| Introduction:The main reason of the difficulty of curing malignant tumors is that they are not easy to be detected early. Once clinical manifestation appears, the majority has reached the middle or advanced stage, with extensive invasion or metastasis and poor clinical treatment effect. At present, the optimal strategy for anti-cancer depends on early detecting and close monitoring for early recurrence to adjust treatment plan reasonably. Development of minimally invasive biomarker assays for early detection and effective clinical management of pancreatic cancer is urgently needed to reduce high morbidity and mortality associated with this malignancy. There has been widespread consensus that bloodstream is considerably suitable for the detection of noninvasive biomarker in population. However, few serum (or plasma) markers with high specificity and sensitivity have been applied. Nasopharyngeal carcinoma (NPC) is seldom seen in western countries, however, it has a high incidence in China and it is also called’Chinese Cancer’. The morbility of nasopharyngeal carcinoma is very concealed without obvious clinical manifestation in earlier period. It is not easy to be detected and the diversion of lymphatic gland happens early. Therefore, early diagnosis of nasopharyngeal carcinoma is very urgent in China. At present, the detecting of the infection status of EBV (eg. EBV-DNA, BV-VCA-IgA, etc) has been commonly used in clinical screening for nasopharyngeal carcinoma. However, nasopharyngeal carcinoma is not completely related with EBV. People with other diseases (eg. lymphoma, gastric carcinoma, breast cancer, pharyngitis, acute lymphadenosis, etc) have EBV infection, and a proportion of healthy individual also has EBV infection. A large number of clinical practice demonstrate that the sensitivity and specificity of EBV-related detection for diagnosing nasopharyngeal carcinoma are poor, which can not meet the requirements of early diagnosing.It had been demonstrated that cancer is a product of the tissue microenvironment. Serum contains a large number of low molecular weight (LMW) peptides which are "records" for the cellular and extracellular events that take place at the level of the cancer-tissue microenvironment. They could be a rich source of cancer-specific diagnostic information. The blood peptidomic were a higher dimension of information content for cancer biomarker discovery. There is a growing interest in the research of using serum peptidome as markers in a series of tumor.MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level by degrading or blocking translation of messenger RNA (mRNA) targets. MiRNAs play important regulatory roles in a variety of cellular functions as well as in several diseases, including cancer. MiRNA-specific expression profiles have been reported for several pathological conditions. Recently, the discovery of miRNAs in serum opens up the possibility of using miRNAs as biomarkers of disease. The potential of microRNAs (miRNAs) as novel tumor markers has been the focus of recent scrutiny because of their tissue specificity, stability, and association with clinicopathological parameters. Therefore, circulating microRNAs (serum miRNAs/plasma miRNAs) were looked as stable blood-based markers for cancer detection and circulating microRNAs have been reported as potential biomarkers for the noninvasive diagnosis of cancer.Objectives:The aim of this study was to compare serum peptide profiling and serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma; to screen for serum molecular biomarkers(serum peptide pattern or serum miRNA) and establish diagnosis model to improve early diagnosis, disease monitoring and prognosis evaluation; and to provide possible target for treatment of nasopharyngeal cancer.Methods:1. Formulate standard procedure for serum sampling, extensive serum specimen of tumor free population and patients with nasopharyngeal carcinoma were gathered (including prior treatment, after one course of treatment), basic and clinical data were consummated, and the storeroom of serum specimen was established; 2. EBV-VCA-IgA ELISA was used to detect the infection of EBV in all serum specimen.3. WCX Magnetic bead combined with MALDI-TOF mass spectrometry were performed to obtain serum peptide profiling of tumor free population and patients with nasopharyngeal carcinoma, and these two groups of serum peptide profiling were compared. Clinpro Tools and CARS were used to establish diagnosis model of serum peptide pattern of nasopharyngeal carcinoma, the variation in different clinical stages was observed, and the relation between the prognosis and serum peptide profiling was analyzed.4. TaqMan Low-Density array (TLDA) was used to obtain serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma, quantity real-time PCR was used to verify and screen for serum miRNAs with differential expression, the diagnosis model of serum miRNAs of nasopharyngeal carcinoma was established, and its diagnostic efficiency was verified, and the relation between the prognosis and serum miRNAs with differential expression was analyzed using Kaplan-Meier.Results:1. Serum specimen of tumor free population and patients with nasopharyngeal carcinoma were gathered, and the storeroom of serum specimen with complete information was primarily established.2. The positive rate of serum EBV-VCA-IgA was 11.4% in tumor free population, and 66.2% in patients with nasopharyngeal carcinoma. The latter was significantly higher than the former.3. A series of experiment condition affecting results were discovered and a reasonable and stable experiment flow-sheet was formulated by exporing the experiment condition (parameter) of the magnetic bead combined with MALDI-TOF mass spectrometry.4. The result showed that age had great influence on the serum peptidome profiling, but gender had little influence compared with age.5. Serum peptide profiling of tumor free population and patients with nasopharyngeal carcinoma were obtained based on WCX magnetic bead and MALDI-TOF mass spectrometry. 6. The diagnosis model of serum peptide profiling of nasopharyngeal carcinoma was established by using Clinpro Tools. The specificity was 85.7% and the sensitivity was 80.7%.7. The discrimination equations of diagnosis of nasopharyngeal carcinoma was established by using CARS algorithm:Y=0.2884X1+0.2073X2-0.2223 X3+0.3985 X4+0.2446 X5 (X1:1741.10Da; X2:2991.11Da; X3:3192.36Da; X4:3890.71Da; X5: 5064.67Da)The sensitivity was 93.50% and the specificity was 84.08%, AUC was 0.9425.8. The variation of serum peptide profiling happened in the early stage of nasopharyngeal carcinoma, but there are not evident variation among different clinical stages of nasopharyngeal carcinoma, by observing the variation of serum peptide profiling in different stages.9. The variation of serum peptide profiling was a litttle between pre-treatment and post-treatment of nasopharyngeal carcinoma.10. The difference of serum peptide profiling was not obvious in nasopharyngeal carcinoma patients with positive and negative EBV.11. The survival curve suggested that the 3890Da serum peptide was related with prognosis.12. The expression of the 103 and 125 miRNAs were detected by using TaqMan Low-Density array(TLDA) to obtain serum miRNAs expression profiling of nasopharyngeal carcinoma patients and tumor free population.13. The expression of miR-17、miR-20a、miR-486-3p increased, and the expression of miR-29c、miR-223 decrease in serum of nasopharyngeal carcinoma by analyzing and verifying the serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma.14. The diagnosis model of serum miRNAs of nasopharyngeal carcinoma was established A=(CtmiR-29c+CtmiR-223)-(CtmiR-17+CtmiR-20a)>-3.30 (A>-3.30:nasopharyngeal carcinoma; A<-3.30:tumor free), and its sensitivity and specificity were more than 96%. 15. The survival curve showed serum miR-20a was related with prognosis of nasopharyngeal carcinoma.Conclusion:1. Serum peptide was affected by disease and many other factors, so the experiment condition in research of serum peptidome should be standardized strictly.2. There were difference in serum peptide profiling and serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma.3. The variation of serum peptide pattern was an early incident of nasopharyngeal carcinoma.4. The reliability of the diagnosis model of serum peptide profiling and serum miRNAs of nasopharyngeal carcinoma was significantly higher than the EBV-VCA-IgA detection. The diagnosis model could be applied in clinical diagnosis of nasopharyngeal carcinoma, and could provide reference for evaluating the prognosis of nasopharyngeal carcinoma. |
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