Objective: To investigate the effects of miR-200a on the proliferation of lung cancer cells and to identify its direct target genes. Methods:Real-time PCR was performed to analyze the miR-200a expression in 15 paired clinical specimens of non-small cell lung cancer and adjacent noncancerous tissues, human lung cancer cell lines (A549, NCI-H520, and SK-MES-1), and one human normal lung bronchial epithelial cell line (16HBE). The effects of miR-200a on the proliferation of A549 lung cancer cells were detected through CCK-8 method. The candidate target genes of miR-200a were identified by bioinformatics screening and then verified by dual luciferase reporter gene assay, real-time PCR, and Western blot. The effects of YAP1 downregulation on the proliferation of A549 lung cancer cell line were also observed through CCK-8 method. Results:The miR-200a expression in non-small cell lung cancer tissues and lung cancer cell lines was significantly decreased (P<0.01). The upregulation of miR-200a expression could significantly inhibit the pro-liferation of A549 lung cancer cells (P<0.01). Dual luciferase reporter gene indicated that miR-200a could directly affect the 3′-untrans-lated region of the YAP1 gene to inhibit luciferase activity (P<0.01). Real-time PCR and Western blot revealed that the upregulation of miR-200a expression could significantly reduce the mRNA and protein expression levels of YAP1 in A549 lung cancer cells (P<0.01). CCK-8 method indicated that the downregulation of YAP1 could significantly prevent the proliferation of A549 lung cancer cells (P<0.01). Conclusion:MiR-200a inhibits the proliferation of lung cancer cells by targeting YAP1. Thus, miR-200a elicits tumor suppression effects.

Objective: To observe the expression of hMLH1 protein in esophagus squamous cell carcinoma, atypical hyperplasia tissue and normal esophagus tissue, so as to discuss the relationship between hMLH1 expression and esophagus carcinogenesis. Methods: The specimens of esophagus squamous cell carcinoma, atypical hyperplasia and normal esophagus tissue were obtained from 92 esophagus carcinoma patients. Immunohistochemistry (IHC) staining technique was used to detect expression of hMLH1 protein. The relationship between hMLH1 expression with clinical parameters, such as gender, age, cancer differentiation level, infiltration depth, tumor stage, lymphatic metastasis, was analyzed. Results: The positive rates of hMLH1 protein in esophagus squamous cell carcinoma, and atypical hyperplasia tissue and normal esophagus tissue were 36.96%,56.52%, and 84.78%,respectively,with the former 2 significantly lower than the latter (P

Objective To investigate the effects of ginsenoside Rb1 pretreatment on the expression of brain derived neurotrophic factor (BDNF) in hippocampus of rat models under acute immobilization stress.Methods Eighteen Sprague-Dawley (SD) rats were randomly divided into three groups (each n =6):normal control group,acute immobilization stress model group,and ginsenoside Rbl group.The rats in acute immobilization stress model group and ginsenoside Rb1 group were exposed to acute immobilization for 2 hours.Thirty minutes before the modeling,ginsnoside Rb1 (40 mg/kg) was injected intraperitoneally into rats in the ginsenoside Rbl group,and the control group was not treated.The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of plasma cortisol (CORT) and adrenocorticotropic hormone (ACTH).The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was applied to examine the expression of BDNF mRNA in rat hippocampus and its expression of BDNF protein was measured by Western Blot.Results In acute immobilization stress model group,compared with those before modeling,the plasma CORT and ACTH concentrations were significantly higher after modeling [CORT (μg/L):3.79 ± 0.50 vs.2.06 ± 0.35,ACTH (μg/L):1.69 ± 0.12 vs.0.94 ± 0.12,both P ＜0.05];compared with the normal control group,the mRNA and protein expressions of BDNF in hippocampus in the acute immobilization stress model group were decreased significantly [BDNF mRNA (A value):42.87 ± 5.56 vs.109.39 ± 9.11,BDNF protein (grey value):0.94 ± 0.02 vs.1.02 ± 0.03,both P ＜ 0.01];compared with acute immobilization stress model group,the mRNA (113.73 ± 6.24 vs.42.87 ± 5.56) and protein expressions (1.04 ± 0.02 vs.0.94 ± 0.02) of BDNF in hippocampus of pre-treatment groups were significantly higher (all P ＜ 0.05).Conclusions The results suggest that pretreatment with ginsenoside Rb1 alleviate hippocampus lesion induced by acute immobilization stress through regulating the BDNF mRNA and protein expressions in hippocampus.

