Efficient replication of blood-borne hepatitis C virus in human fetal liver stem cells.

The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION: Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).

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.

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.

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.

microRNA-92a promotes lymph node metastasis of human esophageal squamous cell carcinoma via E-cadherin.

microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. Recently, we examined the global miRNA expression profile of esophageal squamous cell carcinoma (ESCC) and demonstrated that miR-92a was highly expressed in tumor tissues. In this study, we found that the up-regulation of miR-92a was significantly correlated with the status of lymph node metastasis and TNM stage in 107 ESCC patients. Moreover, the up-regulation of miR-92a was associated with poor survival of ESCC patients and might be used as an independent prognostic factor. Next, we investigated the role and mechanism of miR-92a in ESCC cells, and found that miR-92a modulated the migration and invasion but not apoptosis and proliferation of ESCC cells in vitro. We further demonstrated that miR-92a directly targeted the CDH1 3'-UTR and repressed the expression of CDH1, a tumor metastasis suppressor. In addition, restoring of miR-92a-resistant CDH1 expression in miR-92a-overexpression cells recovered the pro-metastasis activity of miR-92a. Taken together, we demonstrated that miR-92a promotes ESCC cell migration and invasion at least partially via suppression of CDH1 expression, and patients with up-regulated miR-92a are prone to lymph node metastasis and thus have poor prognosis.

The 40-80 nt region in the 5'-NCR of genome is a critical target for inactivating poliovirus by chlorine dioxide.

Chemical disinfection is the most common method used to inactivate viruses from drinking water throughout the world. In this study, cell culture, ELISA, RT-PCR, and spot hybridization were employed to investigate the mechanism underlying chlorine dioxide (ClO(2) )-induced inactivation of Poliovirus type 1 (PV1), which was also confirmed by recombinant viral genome RNA infection models. The results suggested that ClO(2) inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ClO(2) degraded specifically the 40-80 nucleotides (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 RNA lacking this 40-80 nt region was not infectious. This study not only elucidated the mechanism of PV1 inactivation by ClO(2), but also defined the critical genetic target for the disinfectant to inactivate Poliovirus. This study also provides a strategy by which rapid, accurate, and molecular methods based on sensitive genetic targets may be established for evaluating the effects of disinfectants on viruses.

A survey of drug resistance bla genes originating from synthetic plasmid vectors in six Chinese rivers.

Antibiotic resistance poses a significant challenge to human health and its rate continues to rise globally. While antibiotic-selectable synthetic plasmid vectors have proved invaluable tools of genetic engineering, this class of artificial recombinant DNA sequences with high expression of antibiotic resistance genes presents an unknown risk beyond the laboratory setting. Contamination of environmental microbes with synthetic plasmid vector-sourced antibiotic resistance genes may represent a yet unrecognized source of antibiotic resistance. In this study, PCR and real-time quantitative PCR were used to investigate the synthetic plasmid vector-originated ampicillin resistance gene, ß-lactam antibiotic (blá), in microbes from six Chinese rivers with significant human interactions. Various levels of blá were detected in all six rivers, with the highest levels in the Pearl and Haihe rivers. To validate the blá pollution, environmental plasmids in the river samples were captured by the E. coli transformants from the community plasmid metagenome. The resultant plasmid library of 205 ampicillin-resistant E. coli (transformants) showed a blá-positive rate of 27.3% by PCR. Sequencing results confirmed the synthetic plasmid vector sources. In addition, results of the Kirby-Bauer disc-diffusion test reinforced the ampicillin-resistant functions of the environmental plasmids. The resistance spectrum of transformants from the Pearl and Haihe rivers, in particular, had expanded to the third- and fourth-generation of cephalosporin drugs, while that of other transformants mainly involved first- and second-generation cephalosporins. This study not only reveals environmental contamination of synthetic plasmid vector-sourced blá drug resistance genes in Chinese rivers, but also suggests that synthetic plasmid vectors may represent a source of antibiotic resistance in humans.

[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.

[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.

[Resveratrol attenuates cardiac function impairment in plateau hypobaric hypoxia rats].

