Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, IA, IB and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer.

Serum microRNAs have been identified as potential cancer biomarkers. However, the detailed mechanism by which expression of microRNAs contributes to the development and diagnosis of NSCLC remains unknown. This study was to identify specific miRNAs for diagnosing or predicting the prognosis of NSCLC patients and their correlation between miRNA expression in tissues and serums. Six matched cancer and noncancerous tissues from NSCLC patients were analyzed by miRNA microarray. Among these, three miRNAs (miR-21, miR-141, and miR-200c) were examined in 70 NSCLC paired samples (cancer, normal tissue, and serum) and 44 serum samples of normal volunteers by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Consisting with the microarray results, the expression levels of miR-21, miR-141, and miR-200c in NSCLC were higher than those in normal tissues. While the level of serum miR-21 was increased in cancer patients as compared with that in normal counterpart, expression of miR-141 and miR-200c showed lower levels in serums from cancer patients. Overexpression of serum miR-21 was strongly associated with lymph node metastasis and advanced clinical stage of NSCLC. Finally, log-rank and Cox regression tests demonstrated that high expressions of tumor miR 21 and miR-200c or serum miR-21 were associated with a poor survival in NSCLC patients. Our results suggest that tumor miR-21, miR-141, miR-200c, and serum miR-21 may be potential novel biomarkers for the diagnosis of NSCLC. In addition, this study, for the first time, identifies a significant role of the tumor miR-200c played in predicting prognosis in patients with NSCLC.

Nicotine inhibits cisplatin-induced apoptosis in NCI-H446 cells.

Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by certain agents including chemotherapeutic drugs. Here, we first showed that nicotine inhibits cisplatin-induced apoptosis in NCI-H446 cells. An MTT assay, Annexin V-FITC staining, RT-PCR, and Western blot were applied to identify the viability of cells, stages of apoptosis, mRNA and signaling proteins expression, respectively. First, we observed that nicotine induced no significant apoptosis when used alone and promoted cell proliferation at a low concentration or for a short time, but the opposite was observed at a high concentration or for a long time. In addition, an increase in XIAP and Survivin mRNA or protein was observed. Next, when combined with cisplatin, growth inhibition rates were concentration dependent, decreased to the lowest level at first, but later climbed to the highest point. Furthermore, nicotine inhibited apoptosis induced by cisplatin and caused a concentration-dependent increase in both XIAP and Survivin mRNA or protein. Moreover, the apoptotic effect of the combination group was obviously higher than that of nicotine used alone at the same nicotine concentration and lower than that of cisplatin used alone at the same cisplatin concentration. These studies suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutics.

[Promoter methylation status of hPer3 gene in AML patients and the in vitro effect of decitabine on the status].

OBJECTIVE: To investigate the clinical significance of promoter methylation status of hPer3 gene in acute myeloid leukemia (AML) patients and the in vitro effect of decitabine (DCA) on AML cell lines HL-60 and U937. METHODS: The promoter methylation status of hPer3 gene and mRNA expression levels in bone marrow of 206 AML and 40 iron deficiency anemia (IDA) patients (as control) were detected by methylation specific PCR (MS-PCR) and real-time PCR (RT-PCR). The HL-60 and U937 cell lines were treated with different concentrations of DCA for 48 and 72 h. The inhibition rates of cell proliferation were detected by methyl thiazolyl tetrazolium (MTT); the early apoptosis rates by staining with Annexin V and PI; the CD14 and CD11b expressions by flow cytometry (FCM); the promoter methylation status of hPer3 gene by MS-PCR; and the hPer3 mRNA expressions levels by RT-PCR. RESULTS: The promoter methylation rates of hPer3 in newly diagnosed (ND) group, partial remission(PR) group, complete remission (CR) group, relapse (R) group and control group were 93.65% (59/63), 54.39% (31/57), 24.66% (18/73), 61.54% (8/13) and 0% (0/40), and the hPer3 mRNA expression levels were 0.19 ± 0.08, 6.28 ± 2.11, 52.76 ± 14.17, 8.18 ± 4.36, 75.03 ± 18.16, respectively. There was a significant statistic difference between any two group (P < 0.01) excepting for between PR and R group (P > 0.05). After DCA treatment, the promoter hypermethylation status of hPer3 was reduced and the mRNA and CD14, CD11b expression levels were up regulated in a dose dependent manner with an induction of cell apoptosis. CONCLUSIONS: Promotor methylation status and mRNA expression of hPer3 gene may be indicators for evaluating AML. DCA can induce the expression of hPer3 gene and cells apoptosis in AML.

[Comparative study of serum levels of BMP-2 and heterotopic ossification in traumatic brain injury and fractures patients].

