1.366-7.072, p=0.005), and SNP rs2956540 in the LOX gene may confer a reduced risk of KC with a borderline p value in our population (OR=0.664, 95% CI=0.447-0.986, p=0.042). No significant difference was observed between patients and controls in the other eight SNP genotypes and allele frequencies. CONCLUSIONS: The replication association of rs1324183 (MPDZ-NF1B) with KC in our population and the results, which are identical to those in different populations, suggest that rs1324183 (MPDZ-NF1B) is a common genetic risk for KC and should be further investigated.

A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

The physiological effects of resveratrol and its potential application in high altitude medicine.

Resveratrol, as a natural polyphenolic compound, has a wide range of beneficial effects, which includes anti-tumor, cardiovascular protection, anti-oxidant and estrogen-like effects, and so on. Its various physiological properties are closely related to the therapeutic principle for prevention and treatment of high altitude hypoxia injury. Resveratrol may play an important role in relieving or curing high altitude diseases, especially high altitude polycythemia(HAPC). However, the literature about study and application of resveratrol in plateau medicine field is rarely reported up to now. In this review, we summarized the physiological effects of resveratrol, discussed the possible main principle of resveratrol for HAPC therapy, and looked forward to resveratrol's perspective or potential application in high altitude medicine.

Genetic landscape of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers. We performed exome sequencing on 113 tumor-normal pairs, yielding a mean of 82 non-silent mutations per tumor, and 8 cell lines. The mutational profile of ESCC closely resembles those of squamous cell carcinomas of other tissues but differs from that of esophageal adenocarcinoma. Genes involved in cell cycle and apoptosis regulation were mutated in 99% of cases by somatic alterations of TP53 (93%), CCND1 (33%), CDKN2A (20%), NFE2L2 (10%) and RB1 (9%). Histone modifier genes were frequently mutated, including KMT2D (also called MLL2; 19%), KMT2C (MLL3; 6%), KDM6A (7%), EP300 (10%) and CREBBP (6%). EP300 mutations were associated with poor survival. The Hippo and Notch pathways were dysregulated by mutations in FAT1, FAT2, FAT3 or FAT4 (27%) or AJUBA (JUB; 7%) and NOTCH1, NOTCH2 or NOTCH3 (22%) or FBXW7 (5%), respectively. These results define the mutational landscape of ESCC and highlight mutations in epigenetic modulators with prognostic and potentially therapeutic implications.

Development of a novel filter cartridge system with electropositive granule media to concentrate viruses from large volumes of natural surface water.

Exposure to various infectious viruses in environmental drinking water can constitute a public health risk. However, it is difficult to detect viruses in water due to their low concentration. In this study, we have developed a novel filter cartridge system containing electropositive granule media (EGM). Viruses present in large volumes of environmental samples were adsorbed onto the EGM, and then recovered by elution and poly(ethylene glycol) (PEG) concentration. To evaluate the system's efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially contaminate river water samples which were then assayed by quantitative real-time PCR. To optimize the concentration procedure, the eluent type, water flow rate and properties (e.g., pH, bacterial, and viral loads), were evaluated. The highest virus recovery was obtained by pumping river water at a flow rate of 300 mL/min and then pushing 3 L of an eluent containing 3× broth [1.5% (w/v) NaCl, 3% (w/v) tryptone, 1.5% (w/v) beef powder] with 0.05 mol/L glycine through the filter. Using this procedure, the recovery efficiencies of PV-1 from 10 to 100 L of spiked river water were up to 99%. In addition, this method is virus load and pH dependent. Virus recovery was maximal at a load of between 10(3.5) and 10(5.5) TCID50 and a pH ranging from 5 to 7. The bacterial load in the water has no effect on virus recovery. Different types of viruses and surface water were tested to validate the system's applicability. Results revealed that the EGM filter cartridge was able to concentrate PV-1, human adenoviruses (HAdVs) and noroviruses (HuNoVs) with high efficiency from river, lake, and reservoir water. Furthermore, it showed more efficient recovery than glass wool and 1MDS filters. These data suggest that this system provides rapid and efficient virus recovery from a large volume of natural surface water and, as such, could be a useful tool in revealing the presence of viruses in surface water.

Overexpression of Cdc25C predicts response to radiotherapy and survival in esophageal squamous cell carcinoma patients treated with radiotherapy followed by surgery.

Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy. To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery, we evaluated the expression of cell cycle checkpoint-related proteins Chk2, Cdc25C, and Cyclin D1. A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery. Pretreatment tumor biopsy specimens were analyzed for Chk2, Cdc25C, and Cyclin D1 expression by immunohistochemistry. High expression of Chk2, Cyclin D1, and Cdc25C was observed in 44 (78.6%), 15 (26.8%), and 27 (48.2%) patients, respectively. The median survival was 16 months (range, 3-154 months), with a 5-year overall survival rate of 19.6%. Overexpression of Chk2 was associated with smoking (P = 0.021), overexpression of Cdc25C was associated with patient age (P = 0.033) and tumor length (P = 0.001), and overexpression of Cdc25C was associated with pathologic complete response (P = 0.038). Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival. In multivariate analysis, Cdc25C was the most significant independent predictor of better survival (P = 0.014) for patients treated with radiotherapy followed by surgery. Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery. These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients. Thus, immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.

[Relationship between ROCK2 expression and clinicopathologic characteristics of esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression and relationship of Rho-associated protein kinase 2 (ROCK2) and clinical characteristics in esophageal squamous cell carcinoma (ESCC). METHODS: Immunohistochemistry was performed to assay the expression of ROCK2 in tumor tissues and adjacent normal epithelium from 118 ESCC patients in tissue microarray. The relationship between ROCK2 level and clinicopathologic profiles such as age, gender, location, smoking, differentiation degree, T stage, lymph node metastasis and TNM stage were analyzed. RESULTS: The ROCK2 expression was up-regulated in 54 of 118 (45.76%) ESCC tissues. The up-regulated expression of ROCK2 was observed in 55.74% (34/61) ESCC tissues of patients under 61 years old. And it was significantly higher than that in 35.09% (20/57) of patients over 61 years old (χ(2) = 5.062, P = 0.024). In addition, the rate of up-regulation of ROCK2 was significantly higher in high-grade differentiation group (58.70%, 27/46) than that in moderate-grade and low-grade differentiation group (37.50%, 27/72) (χ(2) = 5.080, P = 0.024). CONCLUSION: The up-regulated expression of ROCK2 is correlated with patient age and differentiation grade of ESCC.

Vitamin E modulates cigarette smoke extract-induced cell apoptosis in mouse embryonic cells.

Vitamin E (VE) can effectively prevent occurrence of lung cancer caused by passive smoking in mice. However, whether VE prevents smoking-induced cytotoxicity remains unclear. In this study, a primary culture of embryonic lung cells (ELCs) was used to observe the cytotoxic effects of cigarette smoke extract (CSE), including its influence on cell survival, cell cycle, apoptosis, and DNA damage, and also to examine the effects of VE intervention on CSE-induced cytotoxicity. Our results showed that CSE could significantly inhibit the survival of ELCs with dose- and time-dependent effects. Furthermore, CSE clearly disturbed the cell cycle of ELCs by decreasing the proportion of cells at the S and G2/M phases and increasing the proportion of cells at the G0/G1 phase. CSE promoted cell apoptosis, with the highest apoptosis rate reaching more than 40%. CSE also significantly caused DNA damage of ELCs. VE supplementation could evidently inhibit or reverse the cytotoxic effects of CSE in a dose- and time-dependent manner. The mechanism of CSE effects on ELCs and that of VE intervention might involve the mitochondrial pathway of cytochrome c-mediated caspase activation. Our study validate that VE plays a clearly protective effect against CSE-induced cytotoxicity in mouse embryonic lung cells.

