1.Biological And Molecular Markers For Radiotherapy Response Of Nasopharyngeal Carcinoma 2.The Abnormal Expression Of OLC1 Gene In Bladder Cancer And Its Biomedical Significance

Chapter 1:Biological and molecular markers for radiotherapy response of nasopharyngeal carcinomaHistopathological diagnosis on endoscopic biopsy is the golden standard for the diagnosis of nasopharyngeal carcinoma(NPC).Radiotherapy is the first choice and the most effective treatment for primary nasopharyngeal carcinoma.Since about a quarter of patients is not sensitive to radiotherapy,it will be helpful to develop more individualized therapeutic plan and improve the treatment effect for them,if the response could be estimated before the radiotherapy.Therefore,it is important to discover and identify the biological and/or molecular markers for the radiotherapy response of NPC.In this study,we firstly investigated the correlation between the tumor microvessel exposure under endoscopy and the tumor regression after radiotherapy,with a cohort of 100 cases of NPC.We subsequently screened the microRNAs(miRNAs)and mRNAs associated with the radiotherapy response of NPC.With review of the clinical data of 100 cases of NPC,we found that the microvessel exposure on the tumor surface under the narrow band imaging(NBI)endoscopy was positively correlated with the tumor shrinkage after radiotherapy with 50Gy(p=0.006,X2=19.268).Based on this finding,VEGF,EGFR and Ki67 proteins in the tumor tissues of 89 NPC patients were checked using immunohistochemical analysis,and the results showed that the expression status of the proteins is consistent with the microvessels exposure on the tumor surface revealed by the NBI endoscopy.With gene microarray technique,the miRNA expression profiles were generated from the tissue samples of 10 NPC patients with sensitive radiotherapy response and 10 NPC patients with insensitive radiotherapy response.Based on bioinformatic analysis,78 and 41 differentially expressed miRNAs were screened out from the tumor vs adjacent tissue group and the high radiosensitivity vs low radiosensitivity group.The miRNAs’ target gene prediction was performed using 3 public databases:miRBase,TargetScan and PicTar.The target genes were predicted in all 3 databases for the tumor vs adjacent group,and in at least two databases for the high radiosensitivity vs low radiosensitivity group.Panther software was used to analyze the biological function of the target gene;and Angiogenesis pathway was identified both in the tumor vs adjacent group,and the high radiosensitivity vs low radiosensitivity group.Using next generation sequencing technique,the mRNA expression profiles were generated from the tissue samples of 5 NPC patients with sensitive radiotherapy response and 5 NPC patients with insensitive radiotherapy response.There are 3432 up-regulated genes and 4453 down-regulated genes between the tumor vs adjacent tissues,379 up-regulated genes and 392 down-regulated genes between the microvessels exposed on the tumor surface vs the non exposed group,115 up-regulated genes and 132 down-regulated genes between high radiosensitivity vs low radiosensitivity group.A total of 105 common genes were detected between these three groups.Signal pathway enrichment analysis showed that in the NPC cases investigated,Jak-STAT pathway and VEGF signalling pathway were associated with radiotherapy response,which is consistent with the clinical observation about the micro vascular exposure on the tumor surface derived from 100 cases of NPC.In addition,the transcription sequencing data showed that the expression of some Epstein-Barr virus(EBV)genes,such as EBER1 and EBER2,were highly expressed in the NPC tissues,and associated with the tumor shrinkage after radiotherapy.In conclusion,the status of microvessel exposure on the tumor surface is correlated with the tumor shrinkage after radiotherapy of NPC.This phenomenon could be interpreted by the findings that some abnormally expressed miRNAs and genes associated with the radiotherapy response of NPC involved in the Angiogenesis pathway.EBV encoding genes also play an role in the radiotherapy response.The microvessel exposure on the tumor surface under the NBI endoscopy,and abnormality of some genes from both host and EBV may serve as potential biomedical and molecular markers for predicting the radiotherapy response of NPC,before the treatment.Chapter 2:The abnormal expression of OLC1 gene in bladdercancerand its biomedical significanceThe OLC1(Over-expressed in Lung Cancer 1)gene was cloned and identified firstly in this laboratory,and then was found to be abnormal in a variety of malignant tumors.In present study,we analyzed the expressive status of OLC1 in bladder urothelial carcinoma and the clinical significance,then investigated its biological function.On mRNA level,RT-PCR analysis showed that the expression of OLC1 in the tumor tissues was significantly higher than that in normal bladder mucosa tissues(p<0.001),and that in the muscle invasive bladder cancer(MIBC,stage T2-T4;41/70)was higher than that in the non-muscle invasive bladder cancer(NMIBC,stage T0-T1;29/70)(p<0.05),and that in smokers(24/70)is higher than that in non-smokers(46/70)(p<0.01).On protein level,immunohistochemical analysis showed that high level OLC1 was detected in 37.7%(43 cases)of the tumor tissues;among them,52.7%(29 cases)of MIBC,and 23.7%(14 cases)of NMIBC showed the high expression.The expression level of OLC1 protein was significantly correlated with the pathological stage(p<0.001)and the smoking history of the patients(p=0.022)of bladder cancer.Kaplan-Meier analyses were performed to determine the association between OLC1 protein expression and progression-free survival(PFS)and overall survival(OS)of 106 bladder cancer patients.The 5-year OS rate for the patients with high level OLC1 in the tumor was statistically significantly lower than those with low level OLC1(p=0.04).Additionally the 5-year OS rate for the smoking patients with high level OLC1 protein was significantly lower than those of non-smoking patients with low level OLC1(p=0.01).In vitro experiments show that stable over-expression of exogenous OLC1 gene increased proliferation of bladder cancer cells T24 and 5637,and also enhanced the migrating and invasive ability of the cells.In conclusion,OLC1 gene was over-expressed in bladder cancer,which correlated with the tumor invasion,5-year OS and cigarette smoking status of the patients.The OLC1 could be a potential prognosis biomarker for bladder cancer.