| Background and ObjectivesNasopharyngeal carcinoma (NPC) is one of the most common carcinomas in certain regions of Southern China and Southest Asia, with obvious regional distribution characteristics. It is a tumor with high degree of malignancy in which cervical lymph node metastasis and distant metastasis occurs early, and is closely associated with Epstein Barr virus (EBV). Gene susceptibility, environmental risk factors, specific dietary habit, EBV infection and gene mutation are all involved in the pathogenesis of NPC. The initiation and development of NPC involves multiple genes and signaling pathways, and the molecular basis of NPC is the activation of oncogenes and inactivation of tumor suppressor genes and the imbalance between them. Therefore, study on the primary genes and signaling pathways involved may contribute to the exploration of pathogenesis of NPC.PDCD4(programmed cell death4) is identified as being up-regulated after initiation of programmed cell death. It is localized to human chromosome band 10q24. PDCD4comprises469amino acids, and is expressed ubiquitously in normal tissues with highest levels in liver. A frequent down-regulation or loss of Pdcd4was found not only in lung tumours, but also in other cancers, such as glia-, lung-and renal-derived tumors, tongue tumors, hepatoma tissue, invasive ductal breast carcinoma, skin cancer and human glioma, and the expression of PDCD4is closely related to the progression of malignant tumors, suggesting down-regulation or loss of PDCD4plays an important role in the occurrence and development of NPC. Researches show that PDCD4, a new tumor suppressor, inhibits cell proliferation, transformation, invasion and migration, and induces cell apoptosis, but the molecular mechanism is still unknown. In previous investigation, we used cDNA microarray to examine differentially expressed genes among NPC tissues and non-cancerous nasopharyngeal tissues. By means of the analysis of BRB-array tools, the expression of PDCD4, was shown to be markedly decreased in NPC tissues, suggesting a possible role of PDCD4in suppressing the pathogenesis of NPC. The research on the function and mechanism of this gene in NPC hasn’t been reported yet.MicroRNA(miRNA) is a endogenous non-coding small RNA found in recent years which functions in transcriptional and post-transcriptional regulation of gene expression, is about21to25nucleotides long. Mature miRNA is made from long transcriptional products after a series of processing, and is assembled into RNA induce silence complex (RISC), thus induces the degradation or repression of target gene by means of base-pairing complementary sequences within mRNA molecules. It is shown that miRNA plays an important role in a series of life activities, for example, development of tissues and organs, virus defense, metabolism, cell proliferation, cell differentiation and apoptosis. In addition, miRNAs are closely associated with tumor development. Studies have shown that about50%of miRNAs locate in fragile site associated with tumors, its expression easily changes in a wide variety of tumors. And miRNAs participate in the process of cell proliferation, differentiation, migration, invasion and metastasis, through regulating target genes of transcription and translation. Whether PDCD4in nasopharyngeal carcinoma can be regulated by miRNAs, which miRNAs can in be regulated by PDCD4, and which miRNAs regulated by PDCD4can in turn modulate PDCD4in nasopharyngeal carcinoma, these questions are worthy of our study.Phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) is one of the important signaling pathways that participates in many kinds of the regulation of cellular functions, such as cell growth, proliferation, differentiation, apoptosis, invasion, metastasis, angiogenesis, glucose transporter, degradation of extracellular matrix and so on. Akt is the key of PI3K/Akt signaling pathway, can activate or inactivate a variety of enzymes, kinases and transcription factors through the activation or inactivation of Akt phosphorylation, and then adjusts the function of cells. It is shown that abnormal expression of PI3K/Akt signal pathway was found in a variety of tumors. And the abnormal activation of PI3K/Akt signaling pathway, is closely related to the biological behaviors, for example, cell growth, proliferation, differentiation, angiogenesis, invasion and metastasis, and resistance to apoptosis.Cell cycle, refers to the series of events that take place in a cell leading to its division and duplication, including G1phase and S phase (DNA synthesis), G2phase, M phase (mitosis) and GO phase. Normal operation of cell cycle is the result of the perfect interaction of a series of genes. Tumor growth is out of control cell proliferation. So studies on tumors should focus