[The relationship between RACK1 expression and clinicopathologic information of esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression and relationship of receptor for activated C kinase 1 (RACK1) and clinical characteristics in esophageal squamous cell carcinoma (ESCC). METHODS: Western Blotting was conducted to detect the RACK1 expression in ESCC cell lines. Immunohistochemistry was performed to assay the expression of RACK1 and Ki67 in tumor tissues and adjacent normal epithelium from 113 ESCC patients in tissue microarray. The relationship between the RACK1 level and such clinicopathologic profiles as age, gender, location, smoking, differentiation degree and TNM (tumor, node, metastasis) stage were analyzed. RESULTS: The expression of RACK1 protein was significantly down-regulated in ESCC tissues as compared with the normal adjacent epithelium (χ(2) = 63.363, P < 0.01). An upregulated expression of RACK1 was observed in 72.5% (29/40) ESCC tissues of patients without a smoking history. And it was significantly higher than that in 46.6% (34/73) of patients with a smoking history (χ(2) = 7.040, P = 0.008). In addition, the rate of up-regulated of RACK1 was significantly higher in stage I and II group (63.8%, 44/69) than that in stage III group (43.2%, 19/44) (χ(2) = 4.616, P = 0.032). Moreover, the ESCC tissues with a higher Ki67 score showed a lower level of RACK1 than that with a lower Ki67 score (χ(2) = 8.261, P = 0.016). CONCLUSION: The expression of RACK1 is down-regulated in ESCC tissues and associated with smoking. The expression of RACK1 was associated with smoking, TNM staging and Ki67 score of ESCC.

miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

Cyclin E is reported to be an important cell cycle regulator, and its dysregulation is implicated in tumorigenesis including esophageal squamous cell carcinoma (ESCC). MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and play important roles in tumor initiation and progression. However, the regulation of cyclin E by miRNAs is still unclear in ESCC. In the present study, we found that overexpression of miR-29c inhibited cyclin E expression by targeting 3' untranslated region of cyclin E messenger RNA in ESCC cells. Moreover, overexpression of miR-29c induced cell cycle G(1)/G(0) arrest through suppression of cyclin E expression, without affecting other G(1) phase-related proteins level, such as cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 2 and CDK6. Furthermore, we demonstrated that overexpression of miR-29c inhibited proliferation of ESCC cells in vitro and in vivo. In addition, we detected miR-29c expression in 26 pairs of esophageal tumor-in-site-tissues and 60 pairs of ESCC tissues. The result showed that miR-29c level significantly decreased in ESCC tumor tissues and cell lines compared with normal esophageal epithelia. Taken together, our findings indicated that miR-29c was frequently downregulated in ESCC tissues and cells and suppressed tumor growth by inducing cell cycle G(1)/G(0) arrest mainly through modulating cyclin E expression.

Overexpression of Grb2-associated binder 2 in human lung cancer.

OBJECTIVE: Grb2-associated binder 2 (Gab2), a member of the family of Gab scaffolding adaptors, transmits and amplifies the signals from receptor tyrosine kinases. A recent study demonstrated that Gab2 was over-expressed in breast cancers and metastatic melanomas, and Gab2 was an oncogenic protein. However, the roles of Gab2 in lung cancers are largely unknown. METHOD: In this study, to investigate whether Gab2 expression could be a characteristic of lung cancers, we analyzed the expression of Gab2 in 88 lung frozen tissue samples and 122 paraffin-embedded tissue specimens, using quantitative real-time-PCR, immunohistochemistry and western blot. RESULTS: We found that the positive expression rate of Gab2 in the tumor tissues, as detected by immunohistochemistry, 62.5% in squamous cell cancers, 51.35% in adenocarcinomas, and 75% in other types of lung cancers, was significantly higher than that (12%) in normal lung tissues. The mRNA expression detected by quantitative real-time-PCR and protein expression detected by western blotting in different groups were consistent with the immunohistochemical results. CONCLUSION: Our data indicate that Gab2 is over-expressed in malignant lung tissues compared with that in normal lung tissues, and suggest that Gab2 expression may play a role in lung cancer development.

