| Background and ObjectivesNasopharyngeal carcinoma (NPC) is one of the commonest carcinomas with a high degree of malignant phenotype in Southern China and Southeast Asia. Both the geographic pattern and familial aggregation of incidence are two main characteristics of NPC. The carcinogenesis of nasopharyngeal epithelium is a multi-stage, multi-path and multi-mechanism process, which is involved in the effects of several factors including genetic predisposition, enviromental carcinogen and Epstein barr virus (EBV). The changes of various environmental factors, EBV infection, genetics 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 finally be transformed to NPC.Carcinogenesis is associated with the participation of expression alteration of multiple genes. The expression change of any single gene does not have the ability to drive the malignant tranformation of normal epithelium. In the past few years, although the knowledge of molecular mechanisms about NPC has greatly increased, its pathogenesis remains to be clarified.NESG1 gene (Genbank AF094758), official name CCDC19 (NM-012337.1), was found by Dr Zhongkui Li who is guided by Professor Kaitai Yao in 1999. Dr Li used mRNA differential display combined with the techniques of 3'-RACE and 5'-RACE to amplify respectively the 1.4kb and 0.7kb fragments (There were 174bp 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 consecutive 1850bp fragment was discovered. This gene is located at the chromosome 1q22. Predicted coding sequence (CDS) of this gene was 1161 bp and encoded 386 amino acids. Molecular weight of this protein was 46252Da and its isoionic point was 9.99. Online prediction of SOSUI and PSORTâ…¡showed that deduced NESG1 was a soluble nucleoprotein. Using NCBI's BLAST tool, he found that cDNA of NESG1 was homologous to two ESTs from human fetal lung cDNA library and Stratagene lung carcinoma cDNA library, respectively. Northern blot analysis of NESG1 transcript 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 NESGl is specifically expressed in nasopharyngeal epithelium and columnar ciliated epithelium of trachea.Based on the above investigation, we re-analyzed the sequence of NESG1 and revised the CDS region of NESGl. Further, we identified the function of this revised gene and analyzed its molecular basis in NPC preliminarily. This may provide new clues for further elucidating the NPC pathogenesis.Contents and methods1. Sequence-revision of NESG1 gene and its bioinformatics analysisOriginal NESG1 was recloned and analyzed by sequence identification. Its sequence was revised and CDS was repredicted. Then, revised NESG1 gene was analyzed by bioinformatics.2. Detection of NESG1 expression level in NPC(1). Expression level of NESGl mRNANESG1 mRNA expression was measured by RT-PCR and Real-time RT-PCR in NPC cells, NPC tissues and noncancerous nasopharyngeal tissues. In situ hybridization was used to locate the mRNA expression of NESG1 in nasopharyngeal tissues.(2). Preparation of rabbit anti human NESG1 poly-clonal antibody and analysis of NESG1 protein expressionThe open reading frame of revised NESG1 was cloned into prokaryotic expression vector pGEX-4T-1. Subsequently, recombinant GST-NESG1 fusion protein expression was induced using IPTG and the resulting protein was purified using GSH-sepharose. The purified protein was used to immunize rabbits. After 100 days, serum of rabbit was taken and the antibody was purified by affinity chromatography. The antigenicity of this fusion protein was validated using western blotting assay with rabbit anti-GST antibody. Subsequently, protein expression of NESG1 was determined in NPC and noncancerous nasopharyngeal tissues by Western blot and immunohistochemistry using NESG1 antibody. Furthermore, the expression pattern of NESG1 protein was also determined in other human tissues by immunohistochemistry.3. Functional analysis of NESG1 in NPC(1). Effects of NESG1 overexpression on biological characteristics in 5-8F NPC cellsThe NESG1 open reading frame was cloned into a pGC-FU-GFP lentivirus vector. The resulting lentivirus vector together with pHelperl and pHelper2 vectors were co-transfected into 293FT cells to generate lentiviral stock. An "empty" vector pGC-FU-GFP was utilized as a negative control. Lentiviral particles were used to infect NESG1-negative 5-8F cells, an NPC cell line with high tumorigenic and metastatic potential. Colonies with GFP expression were selected by limiting dilution assay of 96-well plate to expand culture. Finally,5-8F NPC cells with NESG1 overexpression and negative empty vector 5-8F cells were respectively established for further investigation. MTT, colony formation, FACSCaliber cytometry, Transwell and Boyden chamber were utilized to determine the ability of cell growth, cell cycle, cell migration and invasion in vitro. In vivo subcutaneous tumorigenesity in nude mice was used to evaluate the effect of overexpressed NESG1 on NPC cells.