Effect Of ShRNAmir Lentiviral Vector-mediated COX-2 Silencing On The Proliferation, Metastasis And Radiochemosensitivity Of Nasopharyngeal Carcinoma Cells

BACKGROUND AND OBJECTIVENasopharyngeal carcinoma(NPC) is a common malignant epithelial tumor in China, with significant features of distinct endemic distribution, marked tendency to metastasis and close relationship with the EB virus. Radiotherapy is the mainstay treatment for this tumor. But due to its serious complications and sequela, treatment results are unsatisfactory and 5-year overall survival rate has remained at about 50%. Therefore, exploring the molecular mechanism of pathogenesis of NPC and searching for new therapeutic targets to enhance the efficacy of existing strageties is the urgent task of contemporary oncology investigation.COX-2 is one of the two cyclooxygenase isoforms, which is usually absent from most normal cells and tissues, but is induced by growth factors, tumor promoters and cytokines. Inhibiting the expression of COX-2 can enhance tumor response to radio-or chemotherapy. ShRNAmir is a hairpin small interfering RNA, which is reconstructed on the base of the microRNA structure, representing the latest technology of artificial induction of RNA interference and silencing gene expression by utilizing the shRNAmir vector in order to transcribe into shRNAmir which is similar to miRNA,more safely and effectively than the traditional one.In our previous study, we have silenced the COX-2 expression in NPC cells with the application of RNA interference technology, causing the cell cycle arrested in G0/G1 phase and proliferation decreased. With the application of RNA interference technology based on the structure of miR-155, we successfully constructed Anti-COX-2 shRNAmir lentiviral vector. After 48-72h infection of lentivirus to NPC cells, the positive cells emit green fluorescence and the infection efficiency can up to 90.5%.Then selected by flow cytometry, the proportion of fluorescent cells still over 99% after 3 months of serial subculture. COX-2 mRNA gene expression can nearly undetectable by RT-PCR and the NPC subline which has a stable inhibition of COX-2 expression is obtained. It proved that shRNAmir can produce an increased and more consistent knockdown compared with anti-COX-2 siRNA. Application of this technology to the study of the molecular mechanism of NPC is laying foundations for further molecular targeted therapy and gene-radiotherapy.On the basis of the previous study, we aim to explore the possible role of COX-2 gene in the proliferation, metastasis and sensitivity to chemo- or radiotherapy for nasopharyngeal carcinoma and provide a theoretical foundation for comprehensive treatment of nasopharyngeal carcinoma patients.Methods:1. The influence of COX-2 gene silencing on the growth characteristics of nasopharyngeal carcinoma cellsExponential phase cells (2×103cells/well) were planted in the 96-well plate with 100ul cell suspension per well. At 1,2,3,4 and 5d after seeding, cells were treated with 10ul CCK-8 solution for 1h at 37℃. Spectrometric absorbance at wavelength of 450nm was measured on a microplate reader.2. Wound migration assayEqual numbers of cells (2×105) were seeded into 6-well culture plate, each group has three duplicate wells. The injury line was made with a sterile plastic pipette tip on cells cultured in plates at 90% confluency. After being rinsed with phosphate-buffered saline (PBS), cells were cultured in complete medium and migration of cells into the wound was observed at different time points, and then photographs were taken under a 40×objective.3. Sensitivity detection of COX-2 silencing NPC cells to cisplatinCCK-8 assay was performed to assess the sensitivity of cell to cisplatin. Cell were plated in 96-well culture plates at a density of 5×103 cells/well, with 100ul cell suspension per well.24h after seeding, the medium was replaced with fresh complete medium containing various concentrations of cisplatin. After 24h culture in the presence of cisplatin, lOul of CCK-8 was added to each well for 1h incubation. The OD value was read with microplate reader at a wavelength of 450nm. Growth inhibition rates were calculated and IC50, which was the concentration inducing 50% inhibiton of cells, were analyzed with SPSS 13.0 sofeware.4. Cell cycle assaysAfter 24h incubation in the presence of cisplatin at the concentration of 6ug/ml, or after 24h and 48h irradiation at the dose of 2Gy, cells were harvested, washed in PBS, and then fixed with ice-cold 70% ethanol at 4℃overnight. When ready to stain with, cells were rinsed twice in PBS. Then cells were stained with PI at room temperature for 30min. Cell cycle phase distribution was analyzed by flow cytometry.5. Clonogenic survival assay Predetermined number of cells were seeded in 6-well culture plates, and exposed to 0,1,2,4,6 and 8 Gy of irradiation. Then cells were incubated at 37℃,5%CO2 for growth. After two weeks of incubation, when colonies were visible, they were fixed with methanol and stained with 