| BACKGROUND AND OBJECTIVESNasopharyngeal carcinoma has occurred in the nasopharyngeal mucosa malignancies, and the carcinogenesis is a multi-stage, multi-path and multi-mechanism process. The high incidence of NPC is mainly in southern China provinces, Hong Kong, Taiwan and some Southeast Asian countries and regions,with two main characteristics of the geographic pattern and familial aggregation of incidence. The changes of EBV infection, various environmental factors, genetic predisposition and epigenetics cause the overexpression of oncogenes and downregulated or null expression of tumor suppressors in nasopharyngeal epithelium, which will lead to the transition of epithelium to precancerous lesion and then be transformed to nasopharyngeal carcinoma, and finally lead to death since metastatic tumor cells. Since the NPC is very complex, involving a number of factors, more abnormal gene expression and regulation, and changes in multiple signaling pathways, therefore,the fully understood of specific mechanism of pathogenesis has been a pressing problem, and has become a central issue to improve the prevention and treatment of nasopharyngeal carcinoma. Many scholars have continued efforts to explore and resolve.NESG1gene（Genbank AF094758）, official nameCCDC19（NM012337.1）,was found by Dr Zhongkui Li who is guided by Professor Yao in1999. Dr Li used mRNA differential display combined with the techniques of3’-RACE and5’-RACE to amplify respectively the1.4kb and0.7kb fragments （There were174bp overlap sequence in the two fragments, which was utilized to confirm the specificity of amplification）.After the two fragments were spliced, a full-length cDNA with consecutive1850bp fragment was discovered（Since that time the human genome sequence has not yet announced, and the limitations of sequencing technology level, the new cloned sequence is correct or not is difficult to determine exactly）. This gene is located at the chromosome1q22. Predicted coding sequence （CDS） of this gene was1161bp and encoded386amino acids. Molecular weight of this protein was46252Da and its isoionic point was9.99. Online prediction of SOSUI and PSORTII showed that deduced NESG1was a soluble nucleoprotein. Using NCBI’s BLAST tool, he found that cDNA of NESG1was homologous to two ESTs from human fetal lung cDNA library and Stratagene lung carcinoma cDNA library, respectively. Northern blot analysis of NESG1transcript in human multi-tissues including nasopharyngeal epithelium （NE）, trachea （TR）, esophagus （ES）, cerebrum （CR）, heart （HE）, bladder（BL）, large intestine （IN）, liver （LI）, stomach （ST）, kidney （KI）, lung （LU） and thymus （TH） has revealed that NESG1is specifically expressed in nasopharyngeal epithelium and columnar ciliated epithelium of trachea.After cloning the original NESG1, our group had tried to study its function, but there has been no progress. The second half of2005, we re-cloned and sequenced original coding region of the gene. The sequence analysis of newly cloned NESG1showed that, comparing to formerly suggested NESG1sequence, a G base in1181position was replaced by an A base and an A base inserted in905position led to a frame shift mutation. The newly cloned sequence also displayed the consistency with the human genome sequence. NESG1gene coding sequence changed comparing with the previous one because of a G base in1181position was replaced by an A base and an A base inserted in905position. we repredicted its CDS and found that the CDS of NESG1was1656bp and encoded551aa, which is the same with the prediction of NESG1of Macaca fascicularis testis cDNA clone （AB168450） in CDS region. Subsequently, we submitted the application of revised NESG1sequence to department of NCBI reference sequence. They accepted our request and updated the NESG1 version from NM012337.1to NM012337.2.Dr. Liu Zhen researched function and molecular mechanisms of the new candidate tumor suppressor gene NESG1in nasopharyngeal carcinoma,and found that NESG1gene was likely to play a role of tumor suppressor gene in NPC and mediated an important inhibitory signal transduction pathways.The role of NESG1in carcinogenesis of NPC has been identified,however, the signal pathways NESG1participating in is not clear. Therefore,we selected NESG1interacting proteins for the study, demonstrated which of proteins were interacting with NESG1by a large number of experiments, and finally elucidated the biological function and molecular mechanism of NESG1interacting proteins in NPC.We eventually hope to reveal the signaling pathways NESG1participating in and its affect in development of NPC,and provide a theoretical basis for the early diagnosis and treatment of nasopharyngeal carcinoma.METHODS1.Screening and prediction of NESG1-interacting proteins（1） Bioinformatics prediction of NESG1-interacting proteinsNESG1-interacting proteins were predicted by on-line analysis software and data.bases,and its possible interacting proteins were analyzed.