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>

Long non-coding RNA influences radiosensitivity of colorectal carcinoma cell lines by regulating cyclin D1 expression / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism.</p><p><b>METHODS</b>Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines(RKO, Lovo) was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR.</p><p><b>RESULTS</b>Lovo(SF2=0.47) was more sensitivity to radiation than RKO(SF2=0.53) according to the outcome of colony formation assay. High-throughput lncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different (38.6%). There was a significant relationship between expression of several lncRNAs and CCND1 gene. Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines(P>0.05), while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines(P<0.05). After exposed to 2 Gy doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines(P<0.05), while P53 and P21 expressions were not different(P>0.05).</p><p><b>CONCLUSION</b>The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.</p>

Impacts of preoperative radiochemotherapy on operation and postoperative complications in patients with mid-low rectal carcinomas / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the impact of preoperative radiochemotherapy on postoperative complications in patients with mid-low rectal carcinomas.</p><p><b>METHODS</b>Clinicopathologic data of T3 and T4 patients with mid-low rectal carcinomas in the Department of Colorectal Surgery at the Changhai Hospital of The Second Military Medical University from January 2009 to December 2010 were analyzed retrospectively. This cohort included 81 patients treated with preoperative radiochemotherapy followed by operation(radiochemotherapy group) and 93 cases who underwent surgery alone(control group).</p><p><b>RESULTS</b>Both resection rate and sphincter preservation rate were higher in the radiochemotherapy group(100% and 86.4%) than those in the control group(94.6% and 73.1%), and the difference in sphincter preservation rate was statistically significant(P=0.039). There were no significant differences in the mean operative time [(130±15) min vs.(125±20) min, P>0.05] and mean amount of bleeding [(100±15) ml vs. (95±10) ml, P>0.05] between the two groups. The overall incidence of postoperative complications was similar(9.9% vs. 9.7%, P>0.05).</p><p><b>CONCLUSIONS</b>Preoperative radiochemotherapy can significantly increase sphincter preservation rate of mid-low rectal carcinomas, and does not increase the difficulty in surgical procedure and postoperative complications.</p>

Preliminary study on relationship between multi-drug resistance-associated protein 4 and radiosensitivity of rectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the correlation between multi-drug resistance-associated protein 4(MRP4) and the sensitivity of rectal cancer to radiation.</p><p><b>METHODS</b>A total of 95 patients with advanced rectal cancer and received radiation therapy between January 2000 and January 2009. MRP4 and P53 protein expression in the paraffin-embedded specimen were detected by immunohistochemistry. Logistic regression analysis was used to evaluate factors associated with the sensitivity of rectal cancer to radiation.</p><p><b>RESULTS</b>Forty patients(42%) were sensitive to radiation therapy, of whom 10(11%) achieved pathological complete remission. Fifty-five patients were (58%) not responsive to radiation. Patients with low expression of MRP4 had a 66.7%(24/36) response rate, significantly higher than that of patients with high MRP4 expression (29.1%,16/59)(P<0.05). Patients with low expression of P53 had a 63.9%(23/36) response rate, significantly higher than that of patients with high P53 expression(28.8%,17/59)(P<0.01). The response rate after long course radiation therapy was 83.3%(20/24), significantly higher than that of patients who underwent short and medium course radiation[(31.3%, 5/16) and(27.3%,15/55)](P<0.01). Multivariate Logistic regression analysis showed radiation regimen, the expression of P53 and MRP4 protein were independently associated with the sensitivity of rectal cancer to radiation(P<0.05).</p><p><b>CONCLUSION</b>MRP4 may serve as a predictive marker for the sensitivity of rectal cancer to preoperative radiation.</p>