The Expression Of Clock Gene CLOCK And Its Clinical Significance In Nasopharyngeal Carcinoma

Objective:To observe the expression and rhythm of the clock gene CLOCK in nasopharyngeal carcinoma through molecular biology detection methods.In this study,we explored the relationship between CLOCK gene m RNA / protein expression levels and patient clinical characteristics,which including: tumor stage,therapeutic effect and survival data.This study plays an important role in clinical evaluation for the therapeutic effect and prognosis of patients.Besides,this study could provide a certain theoretical and research data in the targeted therapy.Methods:The nasopharyngeal carcinoma cell lines CNE1,CNE2,5-8F and the normal nasopharyngeal cell line NP69 were Cultured.Real-time fluorescent quantitative PCR and Western blot(WB)were used to detect the m RNA and protein expression level of CLOCK.At the same time,CNE1,CNE2,5-8F,and NP69 cell lines were cultured in vitro in dark conditions.Real-time fluorescent quantitative PCR was used to detect the clock gene CLOCK in each cell line at the time points of ZT2,ZT6,ZT10,ZT14,ZT18,and ZT22.The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma.33 cases of nasopharyngeal carcinoma and 17 cases of chronic inflammation of the nasopharyngeal fresh specimens was collected.By using real-time fluorescent quantitative PCR method we detected the expression of CLOCK m RNA.At the same time,68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue wax blocks were collected,and the expression of CLOCK protein was detected by using immunohistochemical method.The expression levels of CLOCK m RNA and protein in nasopharyngeal carcinoma tissues were analysized by relative quantitative method and graphical analysis software.Besides,we also explored their relationship with different clinical features and prognosis value.All patients in this study received 2 to 3 cycles of platinum-based induction chemotherapy combined with concurrent chemotherapy and radiotherapy.Induction chemotherapy regimen: TPF(specific regimen: docetaxel(T)75 mg/m2,d1,intravenous infusion;cisplatin(P)75 mg/m2,d1-5,continuous intravenous pump infusion;5-fluorouracil(F)750 mg/m2/d,d1-5,continuous intravenous pump infusion)or TP(Docetaxel(T)75 mg/m2,d1,intravenous infusion;cisplatin(P)75 mg/m2,d1-5,continuous intravenous pump infusion)or GP(gemcitabine(G)1000mg/m2,d1,d8,intravenous infusion;cisplatin(P)80mg/m2,d1,continuous intravenous pump infusion),21 days/cycle,A total of 2-3 cycles.Concurrent chemotherapy regimen: paclitaxel 135mg/m2,intravenous infusion,d1 or cisplatin 100mg/m2,intravenous infusion,d1,21 days/cycle,a total of 2-3 cycles.Radiotherapy: Intensity-modulated radiotherapy is used.The primary nasopharyngeal lesions were DT69.96～73.92Gy/33 f,positive lymph nodes DT69.96Gy/33 f,cervical lymph node high-risk drainage area DT60.06Gy/33 f,low-risk drainage area DT50.96Gy/28 f.The short-term efficacy evaluation was conducted in accordance with the 2000 evaluation criteria for therapeutic efficacy of solid tumors(RECIST1.1).Effective is complete remission + partial remission(CR+PR),and invalid is stable + progress(SD+PD).Results:The expressions level of CLOCK m RNA and protein in nasopharyngeal carcinoma cell lines CNE1,CNE2,5-8F were significant lower than those of normal nasopharyngeal cell line NP69(P<0.05).RT-PCR results showed that the expression level of clock gene CLOCK m RNA in nasopharyngeal carcinoma cell lines CEN1,CNE2,5-8F and normal nasopharyngeal cell line NP69 was different at different time points,and there were temporal fluctuations.CLOCK gene expression in CNE1 cells fluctuates in a cycle of 16 hours,and its peak and trough times are at ZT10:40 and ZT18:40,respectively.CLOCK gene expression in CNE2 cells fluctuates in a cycle of14 hours,and its peak and trough times are at ZT10 and ZT3,Respectively.CLOCK gene expression in 5-8F cells fluctuates in a cycle of 22 hours,and the peak and trough times are ZT14:30 and ZT3:30,respectively.CLOCK gene expression in NP69 cells fluctuates in a cycle of 24 hours the peak and trough times are ZT12:39and ZT0:39,respectively.The relative expression level of CLOCK m RNA in nasopharyngeal carcinoma tissues were 0.367±0.205,which were significantly lower than that in rhinitis chronic inflammation tissue(1.000±0.000)with statistically significant(P<0.001).The expression of CLOCK protein in nasopharyngeal carcinoma tissue was low,and the optical density value is 0.201±0.261,which was also significantly lower than chronic nasopharyngeal inflammation(0.512±0.414)(P=0.000).In nasopharyngeal carcinoma tissues with high expression of CLOCK protein,the OS at 1,3,and 5 years was significantly higher than that in the low expression group,which were 96.2% vs.92.9%,92.1% vs.78.6%,80.1% vs.57.1%,respectively.The difference between these groups was statistically significant(P=0.009).The 1,3,and 5-year PFS of the CLOCK protein high expression group and the low expression group were 96.2 vs.92.7%,87.8% vs.82.2%,and 87.7% vs.70.8%,respectively.The PFS of the high expression group has an increased situation in the lower expression group,but the difference between these two groups was not statistically significant(P=0.105).The 1,3,and 5-year RFS of in high expression protein level group of CLOCK and the low expression group were 100.0% VS.100.0%,95.7% VS.96.9%,and 95.7% VS.90.0%,respectively.However,there was no statistically significant difference between the two groups.(P=0.514).The 1,3,and 5-year DMFS of in high expression protein level group of CLOCK and the low expression group were 96.2% VS.92.7%,92.0% VS.82.2%,92.0% VS.79.3%,respectively.The DMFS of the high expression group has an increased situation in the lower expression group,but the difference between the two groups was not statistically significant(P=0.136).Cox multivariate regression analysis of CLOCK protein expression in nasopharyngeal carcinoma tissue is an independent prognostic factor that affects OS(P=0.004).The expression level of CLOCK m RNA in nasopharyngeal carcinoma tissue was not related to the patient’s age,gender,tumor T stage,M stage(P>0.05).However,the expression level of CLOCK m RNA in nasopharyngeal carcinoma tissue was correlated with N stage.Besides,the expression level of CLOCK in nasopharyngeal carcinoma patients was much higher in N0+N1stage compared with N2+N3 stage patients(p=0.009).However,the expression level of CLOCK protein in nasopharyngeal carcinoma tissues was not related to the patient’s age,gender and tumor stage(P>0.05).Conclusions:Clock gene CLOCK was expressed in nasopharyngeal carcinoma cell lines and normal nasopharyngeal cell line with rhythmically.Compared with normal nasopharyngeal cell line,the fluctuation period of nasopharyngeal carcinoma cell lines was shortened.This phenomenon may related to the cell rhythm disorder,which leading to an increase in tumor growth rate and leads to a rhythmic expression disorder.Besides,The OS of the CLOCK protein high expression group was significantly higher than that of the low expression group,and there was a tendency to prolong PFS,RFS,and DMFS.Furthermore,the expression level of CLOCK protein is an independent prognostic factors that affect OS.The CLCOK gene in nasopharyngeal carcinoma cell lines and nasopharyngeal carcinoma tissues is low expressed,while the CLCOK gene in normal nasopharyngeal cell lines is highly expressed.Based on the above research results,we speculate that the clock gene CLOCK may be a potential tumor suppressor gene in nasopharyngeal carcinoma.However,we still need further studies to confirm it.