（2） Screening of NESG1-interacting proteins by yeast two-hybrid systemsCDS sequence of NESG1gene was cloned and yeast expression bait vector of pGBKT7-NESGl used in yeast two-hybrid systems was constructed.Universal human cDNA Library was used to screen NESG1-interacting proteins.2.1dentification and bioinformatical analysis of NESG1-interacting proteins Interaction between NESG1and VPS33B （a NESG1-interacting protein） was identified by co-immunoprecipitation assay and co-localization analysis.（1） Co-ImmunoprecipitationTwo eukaryotic expressing vectors expressing NESG1with MYC label and VPS33B with HA label were constructed respectively and co-transfected into HEK293A cells.Cells were lysed,and proteins were precipitated by anti-HA+protein A/G-beads or anti-MYC+protein A/G-beads respectively,Protein complex were analyzed by Western blots with anti-HA or anti-MYC monoclonal antibodies.（2） Co-localization analysisTwo eukaryotic expressing vectors expressing NESG1with MYC label and VPS33B with HA label were constructed respectively and co-transfected into HEK293A cells. Cells were stained with anti-HA or anti-MYC antibodies followed by Dylight488or Dylight594conjugated specific antibodies48hours after transfection and observed under Upright fluorescence microscope.Green fluorescence and red fluorescence of cells were merged to localize the contacting sites of both proteins.（3） Bioinformatical analysis of VPS33B proteinFunctions,expression patterns and possible interaction networks of VPS33B protein were predicted and analyzed by on-line analysis software and databases.3. Expression of VPS33B in nasopharyngeal carcinomas and its clinical significancemRNA and Protein expression of VPS33B in nasopharyngeal carcinoma tissues and noncancerous nasopharyngeal tissues were measured by Real-time RT-PCR and immunohistochemistry.4. A preliminary study of the function and molecular mechanisms of VPS33B gene in nasopharyngeal carcinomaThe recombinant lentivirus vector containing CDS sequence of VPS33B was constructed.293FT cells were co-transfected with recombinant lentivirus vector and adjunctive plasmids.Virus supernatants were collected and virus titer was determined as routine.5-8F and HONE-1NPC cells were infected with virus supernatants obtaining VPS33B gene recombinant lentivirus vectors,5-8F and HONE-1NPC cells infected with empty lentivirus vectors were regarded as negative controls.The stable VPS33B overexpressed NPC cells were screened by Flow Cytometry. Western blot analysis were used to examine the expression of VPS33B gene in VPS33B overexpressed NPC cells. Effects of VPS33B overexpression on cell proliferation was assessed by MTT assay, plate colony formation assay and flow cytometry in vitro. Subcutaneous tumorigenicity in nude mice was used to assess the effect of overexpressed VPS33B on MPC cells in vivo. Effects of VPS33B overexpression on cell invasion was evaluated by Boydeh chamber assay. Western blot analysis was used to test the change of several important proteins associated with cell cycle progression and other important signaling pathway proteins before and after VPS33B overexpression,in order to preliminarily explore the molecular mechanism of VPS33B in suppressing NPC developmentTransfection synthetic siRNA targeting VPS33B gene into NPC cells with VPS33B overexpression.Real time RT-PCR and Western blot analysis were used to examine the effectiveness of RNA interference. MTT and Bpyden chamber assay were used to investigate the changes of the ability of cell growth and invasion in VBS33B silencing;RESULTS1.Screening and prediction of NESG1-interacting proteins（1） Bioinformatics analysis of NESGl-interacting proteinsIt was found that human NESG1gene was homologous to CG11449-PA in drosopbila, and GG11449-PA was an important meeting point among CG1135-PA, CG1487-PA and other fifteen proteins in drosophila. The method of interaction between conserved protein was applicated to forecast human UBR4, ARRB1and other six proteins as NESG1-interacting proteins.（2） Screening of NESGl-interacting proteins by yeast two-hybrid systemsNine positive clones through our yeast two-hybrid systems were found after universal human cDNA Library had been screened. And nine positive clones.were sequenced and confirmed as gene ENKUR,CCDC65,VPS33B,RNF2,NUP155,TEX2, ZRANB1,SPATA22and GOSR1. 2. Identification and bioinformatical analysis of NESG1-interacting proteins（1） Co-ImmunoprecipitationCo-immunoprecipitation showed that protein A/G-beads+Anti-MYC antibodies precipitated MYC-NESG1protein while,HA-VPS33B protein also be precipitated together. Meanwhile, protein A/G-beads+Anti-HA antibodies precipitate HA-VPS33B protein while MYC-NESG1protein also be precipitated.All results indicated that NESG1interacted with VPS33B.