(2). Effects of suppressing the expression of NESG1 on biological characteristics in NESG1-overexpressed NPC 5-8F cellsNESG1-shRNA lentivirus interference vector was constructed and NPC cell with NESG1 silenced by stable RNA interference was established. MTT, colony formation, Transwell and Boyden chamber assay were utilized to detect the alterations of the ability of cell growth, migration, and invasion in NESG1 silencing.4. Priliminary investigation of molecular basis mediated by NESG1 in NPC(1). Affymetrix genome-wide microarray was employed to measure the differentially expressed genes after NESG1 gene was introduced into NPC cells(2). Bioinformatics was used to analyze the signal pathways mediated by these differentially expressed genes(3). Preliminary molecular mechanisms of NESG1 in inhibiting cell cycle progressionOwing to the role of overexpressed NESG1 in blocking the transition of cell cycle from G1 to S phase, we believed that NESG1 is involved in inhibiting cell cycle progression. Based on the microarray data, we verified the expression of several significant genes associated with S phase including CCND1, CCNA1, CDK4, p21, CDK2 and CDC2 and preliminarily explored the molecular mechanism of NESG1 in suppressing cell cycle progression.Results1. Sequence revision of NESG1 and its bioinformatics analysis(1). Sequence revision of NESG1The sequence analysis of newly cloned NESG1 showed that, comparing to formerly suggested NESG1 sequence, a G base in 1181 position was replaced by an A base and an A base inserted in 905 position led to a frame shift mutation. Furthermore, newly cloned sequence also displayed the consistency with the human genome sequence, some human cDNA clone sequences (CR604695, BC089391) and model organism sequences, such as NM-001024882.1 (Rattus norvegicus), XM-513919 (Pan troglodytes) and AB168450 (Macaca fascicularis) et al at 1181 and 905 positions. Based on these data, we updated NESG1 sequence and repredicted its CDS. The CDS of NESG1 was 1656bp and encoded 551aa, which is the same with the prediction of NESG1 of Macaca fascicularis testis cDNA clone (AB168450) in CDS region. Subsequently, we submitted the application of revised NESG1 sequence to department of NCBI reference sequence. They accepted our request and updated the NESG1 version from NM 012337.1 to NM 012337.2(2). Bioinformatics analysis of revised NESG1 geneRevised NESG1 encoded 551aa and its predicted molecular weight was 65729.7Da, the isoionic point was 8.94. Using the prediction of SMART program, we found that NESG1 is a coiled-coil structural protein (157-468aa and 498-527aa) Using blast and distance tree evolution analysis, we found that human NESG1, a relatively conserved gene in evolution, is highly homologous to the sequence of some other model organisms. Furthermore, human NESG1 sequence shares a higher homology with other model organisms with higher evolution level. PROSITE analysis showed that NESG1 contains one N-myristoylation site, four Protein kinase C phosphorylation sites, one N-glycosylation site, seven Casein kinase II phosphorylation sites, two cAMP and cGMP-dependent protein kinase phosphorylation sites and one amidation site. Prediction of hydrophobicity and transmembrane protein indicated that the probability of transmembrane protein of NESG1 was very small. Signal peptide prediction by SignalP 3.0 software displayed that there was no signal peptide sequence in NESG1, so NESG1 is not a secretory protein. Using three different softwares to predict Nuclear Localization Signal of NESG1, we found that there all existed Nuclear Localization Signal in 248aa-258aa and 503aa-521aa. This hinted that NESG1 could be expressed in cell nucleus.2. Measurement of NESG1 expression level in NPC(1). mRNA expression level of NESG1In situ hybridization showed that NESG1 mRNA was expressed in nucleus and cytoplasm of human nasopharyngeal epithelial cells. Using reverse transcription PCR and real-time PCR, we found that comparing to normal nasopharyngeal tissues, NESGl expression was down-regulated in NPC tissues and all of 8 NPC cell lines (t=19,819, P<0.001)(2). Preparation of rabbit anti human NESG1 polyclonal antibody and analysis of NESG1 protein expressionUsing the newly prepared anti-NESG1 rabbit polyclonal antibody, we investigated NESGl protein expression in NPC, NP and other human tissues. Western blotting assay showed a protein band with apparent molecular weight of approximate 66 kD, which is in agreement with the calculated molecular weight based on the revised NESG1 ORF sequence. Western blot revealed that protein expression of NESG1 was significantly downregulated in NPC tissues and NPC cells comparing with noncancerous nasopharyngeal tissues (t=41.116, P<0.001). Immunohistochemistry staining was performed for 40 normal nasopharyngeal tissues and 82 NPC tissues. NESG1 protein was abundantly expressed in nasopharyngeal epithelial cells and mainly localized in cytoplasm (sometimes expressed in nucleus). In contrast, NESGl expression was significantly down-regulated in NPC tissues (Z=6.852, P<0.001). No significant associations were found between NESG1 expression and age (Z=0.506, P= 0.613)and gender (Z=0.717, P=0.474) of NPC patients. Interestingly, we observed that NESGl expression was negatively correlated with N classification (Z=2.110,P=0.035) and clinical stage (x2=14.660, P=0.001) of NPC patients. Furthermore, we also performed the expression pattern of NESG1 protein in other human tissues by immunohistochemistry. The result showed that NESG1 was mainly expressed in cytoplasm of thyroid duct epithelium, breast duct epithelium, squamous epithelium of tongue, bronchiole columnar epithelium, intestinal epithelium and gastric epithelium, furthermore, weak expression of NESG1 was also showed in distal convoluted tubules. Among these, intranuclear expression of NESG1 was also seen in thyroid duct epithelium, gastric pits and bronchiole columnar epithelium. However, NESG1 expression was undetectable in the other tissues including alveolar epithelium, thymus gland, liver, spleen, vermiform appendix, endometrium and squamous epithelium of cervix.3. Functional investigation of NESG1 in NPC(1). Effects of NESG1 overexpression on biological characteristics in 5-8F NPC cellsAfter NESG1 was introduced into NPC cells, we established stable NESG1 overexpressed NPC cells. Two single cell clones 2F4 and 3D8 were chosen for further functional study. Empty vector C6 was selected as a negative control. In 2F4 and 3D8 cells, expression level of NESG1 mRNA in 2F4 cells was about 2 folds higher than that in 3D8 cells, and protein expression level of NESG1 in 2F4 cells was 1.76 folds higher than that in 3D8 cells. Compared to C6 control cells, both two clonal cells showed to be inhibited markedly in MTT cell proliferation assay (F=1404.066, P<0.001), colony formation assay (2F4:F=50.148, P<0.001; 3D8:F=27.559, P<0.001), Transwell migration assay (F=1054.101, P< 0.001) and Boyden invasion assay (F=304.640, P<0.001). In vivo subcutaneous tumorigenesity in nude mice showed that tumor weight and volume of 3D8 and 2F4 were significantly decreased comparing with C6 (weight of 3D8:F=8.478, P=0.005; weight of 2F4:F=2.904, P< 0.001; volume of 3D8:F=22.199, P=0.004, volume of 2F4:F=7.957, P=0.010). Furthermore, cell cycle analysis showed that the S phase population decreased significantly in 2F4 cells in comparison to the NESG1-negative C6 cells (P=0.003). However, in 3D8 cells the change in S phase population was not significant (P=0.796). This might be associated with the fact that 2F4 cells had higher levels of NESG1 than 3D8 cells did.