Objective: To study the protective effects of resveratrol (RSV) on cardiac function in rats with high altitude hypobaric hypoxia and its mechanisms. Methods: Thirty-six rats were randomly divided into control group, hypobaric hypoxia group (HH) and hypobaric hypoxia + RSV group (HH+RSV) according to the random number, 12 rats in each group. Rats in the HH and HH+RSV groups were subjected to chronic long-term high altitude hypobaric hypoxia intervention for 8 weeks in a hypobaric chamber at a simulated altitude of 6 000 m for 20 h / d. The rats of HH + RSV were fed with RSV at a dose of 400 mg/(kg·d). The rats were tested once a week for body weight and twice a week for food intake. Before execution, the rats were tested by blood cell analyzer for routine blood parameters and echocardiogram for cardiac function parameters in each group. The routine blood indexes of each group were measured by blood cell analyzer, the cardiac function indexes of each group were measured by echocardiography, myocardial hypertrophy was evaluated by HE staining, myocardial tissue reactive oxygen levels were evaluated by dihydroethidium (DHE) staining. Oxidative stress was evaluated by serum and myocardial tissue total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD) and malondialdehyde (MDA) content. Results: Compared with the C group, the body mass and food intake of rats were decreased significantly (P＜0.05) in HH group, while compared with the C group, RSV had no significant effects on the body mass and food intake of rats in the HH+RSV group (P＞0.05). Compared with the C group, the levels of erythrocytes and hemoglobin of rats in the HH group were increased significantly (P＜0.05), while the platelet concentration was decreased significantly(P＜0.05); compared with the HH group, the erythrocyte and hemoglobin levels were decreased significantly (P＜0.05) and platelet concentration was increased significantly(P＜0.05) in rats of the HH+RSV group. Compared with the C group, the cardiac coefficient, myocardial fiber diameter and thickness were significantly increased in the HH group (P＜0.05); compared with the HH group, the cardiac coefficient and myocardial fiber thickness were significantly decreased in the HH+RSV group (P＜0.05). Echocardiographic analysis showed a significant increase in ventricular wall thickness (P＜0.05) and a significant decrease in ejection fraction and cardiac output (P＜0.05) in the HH group compared with the C group, and a significant decrease in ventricular wall thickness and a significant improvement in cardiac function (P＜0.05) in the HH+RSV group compared with the HH group. The results of DHE staining showed that myocardial tissue reactive oxygen levels were increased significantly in the HH group compared with the C group (P＜0.05); myocardial tissue reactive oxygen levels were significantly restored in the HH+RSV group compared with the HH group (P＜0.05). The oxidative/antioxidant results showed that the serum and myocardial T-AOC and SOD activities were decreased significantly (P＜0.05) and the MDA level was increased significantly (P＜0.05) in the HH group compared with the C group; the serum and myocardial T-AOC and SOD activities were increased significantly (P＜0.05) and the MDA level was decreased significantly(P＜0.05) in the HH+RSV group compared with the HH group. Conclusion: Long-term plateau hypobaric hypoxia exposure leads to myocardial hypertrophy and reduced cardiac function in rats. Resveratrol intervention significantly improves myocardial hypertrophy and cardiac function in rats caused by altitude hypobaric hypoxia exposure, which is closely related to reducing of reactive oxygen species and improving myocardial oxidative stress levels.

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.

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.

[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.

[Correlations between 14-3-3ε expression and clinicopathological factors and prognosis in patients with esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression of 14-3-3ε in esophageal squamous cell carcinoma (ESCC) and relationship of 14-3-3ε and clinicopathological factors of ECSS patients. METHODS: The 14-3-3ε expression in tumors and adjacent normal epithelia from 72 ESCC patients was detected by immunohistochemical staining. Correlations of 14-3-3ε expression or their subcellular localization and clinicopathological factors were analyzed using Chi-squared test. Survival curves were generated according to the Kaplan-Meier method, and the statistical analysis was performed by Log-rank test. RESULTS: The expression of 14-3-3ε protein was significantly up-regulated in tumors with 77.8% positive and 27.8% strong positive staining, compared with paired adjacent normal epithelia with 43.1% positive and only 2.8% strong positive staining (P < 0.01). In a total of 56 positive staining tumors, 14-3-3ε was detected in cytoplasm of 37 (66.1%), in nucleus of 4 (7.1%), in both cytoplasm and nucleus of 15 (26.8%) cases. Whereas, in a total of 31 positive staining normal epithelia, 14-3-3ε was detected in cytoplasm of 0, in nucleus of 13 (41.9%), in both cytoplasm and nucleus of 18 (58.1%) cases (P < 0.01). Statistical analysis revealed that strong 14-3-3ε expression was correlated with lymph node metastasis (P = 0.028) and lower 5-year survival (P = 0.018). The survival curves calculated by Kaplan-Meier method further showed that the patients with strong 14-3-3ε expression had a shorter survival than patients with negative or weak 14-3-3ε expression (Log-rank, P = 0.031). No correlation was found between subcellular localization of 14-3-3ε in tumor cells and clinicopathologic factors and prognosis of ESCC patients. CONCLUSION: The 14-3-3ε expression is significantly up-regulated in ESCCs. It was usually located in cytoplasm of tumor cells, but nucleus of normal epithelia. Strong expression of 14-3-3ε in tumors is associated with lymph node metastasis and considered as an poor prognostic factor for ESCC.