OBJECTIVE: To investigate the relationship between serum level of bone morphogenetic proteins-2 (BMP-2) and heterotopic ossification (HO) in traumatic brain injury (TBI) and fractures patients, providing the theoretical evidence for the clinical prevention of HO. METHODS: From December 2007 to January 2009, 145 with trama patients were selected. There were 96 closed primary traumatic brain injury patients, 1 penetrating primary traumatic brain injury, 29 fractures of the radius and ulna, 11 fractures of the humerus, 32 fractures of the tibia and fibula, 27 fractures of the femur. All patients were divided into three groups (i.e., group A, group B and group C) by the type of fracture. Fifty-seven patients in group A (TBI only), including 37 males and 20 females, ranged in the age from 29 to 61 years, with an average age of (43.91 +/- 11.09) years. The disease course was from 13 to 67 d, with an average duration of (18.96 +/- 10.46) d. Forty-eight patients in group B (fractures only), 25 males and 23 females, ranged in age from 31 to 54 years, with an average age of (41.73 +/- 8.41) years. The disease course was from 6 to 48 d, with an average duration of (16.02 +/- 8.71) d. Forty patients in group C (TBI combined with extremities fractures), including 23 males and 17 females, ranged in age from 30 to 60 years, with an average age of (45.87 +/- 14.15) years. The disease course was from 18 to 76 d, with an average of (21.28 +/- 13.02) d. Thirty-one extremities fractures with no significant separations or displacements of fragments were treated with traction reductions, cast immobilization or splint fixations. Sixty-eight fractures with significant separations and displacements of fragments were treated with intramedullary nail fixations or screw internal fixations. Sixty-three TBI patients were treated with open-skull surgeries immediately while 34 TBI patients were treated with stanching bleeding, reducing intracranial pressure and improving cerebral blood circulation. All patients were also divided into two groups (group D and group E) according to the 14-to 16-month follow-up X-ray film results. Seventeen patients in group D (HO had been found), including 11 males and 6 females, ranged in age from 29 to 55 years old, with an average age of (46.88 +/- 7.13) years. The disease course was from 6 to 30 d, with an average of (20.18 +/- 9.78) d. All 128 patients in group E (HO had been not found), including 74 males and 54 females, ranged in age from 33 to 61 years, with an average age of (43.31 +/- 12.94) years. The disease course was from 15 to 76 d, with an average of (18.42 +/- 11.58) d. The 49 subjects in group F (normal controls), 29 males and 20 females, ranged in age from 31 to 60 years, average (43.50 +/- 14.40) years. Peripheral blood samples were taken for the determination of BMP-2 in blood serum on 0.5, 3, 15 d and 30 d after fractures by enzyme-linked immunosorbent assay (ELISA). Analysis of variance and least significant difference test were done with the help of SPSS 13.0 statistic software. RESULTS: The incidence rates of HO between the TBI only patients (21.05%, 12/57) and the fractures only patients(4.17%, 2/48) were significant different (chi2=5.05, P<0.05). Serum levels of BMP-2 at 0.5, 3 d and 15 d between group A and group B were significant different. Serum levels of BMP-2 at 0.5, 3, 15 d and 30 d between group D and group E were significant different. Serum levels of BMP-2 at each time in each group were higher than the control group (51.30 +/- 23.41 ng/L) (P<0.01). CONCLUSION: High serum levels of BMP-2 in TBI only group is one of factors in causing HO. Serum level of BMP-2 at 15 d since fractures may be the obvervational index of HO prevention.

[Expression and prognostic value of vacuole membrane protein 1 in non-small-cell lung cancer].

OBJECTIVE: To investigate the expression and prognostic value of vacuole membrane protein 1 (VMP1) in non-small-cell lung cancer (NSCLC). METHODS: The clinical data and survival status of 78 NSCLC patients were collected. Their paraffin-embedded tissues were detected immunohistochemically with a custom-made rabbit anti-human VMP1 polyclonal antibody. And the clinical significance of VMP1 expression and its relationship with the overall survival rate were analyzed statistically. RESULTS: The positive expression of VMP1 could only be observed in cytoplasm of cancer cells. Among 78 patients, 40 (51.3%) patients were stained VMP1-positive. The positive rate of VMP1 had a correlation with clinical stage (χ(2) = 6.829, P < 0.05), but not with gender, age, pathological type, gross classification and differentiation grade (P > 0.05). The overall 1-and 3-year survival rate was 94.9% and 66.3% respectively. The overall estimated survival time was 37.2 months (95%CI: 33.7 - 40.8). The expression of VMP1 had significant influence on the survival rate (χ(2) = 6.192, P < 0.05). The VMP1-positive patients lived shorter with an estimated survival time of 29.0 months (95%CI: 25.0 - 33.0). VMP1 expression and clinical stage were independent risk factors of influencing the survival rate. CONCLUSION: The detection of VMP1 in resected NSCLC tumor tissues may be helpful for prognostic prediction. NSCLC patients with VMP1-positive or late clinical stage have a worse prognosis.