Association of epithermal growth factor receptor expression and its downstream gene mutation status with radiosensitivity of colorectal carcinoma cell lines in vitro / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of epithermal growth factor receptor (EGFR) expression and K-ras, B-raf and PIK3CA mutation status on the radiosensitivity of human colorectal carcinoma (CRC) cell lines in vitro.</p><p><b>METHODS</b>Real-time RT-PCR was used to measure EGFR mRNA expression in nine human CRC cell lines, and K-ras, B-raf and PIK3CA mutation status of each CRC cell line was also identified respectively. After treatment with irradiation at graded dose, the cell viability was measured by clonogenic survival assay. The rate of cell apoptosis and cell cycle distribution were tested by flow cytometry. The cell morphology was observed with hoechst 33258 staining to analyze the correlation between EGFR mRNA expression and radiosensitivity of CRC cell lines.</p><p><b>RESULTS</b>A positive correlation between EGFR mRNA expression and survival fraction of 2 Gy(SF2) was observed (r=0.717, P=0.030). Association was also identified between the mutation status of PIK3CA and radiosensitivity (t=2.401, P=0.047), while mutation status of K-ras and B-raf was not associated with radiosensitivity. At 48-hour after exposing to irradiation, the apoptosis rate of radiosensitive cell line (HCT116) was significantly increased in a dose-dependent manner (P<0.05), while the apoptosis rate of radioresistant cell line (HT29) was significantly increased only when radiation dose increased to 6 Gy. The ratio of G0/G1 phase was reduced significantly with the increase of radiation dose in radiosensitive cell line (HCT116, P<0.05), while this trend was not observed in radioresistant cell line (HT29, P>0.05).</p><p><b>CONCLUSIONS</b>Over-expression of EGFR mRNA is correlated to radioresistance of human CRC cell lines, and mutation status of PIK3CA is closely related with radiosensitivity of CRC cells. The inhibition of apoptosis and G0/G1 arrest may induce the radioresistance of CRC cell lines.</p>

Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.</p><p><b>METHODS</b>The vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.</p><p><b>RESULTS</b>ShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).</p><p><b>CONCLUSIONS</b>Expression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.</p>

Molecular evolutionary analysis of matrix protein and nucleoprotein genes of novel influenza virus A/H1N1 in 2009 pandemic / 第二军医大学学报

Objective: To analyze evolutionary characteristics of the matrix protein (M) and nucleoprotein (NP) genes of influenza virus A/H1N1 in 2009 pandemic. Methods: The M and NP genes of A/H1N1 viruses were downloaded from NCBI database. MEGA4.0 software and NJ method were used for sequence alignment, protein sequence alignment, and the phylogenetic tree construction. Meanwhile, Epi Info software was used to analyze the linear trend of evolutionary distance of the M and NP genes of human H1N1 strains isolated during 1918 to 2009. Results: The M and NP gene sequences were similar among the novel A/H1N1 viruses, but different from those of the previous influenza H1N1 viruses. Using reference sequences of human H1N1 strains isolated during 1918 to 2008, we found that changes in evolutionary distances of the M genes between novel A/H1N1 strains and each of the reference A/H1N1 strains increased with increasing year intervals (Ptrend = 0.001). Compared with the amino acid sequence of M2 protein of reference human A/H1N1 virus strains isolated during 1918 to 2008, the novel A/H1N1 viruses had the amino acid substitutions at 6 sites: 11, 43, 54, 57, 77, and 78. Compared with swine and avian A/H1N1, the novel A/H1N1 virus only had the amino acid substitutions at 43 and 77. Conclusion: The NP gene of novel A/H1N1 virus, which is routinely considered as a conserved sequence, is different from those of the previously isolated human H1N1 influenza viruses; the related mechanisms and consequences on viral activity remain to be elucidated. The substitution to threonine at 11 and 43 amino acids of M2 protein might contribute to amantadine resistance of the novel H1N1 virus pandemic in 2009.

Epidemic situation of emerging influenza virus A/H1N1 in 2009 pandemic and the prevention/control measures / 第二军医大学学报

Since the first reported case of swine influenza A in Mexico, a total of 15 510 cases have been confirmed in 53 countries by May 29, 2009. On April 29, 2009, the World Health Organization (WHO) raised its pandemic alert from grade 4 to grade 5. The virus is described as a new subtype of A/H1N1 and is not detected in pigs or humans previously. The virus is sensitive to oseltamivir and zanamivir, but resistant to amantadine and rimantadine. The genetics of the virus are so novel that humans are unlikely to have much immunity to it. The virus can transmit from human to human; therefore it is necessary to enforce quarantine measure for close contactors because the virus transmits during latency. Precaution methods like covering noses and mouths with a tissue when coughing or sneezing can reduce the transmission opportunity. Hands should be washed frequently with soap, especially after coughing or sneezing. Public places with ventilation conditions, personal health behavior and health condition are critical for the prevention and control of this epidemic.