1% crystal violet. Colonies which contained more than 50 cells were manually counted. Then planting efficiency (PE) and surviving fraction(SF) were calculated as follows:PE=colony number/inoculating cell number×100%. SF=PE (tested group)/PE (OGy group)×100%. Then survival curves were plotted and radiobiological parameters were calculated.6. In vivo studiesFemale BABL/c nude mice, aged 4-6 weeks, weighting 18-22g were housed in a specific pathogen-free animal facility. Mice were injected subcutaneously in the right hind limb with 1×105 cells. During experiment, two orthogonal dimensional diameters of each tumor were measured using vernier caliper every other day. The tumor volume was calculated by the formula L×W2/2. When tumors reached an average volume of about 200mm3, the mice were divided into four groups:COX-2 (+) control group, COX-2(-) control group, each of these two groups received no treatment; COX-2(+) radiation group, COX-2(-) radiation group, each tumor of these two groups were exposed to lOGy X-ray alone. Two weeks later the tumor-bearing nude mice were killed. Tumor inhibition rate was calculated.7. Statistical analysisSPSS 13.0 software package was used to carry out statistical analysis. Results are presented as mean±SD. Comparison of mean was analyzed by One-Way ANOVA or Independent-Sample T Test. Factorial analysis was utilized for analyzing the results of CCK-8 and wound migration assay. Student-Neuman-Keuls method was used for multiple comparing. A P value less than 0.05 was considered statistically significant.Results:1. The influence of COX-2 gene silencing on the abilities of proliferation and migration for nasopharyngeal carcinoma cellsAnti-COX-2 C666-1 and Anti-GL-2 C666-1 cells were inoculated in 96-well plates with continuous observation for 5 days. Growth curves were plotted according to the OD value at 450nm. Factorial analysis showed that the ability of proliferation was significantly decreased in the COX-2 negative group(F=5.927, P=0.019).In vitro wound migration assay was carried out to detect the ability of cell migration in the presence or absence of COX-2 gene expression. Factorial analysis showed that, width of injury line between these two groups was significantly different (F=157.01, P<0.001), indicating that knockdown of COX-2 expression decreased the ability of in vitro migration.2. Sensitivity alteration of COX-2 silencing NPC cells to cisplatinFactorial analysis showed that, after 24h treated of various concentrations of cisplatin, inhibition rate of cell proliferation between two groups were significantly different (F=22.219, P<0.001). IC50 calculated by Anti-COX-2 C666-1 cells and Anti-GL-2 C666-1 cells to cisplatin has statistically differences (t=-3.489, P=0.025), the value were 3.682±0.980 and 8.819±2.354, respectively.3. Alteration of cisplatin-induced cell cycle by silencing COX-2 gene expressionFlow cytometry analysis showed that,before the treatment of cisplatin, no significant difference can be seen in the proportion of both G0/G1 and S phase cells between Anti-COX-2 C666-1 and Anti-GL-2 C666-1 cells. However, a significant difference can be seen at G2/M phase between these two groups. After 24h treatment of cisplatin, S and G2/M peak were increased in both of these two groups. No statistically difference can be seen in the S-phase change between these two groups, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 24h after treatment of cisplatin, indicating that silencing of COX-2 gene expression significantly decreased the cisplatin-induced G2/M phase arrest.4. The influence of Sensitivity of COX-2 silencing NPC cells to radiation in vitroThe values of D0, Dq, SF2 andα/βwith C666-1 cells before and after silencing of COX-2 gene expression were all statistically significant. The ratio of enhanced radiotherapy sensitivity was 1.4014.5. The influence of radiation-induced cell cycle by silencing COX-2 gene expressionFlow cytometry analysis showed that, G2/M peak were increased in both of these two groups 24 and 48h after radiaton, but G2/M arrest decreased dramatically in Anti-COX-2 C666-1 after radiation, indicating that silencing of COX-2 gene expression significantly decreased the radiation-induced G2/M phase arrest.6. The influence of Sensitivity of COX-2 silencing NPC tumor-bearing nude mice to radiationIn the non-radiotherapy groups, the xenograft tumor growth curves of COX-2(+) and COX-2(-) groups were almost overlaped. After radiotherapy of lOGy alone to both of these two radiation groups, the inhibition rates were significantly different, the value were 32.72% and 51.76%,respectively.Conclusion:1.Silencing of COX-2 gene expression decreased the abilities of proliferation and in vitro migration in nasopharyngeal carcinoma cells.2.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to cisplatin and decreased the cisplatin-induced G2/M phase arrest.3.Silencing of COX-2 gene expression enhanced the sensitivity of NPC cells to radiation both in vitro and in vivo, and decreased the radiation-induced G2/M phase arrest.