Primary Screening And Identification And Functional Research Of Serum Biomarker In Nasopharyngeal Carcinoma Patients

Objective: Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially southern of China. The treatment of many NPC patients was delayed before because of the depth of lesion location, the slur of early symptom and the lateness of correct diagnosis. Therefore, it is urgent to screen for a serum biomarker used to early screening and diagnosis of NPC. This study applied surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI) to screen a marker protein early in the serum of NPC patients. Then the differential proteins were identified. Moreover, the function of confirmed Serum amyloid protein A (SAA) was primarily studied in order to clarify the relationship with NPC generation.Methods: Subjects included 113 NPC patients, 67 nasopharyngeal benign disease patients (Benign) and 82 normal controls. Their serum were collected and stored for use at a temperature of -80℃. Protein Chip CM10 was applied to protein detection and the results was analyzed by Biomarker Wizard? 3.2 software. The parameter was set up as following, signal-noise ratio was 5, laser energy was 220, test sensitivity was 9 and the molecular range was 3000-50000D. The protein peak which was different between NPC, Benign and controls was identified by Tricine-SDS-PAGE gel and spectrometry identification. And the expression of the protein in the serum of NPC, Benign and Controls was further tested by Western-blotting. Meanwhile, protein expression in CNE2, HNE1 and normal nasopharyngeal epithelial cell (NEC) was detected by RT-PCR and Western-blotting. The gene sequence of the screened biomarker protein was looked up on NCBI (National Center for Biotechnology Information). The pSIREN-Shuttle (Clontech) was used to RNA interference (RNAi) expression vector and 3 target gene regions and a negative control gene region were selected according to the guide of pSIREN-Shuttle. Four RNAi expression vectors were constructed and transferred into CNE2. The interference effect of the target protein expression was tested by RT-PCR and Western-blotting. Then, the positive CNE2 whose target protein expression was suppressed was screened. And the cell growth speed, proliferation and cell colony forming of RNAi positive CNE2 was compared with that of RNAi negative CNE2 by MTT, flow cytospectometry and cell colony forming rate calculation.Results: (1) There was a protein peak (11.72km/z) in 102 serums of NPC patients (positive rate 90.27%). The intensity of the protein peak was significantly higher than that of Benign patients and Controls (t=6.34, P<0.05). The protein whose protein peak was 11.72km/z was identified as Serum Amypliod Protein A (SAA) by Tricine-SDS-PAGE gel and spectrometry identification. (2)The expression of SAA was detected high in the serum of NPC but low in the serum of Benign and Control by Western-blotting. The results of cell experiments showed that SAA expressed high in two nasopharyngeal carcinoma cell strains CNE2 and HNE1 but low in normal nasopharyngeal epithelial cell. (3)Three siRNA expression vectors were constructed successfully and pSS-SAA2 could effectively suppress the expression of SAA in CNE2. Compared with CNE2, the growth speed of the interference CNE2 was slower, the ability of cell proliferation and colony forming of the interference CNE2 were reduced (t=5.26, P<0.05).Conclusion: (1) A serum indistinct biomarker of NPC was correctly screened and identified as SAA. (2)RNA inference vector pSS-SAA was constructed successfully and could effectively inhibit the expression of SAA in CNE2. (3) SAA overexpressed in serum of NPC patients, which was related closely with the development of NPC.