| BackgroundNasopharyngeal carcinoma (NPC) is one of the most incident and dangerous malignant tumors in southern China(incidence of10-150/1001000) and is one of the most common Head and Neck malignant tumors. The main treatment is radiotherapy or/and induction chemotherapy,which leading to the local or systemic sequelae affect the patient’s quality of life. And, due to that the high risk of nasopharyngeal carcinoma is relatively hidden, the first examination in most patients is associated with lymph node invasion or distant metastasis, therefore, chemo-radiotherapy becomes the standard treatment for patients with advanced nasopharyngeal carcinoma (NPC).but multi-drug resistance become one of the major factors that lead to treatment failure, recurrence and metastasis of NPC and affects the overall survival rate,it was reported that chemotherapy resistance accounted for about75%of malignant tumors. At present, tumor multi-drug resistant mechanism is unclear, and it will be the burning question how to improve the effect and to reverse drug resistance in nasopharyngeal carcinoma therapy.The multi-drug resistance of tumor cells refers to the resistance of drugs with different structure and function, it is divided into primary and acquired drug resistance, the latter is more common. To study the molecular mechanism of MDR is extremely important to improve the survival rate,reduce the recurrence and metastasis in patients with nasopharyngeal carcinoma (NPC).Some studies have shown that telomere length and telomerase activity were involved in the MDR. Our team had a systematic discussion on the role of telomerase in incidence of nasopharyngeal carcinoma (NPC), and found the over-expression of telomerase in nasopharyngeal carcinoma cells and CD133+cells, the activity of proliferation and migration was reduced by inhibiting telomerase in nasopharyngeal carcinoma cells.In recent years, PinXl is found in human and yeast cells as a conservative nucleolar protein interacting with telomere/telomerase,it is also a protein combining with telomerase pin2/TRF1interactions, and is the only protein directly binding with telomerase. The over-expression of PinX1C-terminal fragment can significantly inhibit cell telomerase activity; shorten telomere and inhibite growth of telomerase positive tumor cells, so it is considered by some scholars as a kind of endogenous telomerase inhibitor. Our previous studies have confirmed PinX1can reduce the telomerase activity of nasopharyngeal carcinoma cell lines, and promote the generation of apoptosis. On the basis of prophase research, this study intends to observe the role of PinX1gene in chemosensitization and reversing the drug resistance, and to further explore the role of telomerase, Bcl2and LRP in this process.ObjectiveTo investigate the effect of the combination PinXl and cisplatin on biological characteristics in human nasopharyngeal carcinoma cells, which provide theoretical basis of reduced chemotherapy resistance in nasopharyngeal carcinoma (NPC); To observe the adjustment of telomerase activity, the Bel-2and LRP In the process.Methods1.Construction and identification of recombinant lentivirus: First, connect pinxl to shuttle vector of lentivirus, confirmed by the double enzyme digestion and sequencing identification, shuttle vector and two other auxiliary plasmid co-transfected293T cells, collected and concentrated restructuring poison disease, virus drops degree was calculated by the concentration gradient method.2.to infect nasopharyngeal carcinoma cells, and detect the expression of PinXl:The nasopharyngeal carcinoma cells were respectively infected by Lenti-PinXl or Lenti-Crtl and named Lenti-PinXl-5-8F,Lenti-Ctrl-5-8F. We detected the expression of PinXl by fluorescence quantitative PCR and Western blotting in Lenti-PinX1-5-8F, Lenti-Ctrl-5-8F and5-8F.3.The influence of PinXl or cisplatin or the combination to the biological characteristics of nasopharyngeal carcinoma cell lines:1)measurement of NPC cells proliferation by MTT in different treatment groups: after5-8F and Lenti-PinXl-5-8Fwere cultivated with different concentrations of cisplatin for different time, OD570nm values were detected in different groups by Enzyme standard instrument and five duplicate wells were set up in each group, the OD570nm value of each group was the average of duplicate wells, which was used to calculate the growth inhibition rate and plot curve of growth inhibition, and calculate the interactions. between PinXl and effective according to the following formula: Q=E(A+B)/(EA+EB-EA*EB).2) Hoechst33258dye on cell apoptosis morphological observation:the cells of5-8F and Lenti-PinXl-5-8F at logarithmic phase were adjusted tol×104/ml,500ul cell suspension was added into the well of24-well plate. The cells were attached to the plate after12h cultivation, washed with PBS. according to the experimental group, respectively,500ul whole culture dilution of cisplatin (16ug/ml) or complete medium were added into ever plate, cells were fixed with4%para formaldehyde at room temperature after cultured for48h at37℃in a incubator supplemented with5%C02, stained with lug/ml Hoechst33258dye, then, cell morphological changes were watched through fluorescent microscope.3) Detection of the cell cycle by Flow cytometry instrument:logarithmic phase cells were collect-ed, washed with PBS, resuspended in PBS at1×106/ml, centrifuged at1000RPM for5min, after fixing with pre-cooled75%ethanol at4℃,Cellular precipitation with100ul