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.
Agrobacterium ; Blotting, Southern ; Digestion ; Genome ; Genomics ; Methods ; Microscopy, Fluorescence ; Oxidoreductases ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Wounds and Injuries

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue (1–10 μg) to 20 μl 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at 34 °C for 15 min. Finally, 2 μl of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.

Objective To investigate the risk factors of acute kidney injury (AKI) occurring in patients with critical neurological disease, and the related factors affecting their prognosis. Methods The clinical data of 207 patients with critical neurological disease admitted to the Department of Critical Care Medicine of Anhui Provincial Hospital Affiliated to Anhui Medical University (South District) from January 2016 to March 2017 were analyzed retrospectively, they were assigned into an AKI group (40 cases) and a non-AKI group (167 cases), and according to the prognosis, the patients with AKI were subdivided into a survival subgroup (14 cases) and a death subgroup (26 cases). Clinical data of Glasgow coma score (GCS), acute physiology and chronic health evaluation Ⅱ (APACHEⅡ), blood glucose, white blood cell count (WBC), central venous pressure (CVP), blood sodium, cystatin C, urea nitrogen (BUN) etc. index levels and the proportions of patients using glycerin fructose and furosemide before occurrence of AKI were collected. The indexes with statistical significant differences found in the univariate analysis were analyzed by multivariate logistic regression analysis to screen out the risk factors influencing the occurrence of AKI and the factors related to the prognosis of the AKI patients; the receiver operating characteristic curve (ROC) was drawn to assess the predictive value of risk factors in patients with severe neurological disease to develop AKI. Results The incidence of AKI was 19.3% (40/207) in the patients with critical neurological disease. The hospital mortality in AKI group was significantly higher than that in the non-AKI group [65.0% (26/40) vs. 22.2% (37/167), P < 0.01]. Compared with non-AKI group, GCS (4.44±1.65 vs. 5.39±1.62), CVP [cmH2O (1 cmH2O = 0.098 kPa): 7.69±2.66 vs. 8.98±2.56] were obviously lower in AKI group at admission, APACHEⅡ(24.50±3.67 vs. 20.05±4.42), blood glucose (mmol/L: 12.33±6.53 vs. 9.33±3.26), serum sodium (mmol/L: 144.75±10.85 vs. 140.58±5.23), WBC (×109/L: 16.15±6.25 vs. 12.79±4.22), Cystatin C (mg/L: 1.27±0.74 vs. 0.74±0.26) and BUN (mmol/L: 7.81±3.33 vs. 5.53±3.20) and proportion of male [77.5% (31/40) vs. 59.9% (100/167)], patients with the comorbidity of hypotension [37.5% (15/40) vs. 19.8% (33/167)], use of glycerin fructose [17.5% (17/40) vs. 3.6% (6/167)], or use of furosemide [70.0% (28/40) vs. 13.8%(6/167)] were significantly increased in AKI group, there was a statistically significant difference between the above two groups (all P < 0.05). Multivariate logistic regression analysis showed that the hyperglycemia [odds ratio (OR) = 1.201, 95% confidence interval (95%CI) = 1.01-1.42, P < 0.05] and use of furosemide for treatment (OR = 24.493, 95%CI =4.92-120.36, P < 0.01) were the independent risk factors for occurrence of AKI in critical neurological patients. ROC curve analysis showed that blood sugar had certain predictive value of developing AKI in patients with critical neurological disease, the area under the ROC curve (AUC) of blood glucose was 0.733, when the optimal cut-off value of blood glucose was 9.05 mmol/L, the sensitivity was 77.5% and the specificity was 62.6%. Compared with the survival subgroup in the patients with AKI, the GCS at admission in death subgroup was significantly lower (3.77±0.87 vs. 5.50±2.03), but their levels of blood glucose (mmol/L: 16.51±9.10 vs. 10.09±2.89) and BUN (mmol/L: 10.26±3.07 vs. 6.48±2.70) were obviously higher than those in the survival subgroup (all P < 0.05). Conclusion AKI is one of the common complications in patients with critical neurological disease, hyperglycemia and the use of furosemide are the independent risk factors of occurrence of AKI in such patients; the blood glucose has moderate predictive value; and lower GCS, higher glucose and BUN levels in AKI patients may enhance their risk of death.