[Correlation study of the overexpression of activated Cdc42-associated kinase 1 and the stage and prognosis of esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression and relationship of activated Cdc42-associated kinase 1 (ACK1) and the clinical characteristics of esophageal squamous cell carcinoma (ESCC). METHODS: The ACK1 expression in ESCC cell lines was detected by Western blot. Immunohistochemistry was performed to assay the expression level of ACK1 protein in tumor tissues and adjacent normal epithelium from 105 ESCC patients in tissue microarray and 45 patients in normal tissue slices. Semi-quantitative RT-PCR(reverse transcription-polymerase chain reaction)was performed to determine the expression level of ACK1 mRNA in 45 pairs of ESCC frozen tissues. RESULTS: The expression level of ACK1 protein was significantly up-regulated in 48.6% ESCC tissues as compared with the normal adjacent epithelium in tissue microarray. The overexpression of ACK1 was significantly correlated with the lymph node metastasis and TNM stage of ESCC patients. The results of normal tissue slices were consistent with those of tissue microarray. Furthermore the overexpression of ACK1 was associated with a poor survival of ESCC patients (P = 0.030). The elevated mRNA level of ACK1 in ESCC tissues was correlated with the lymph node metastasis and TNM stage of ESCC patients. And a significant correlation was observed between protein and mRNA level of ACK1 (P = 0.021). CONCLUSION: The up-regulated expressions of ACK1 protein and mRNA are correlated with the progression and prognosis of ESCC.

Expression and unique functions of four nuclear factor of activated T cells isoforms in non-small cell lung cancer.

Nuclear factor of activated T cells (NFAT) is an important family of transcription factors that can be activated by calmodulin and calcineurin in human cells. To investigate the expression and clinical significance of NFAT isoforms and calcineurin in non-small cell lung cancer (NSCLC), we collected tumor and adjacent normal tissues from 159 NSCLC patients and assembled them in a tissue microarray. Protein levels of NFAT1, NFAT2, NFAT3, NFAT4, and calcineurin were determined using immunohistochemistry. Correlations between NFAT and calcineurin expression and clinicopathologic characteristics were analyzed. We found that the positive rates of NFAT1 (52.8%, 84/159), NFAT2 (11.3%, 18/159), NFAT3 (28.3%, 45/159), NFAT4 (47.2%, 75/159), and calcineurin (47.8%, 76/159) expression were significantly higher in tumor tissues than in adjacent normal lung tissues (P<0.001), respectively. The positive rate of NFAT1 expression was significantly higher in patients with adenocarcinoma (63.5%, 47/74) than in those with squamous cell carcinoma (43.5%, 37/85) (χ2=6.340, P=0.012); with lymph node metastasis (61.6%, 53/86) than without lymph node metastasis (42.5%, 31/73) (χ2=5.818, P=0.016); and with stage-II and -III diseases (61.8%, 55/89) than with stage-I disease (41.4%, 29/70) (χ2=6.524, P=0.011). Moreover, the overexpression of NFAT1 was associated with poor survival of NSCLC patients (χ2=5.006, P=0.025). The positive rate of NFAT4 was significantly higher in patients with squamous carcinoma (57.6%, 49/85) than in those with adenocarcinoma (35.1%, 26/74) (χ2=8.045, P=0.005) and with high and moderate differentiation (54.9%, 61/111) than with low differentiation (29.2%, 14/48) (χ2=8.943, P=0.003). Calcineurin overexpression was significantly associated with histologic type (higher in squamous carcinoma than in adenocarcinoma, χ2=8.897, P=0.003), differentiation grade (higher in high-moderation grade than in low grade, χ2=9.566, P=0.002) and gender (higher in male than in female, χ2=5.766, P=0.016). Furthermore, calcineurin expression was significantly correlated with NFAT4 level (r=0.429, P<0.001). These results suggest that NFAT1 expression is associated with lung adenocarcinoma progression, and NFAT4 expression, which was higher in squamous lung cancer, is associated with calcineurin expression and differentiation grade.