RnaseA was incubated in the37℃water bath for30min.then, cell cycle was determined after stained by400ul PI in4℃dark environment for at least30min.4) Cells migration ability of different groups were tested by Transwell Chambers: Logarithmic growth phase cells were inoculated into the24well-plate, and were given16ug/ml cisplatin or complete medium to be cultured for48hours after being adherent, washed with PBS, they were resuspended in serum free basal medium and adjusted to Appropriate concentration.200ul cell suspension was added into the upper chamber of the transwell and500ul DMEM containing20%newborn calf serum was added into the lower chamber of the transwell.The transwell was cultured for12hours at37℃in a incubator supplemented with5%CO2.then,the cells in the upper chamber were removed with a cotton swab and the cells which migrated onto the lower surface of the membrane were fixed with4%paraformaldehyde for20min,washed with PBS and stained with crystal violet for20min. the cell number on the membrane was counted under microscope at400magnification, the number of migrated cells was expressed as the average of five randomly selected fields.5) Healing ability of nasopharyngeal carcinoma cells was tested by scratch assay: the cells of logarithmic phase were inoculated in6-well plate, when monolayer was formed, cells were scratched with a200ul tip,washed with PBS and cultured in16ug/ml n the every hole, and PBS washing off cells, respectively, serum-free culture medium diluted16ug/ml cisplatin or serum free medium.0,12,24,36h after scratching, cells in each well were observed and photographed to calculate the healing rate.4.Telomerase activity, Bcl-2and LRP were detected in nasopharyngeal carcinoma cells:Collect the logarithmic phase cells of Lenti-PinX1-5-8F, Lenti-Ctrl-5-8Fand5-8F, then the telomerase activity was detected by the Stretch PCR, the expression of Bcl-2and LRP were tested by fluorescence quantitative PCR and Western blotting, in order to understand the molecular mechanism that pinxl increases sensitivity to drug in nasopharyngeal carcinoma cells. 5.Statistical analysis:Data was analyzed using SPSS13.0statistical software package. Firstly, homogeneity of variance is tested for parameter, In the case of heterogeneity of variance, the approximate variance F test/Welch method was used. Difference between samples in qRT-PCR, Western Blotting, telomerase activity, Transwell chamber assay, cell cycle assay were tested using single factor analysis of variance and LSD/Dunnett’S T3method for multiple comparisons. Differences between samples in proliferation assay or scratch assay were tested using factorial design analysis of variance and SNK method for multiple comparisons. P value less than0.05was considered as Statistical difference, P value less than0.01was considered as significant Statistical difference.Result1.Construction and identification of recombinant lentivirus:the sequence of recombinant lentivirus was validated to be correct after digested by double enzyme, the poison disease was successfully obtained and virus drop was1.3×107Iu/ml.2.The infection of the nasopharyngeal carcinoma cells and detection of PinX1expression:green fluorescence was observed in nasopharyngeal carcinoma cells,which were infected for48hours by virus containing PinX1or empty vector virus, the group of empty vector virus was relatively strong. with extension of time, the amount of fluorescence increases, and up to the most at96hours after infection, however,uninfected cells always could not see fluorescent expression; QRT-PCR and Western blotting showed the expression of PinX1in Lenti-PinX1-5-8Fcells was more than Lenti-Ctrl-5-8F and5-8F cells.3.PinX1and cisplatin impact on the biological characteristics of nasopharyngeal carcinoma cell lines:determined by MTT test shows PinX1, Cisplatin or combination can inhibit cell proliferation of nasopharyngeal carcinoma (NPC), but inhibition of combined group significantly stronger than other group. PinX1combined8to32ug/ml Cisplatin could be more obvious synergy, and PinX1can increase the sensitivity of the nasopharyngeal carcinoma cells to cisplatin; From the aspects of morphology, Hoechst33258dyeing further stated that karyopyknosis was more obvious in combined group; Flow cytometry instrument to detect the cell cycle show the cell cycle at G0/G1phase is obvious in combined group; Transwell Chamber assay and scratch assay also confirmed that the capability of cell migration and healing ability were significantly lower than other groups.4.The influence of PinXl on telomerase activity, Bcl-2and LRP in nasopharyngeal carcinoma cells:telomerase activity was detected by Stretch the PCR and was obviously reduced in the Lenti-PinXl-5-8F cells compared with the other groups; the expression of Bcl-2or LRP was obviously reduced in Lenti-PinXl-5-8F cells by fluorescence quantitative PCR and Western blotting.Conclusions1.The combination of PinXl gene with cisplatin can increase the sensitivity of nasopharyngeal carcinoma (NPC) cells to chemotherapy, the synergy of PinXl and cisplatin when the concentration of cisplatin was16ug/ml,which show that the combined PinXl and cisplatin could enhance the curative effect of chemotherapy and also reduce the dosage of cisplatin, and it’s significance to reduce the effective systemic side effects.2.PinX1genes may increase the sensitivity of nasopharyngeal carcinoma (NPC) cells (5-8F) to cisplatin through the regulation of telomerase activity, Bcl-2and LRP. |