（2） Co-localization analysisConsistent with our co-immunoprecipitation results, NESG1and VPS33B were found to be colocalized at cytoplasm.Preliminary screening of yeast two-hybrid,co-immunoprecipitation and co-localization analysis demonstrated that NESG1was interacted with VPS33B.（3） Bioinformatical analysis of VPS33B proteinIt has been confirmed that VPS33B gene belonged to one of Sec-1domain family. The results of digital Northern blots showed that VPS33B protein was lower expression in thyroid and lung cancer. Ras oncogene family members RAB11A was predicted as one of VPS33B-interacting proteins by bioinformatical analysis.3. Expression of VPS33B in nasopharyngeal carcinomas and its clinical significanceUsing Real-time RT-PCR, we found that comparing to noncancerous nasopharyngeal tissues,VPS33B expression was down-regulated in NPC tissues （t=-2.412, P=0.029）. Immunohistochemistry showed that VPS33B protein was mainly localized in cytoplasm and significantly down-regulated in NPC tissues comparing with noncancerous nasopharyngeal tissues （χ2=18.721, p<0.001）. The expression of VPS33B protein closely related to clinical stage of NPC patients:the clinical stage sooner, VPS33B express stronger; while the later clinical stage, VPS33B express weaker or negative.4. A preliminary study of the function and molecular mechanisms of VPS33B gene in nasopharyngeal carcinoma （1） Establishment of the stable NPC cells of VPS33B overexpressionAfter VPS33B was introduced into5-8F and HONE-1NPC cells, we screened the cells by Flow Cytometry and established stable VPS33B overexpressed NPC cells, which were chosen for further function and molecular mechanism study.（2） A preliminary study of the function and molecular mechanisms after VPS33B overexpression in NPCCompared to5-8F-NC and HONE-1-NC negative control cells, both5-8F-VPS33B and HONE-1-VPS33B cells showed to be inhibited significantly in MTT cell proliferation assay （5-8F:F=134.393, P＜0.001; HONE-1:F=134.761, P＜0.001）, plate colony formation assay （5-8F:t=6.037, P=0.004; HONE-1:t=7.846, P-0.001）.Furthermore, cell cycle analysis showed that the G1phase increased significantly and S phase decreased markedly in5-8F-VPS33B and HONE-1-VPS33B cells in comparison to5-8F-NC and HONE-1-NC negative control cells （5-8F:t=3.662, P=0.022; HONE-1:t=4.054, P=0.015）.However,5-8F-VPS33B and HONE-1-VPS33B cells had not markedly reduced invasion comparing with5-8F-NC and HONE-1-NC negative control cells by Boy den chamber assay in vitro（5-8F-VPS33B:t=0.263, P=0.805; HONE-1-VPS33B:t=0.697, P=0.524; P>0.05）.The VPS33B overexpressed NPC cells and control cells were inoculated subcutaneously in nude mice.After two or three weeks,we found VPS33B overexpression significantly inhibited the proliferation of nasopharyngeal carcinoma cells in vivo.Western blot analysis was used to test the change of several important proteins associated with cell cycle progression and other important signaling pathway proteins before and after VPS33B overexpression. The results showed that the expression of c-myc,CDK4,CCND1,CCDE1,p-Rb and E2F1in5-8F-VPS33B and HONE-1-VPS33B cells was down-regulated, Rb had no significant difference, while p16expression was up-regulated. Western blot also showed that the expression of p-AKT and p-PI3K was down-regulated,but PTEN,AKT and PI3K had no significant change. Furthermore,we examined the expression of E-cadherin、N-cadherin and Vimentin after the overexpression of VPS33B and found that the protein level of E-cadherin> N-cadherin and Vimentin had no marked difference.（3） Effects of Inhibition VPS33B expression on the biological function of NPC cellsChemically synthesized siRNA was transfected into NPC cells5-8F-VPS33B and HONE-1-VPS33B by cationic polymer transfection reagent, and then real-time RT-PCR and Western blot were applied to detect silence effects after48h or72h. The results showed that03had the best interference effect among the three interference sequences,and can be used for further experiment. In comparison to5-8F/VPS33B/NC and HONE-1/VPS33B/NC negative control cells, the proliferation ability of5-8F/VPS33B/si-VPS33B and HONE-1/VPS33B/si-VPS33B cells was markedly increased （5-8F:F=56.419, P＜0.001; HONE-1:F=51.638, P<0.001）,however the ability of invasion had no significant change.CONCLUSIONS1. VPS33B protein can directly interact with NESG1protein in vivo;2. VPS33B gene significantly correlates to proliferation of NPC and suppresses the proliferation ability of NPC cells;3. VPS33B is closely associated with NPC development and may be an important gene correlated with NPC proliferation;4. NESG1-VPS33B related signal pathway plays a role in development of NPC. |
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