(2). Effects of NESG1 expression suppressed on biological characteristics in 5-8F NPC cellsAfter shRNA lentivirus interference vector targeting NESG1 was constructed, we established 2F4 NPC cells with NESG1 silenced by stable RNA interference. Two stably cell clones 1C9 and 1D10 were chosen for functional exploration. Interference efficiency of 1C9 and 1D10 was 97.7% and 97.8% respectively. Compared to a negative control C1 cell, the proliferation ability of shRNA-NESGl 1C9 and1D10 cells was significantly increased (F=3764.751, P<0.001). Furthermore, the ability of migration (F=1162.190, P<0.001) and invasion (F=887.155,P<0.001) in shRNA-NESG1 1C9 and1D10 cells was also markedly increased compared to control cells.4. Preliminary study of molecular basis mediated by NESG1(1). Affymetrix genome-wide expression profile microarrayAffymetrix genome-wide expression profile microarray was used to determine the differential gene expression between NESG1-2F4 NPC cells and control C6 NPC cells. The results showed that 2400 genes were differentially expressed. Among them, 797 genes were upregulated and 1707 genes were downregulated. We analyzed NESG1-mediated pathways by comparing gene expression profiles in 5-8F NPC cells with or without NESG1 expression. Interestingly, there were many pathways determined by KEGG Pathways Database, including Tight junction (P=3.0E-6), MAPK signaling pathway (P=4.0E-6), regulation of Actin cytoskeleton (P=2.42E-4), and Cell cycle (P=3.8E-3) etc. (2). Preliminary molecular mechanisms of NESG1 in inhibiting cell cycle progressionWe verified the expression of several important genes associated with S phase progression by real-time PCR and Western blot based on microarray data. The results showed that the mRNA levels of CCND1 (F=0.017, P=0.983),CDK4 (F=0.579, P=0.589),CDK2 (Welch=0.092, P=0.914), and CDC2 (F=0.769,P=0.504) did not change in spite of the restored expression of NESG1. However, we found that CCNA1 mRNA expression was inhibited in both two clonal cells, especially in 2F4 cell (Welch=213.053, P=0.001). Furthermore, p21 expression level was also increased in 2F4 and 3D8 (F-35.675, P<0.001). Western blot also showed that the protein level of CCND1 (F=0.558,P=0.600) and CDK4 (F=0.470, P=0.646) in 2F4 and 3D8 cells have no significant change after the overexpression of NESG1, accompanied with the decreased CCNA1 (Welch=155.736, P=0.001) and increased p21 (F=86.359, P<0.001) protein expression level. The suppressive effect of NESG1 on cell cycle is greater in 2F4 cells than in 3D8 cells. Considering the fact that 2F4 cells express higher levels of NESG1 than 3D8 cells do, these data suggest that the effect of NESGl on cell cycle is dose-dependent. Taking together, our studies suggest that NESGl is able to retard the progression of cell cycle, and this effect is possibly mediated by the inhibited CCNAl expression and the upregulated p21 expression.Conclusion1. After NESG1 sequence was revised, its version in Genbank database was updated from NM 012337.1 to NM 012337.2.2. Funtional investigations showed that, after NESG1 was introduced into NPC 5-8F cells, the cell proliferation in vivo and in vitro, cell migration and invasion were markedly reduced. Furthermore, in higher NESG1-expressed 2F4 cells, cell cycle progression was blocked from G1 to S phase. After NESG1 expression was inhibited, the cell proliferation, migration and invasion was significantly recovered, which suggest that NESG1 should be a tumor-suppressive associated gene.NESG1 was involved in the regulation of cell cycle progression of NPC cell by inhibiting the expression of CCNA1 and upregulating the expression of p21.Discoveries and innovations of this investigation1. NESG1 gene was revised and its version in Genbank database was updated.2. NESG1 location was confirmed in human tissue. Furthermore, mRNA and protein expression level of NESG1 was down-regulated in NPC. This may provide clues for understanding and investigating the molecular mechanism of NPC pathogenesis.3. NESGl was closely associated with the genesis and development of NPC. Overexpressed NESGl could suppress the ability of cell proliferation, cell migration and cell invasion. Furthermore, Overexpressed NESG1 blocked the cell cycle progression from G1 to S phase.4. Microarray analysis showed that NESGl mediated multi-pathway, such as Tight junction, MAPK and Cell cycle. This may provide key clues for studying the signal pathway mediated by NESGl.5. NESG1 retarded the progression of cell cycle by inhibiting CCNA1 expression and upregulating p21 expression. 6. As a new candidate tumor suppressor, NESG1 was cloned and revised by our own research group. So we possesed the knowledge property right of NESG1 ourselves. Furthermore, we would continue to investigate its role in NPC, which might laid the basis for further elucidating the NPC pathogenesis. |
PDF Full Text Request