[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.

[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.

[Expression of coxsackievirus and adenovirus receptor in non-small cell lung cancer and its significance].

OBJECTIVE: To investigate the mRNA and proten expression of coxsackievirus and adenovirus receptor (CAR) in the corresponding normal lung tissue, para-neoplastic tissue and lung cancer tissue, and the correlation of CAR expression with the carcinogenesis as well as the expression difference in various clinicopathologic parameters. METHODS: The expression of CAR mRNA and protein in the samples from 32 lung cancer patients was determined by RT-PCR and Western blot, respectively. RESULTS: The expression level of CAR mRNA and protein in normal lung tissue, paraneoplastic tissue and cancer tissue were 1.000 +/- 0.012, 1.048 +/- 0.035, 1.282 +/- 0.072, and 0.902 +/- 0.038, 0.944 +/- 0.042, 1.08 +/- 0.052, respectively, with a statistical significance among the groups (P = 0.022, P = 0.007, P = 0.009, P = 0.027). There was a statistically significant positive correlation between expression of CAR mRNA and that of CAR protein (r = 0.448, P = 0.026). The expression levels of CAR were significantly different among different pathological types (P = 0.012), with a high level of CAR in all 7 bronchiolo-alveolar carcinoma (BAC, P = 0.029). However, there was no statistical significance in other clinicopathologic parameters (P > 0.05), including gender, age, smoking or not, tumor size, with or without lymph node metastasis and TNM stage. CONCLUSION: The expression of CAR mRNA and protein in cancer tissue samples are significantly higher than that in the normal and paraneoplastic samples, indicating that CAR might play a crucial role in the carcinogenesis. It may become a new potential prognostic marker for lung cancer patients.

[Effects of Atpif1 gene on hemoglobin synthesis in K562 cells].

OBJECTIVE: To investigate the effects of mitochondrial ATPase inhibitory factor 1 (Atpif1) on hemoglobin synthesis. METHODS: Firstly, the K562 cells were divided into 2 groups, hypoxia-treated group and normoxic control group. The K562 cells in hypoxia-treated group were treated with 2% oxygen. The K562 cells in the two groups were collected after cultured for 24, 48 and 72 hours. The proliferation-inhibitory rates of cells were detected by CCK-8 assay. The apoptosis rates of K562 cells were analyzed by flow cytometry. The hemoglobin synthesis of K562 cells was induced by hemin. The gene expressions of Atpif1, Aladelta-aminolevulinate synthase 2 (Alas2) and nuclear factor kappa B (NF-κB) were detected by qRT-PCR. Then, the K562 cells were cultured in hypoxic incubator and divided into blank control group, negative control group and si-Atpif1 group. After sliencing Atpif1 gene, the hemoglobin synthesis and the levels of NF-κB and Alas2 were determined. RESULTS: Compared with the normoxic control group, the proliferation activity of K562 cells was inhibited, the apoptosis rate was increased, and the hemoglobin synthesis was also increased in hypoxia-treated groups. The expressions of Atpif1, Alas2 and NF-κB mRNA of K562 cells were upregulated. Compared with blank control group and negative control group, the content of hemoglobin was decreased, and the levels of NF-κB and Alas2 mRNA were also decreased in si-Atpif1 group. CONCLUSION: Atpif1 gene is involved in the regulation of hemoglobin synthesis. Exploring its roles in the development of high altitude polycythemia (HAPC) can provide new ideas and therapeutic targets for the prevention and treatment of HAPC.

[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.