on cell cycle, especially the transformation of G0/G1and G1/S, and key genes which regulate cell cycle progression, will contribute to reveal the occurrence, development, prevention and treatment of tumors.Apoptosis refers to independent programmed cell death controlled by genes in order to maintain stable internal environment. Cell apoptosis is one of the basic cell biological phenomena, which plays an important role in the biological evolution, development and stability of internal environment. Apoptosis mainly has two ways: one is the membrane receptor pathway, take Fas-FasL for example. Fas is a transmembrane protein, belonging to the tumor necrosis factor receptor superfamily member, it can bind with FasL, and activates caspase8,3,6,7, PPAR, and then makes cell apoptosis. Another is the mitochondrial apoptosis. bcl2/bax apoptosis related proteins regulate the release of cytochrome C. Once cytochrome C is released, it binds with apoptotic protease activating factor-1(Apaf-1) and ATP, which then binds to pro-caspase-9to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3,6,7and PARP, eventually makes DNA rupture and apoptosis occur. Caspases play a key role in the process of cell apoptosis. A lot of researches have shown that the development of tumor is closely related to cell apoptosis.Based on the current research of PDCD4in NPC, we investigated the function of PDCD4gene, and explored the relationship between PDCD4, miRNA and signaling transduction pathways involved in, to provide a new idea for the research on the molecular mechanisms of NPC.Contents and methods1. Detecting the expression of PDCD4in NPC cells and tissues by immunohistochemisty, quantitative real-time PCR (qPCR) and western blot.2. The effect on cell biology of NPC by PDCD4(1) PDCD4overexpression vector pGC-FU-PDCD4-GFP was constructed. Lentivirus was packaged in293T cells and then infected5-8f cells to establish monoclonal cells with stable overexpression of PDCD4. MTT, plate clone formation, nude mice subcutaneously tumorigenesis, cell cycle and cell apoptosis were used to observe the ability of cell growth, proliferation and apoptosis of5-8f cells with PDCD4overexpression, and the function of PDCD4gene in NPC cells were determined.(2) PDCD4interference plasmid was contructed. And lentivirus was packaged in293T cells and infected HONE1and SUNE1cells to establish polyclonal cells with PDCD4knockdown. MTT, plate clone formation, cell cycle and cell apoptosis were used to observe the ability of cell growth, proliferation and apoptosis of HONE1and SUNE1cells with PDCD4knockdown, and the function of PDCD4gene in NPC cells were further determined.3. Function and target genes of differentially expressed miRNAs detected by miRN A array(1) Using miRN A array to detect differentially expressed miRNAs in cells with PDCD4knockdown, and detecting the expression of miR-184by qPCR in cells with PDCD4overexpression or knockdown, which was downregulated the most obvious after PDCD4knockdown.(2) Choosing miR-184as the subject of study, and using MTT and cell apoptosis to observe the ability of cell growth and apoptosis in SUNE1and5-8f cells infected with miR-184mimics, further to determine the function of miR-184in nasopharyngeal carcinoma cells; using MTT to observe cell viability of PDCD4-suppressed cells infected with miR-184mimics, further to determine the relationship between miR-184and PDCD4.(3) Using miRwalk and RNAhybrid to predict target genes of miR-184.(4) Constructing dual-luciferase report gene carrier psicheck-2/BCL23’UTR and psicheck-2/C-MYC CDS, and the mutant carrier psicheck-2/BCL2mt3’UTR and psicheck-2/C-MYC mt CDS, which were then cotransfected into SUNE1cell with miR-184mimics or inhibitor, separately. And the activity of firefly and renilla luciferase was detected48h after transfection. So according to the ratio of luciferase, whether BCL2and C-MYC were directly regulated by miR-184could be easily confirmed.(5) Detecting the expression of BCL2and C-MYC after transfection cells with miR-184mimics/inhibitor by western blot, to further determine whether BCL2and C-MYC were target genes of miR-184.(6) Detecting the expression of PDCD4after transfection cells with miR-184mimics/inhibitor by western blot, to further determine whether miR-184could in turn regulate PDCD4.4. The molecular mechanism of PDCD4and miR-184which inhibited cell proliferation and promoted apoptosis in nasopharyngeal carcinoma (NPC)(1) Detecting the expression of proteins involved in PI3K/Akt signaling pathways by western blot in PDCD4overexpression/interference cells, such as PI3K, pPI3K, pAkt, JNK1and C-JUN.(2) Detecting the expression of proteins involved in PI3K/Akt signaling pathways by western blot in SUNE1and5-8f cells transfected with PI3K inhibitors Ly294002, such as PI3K, pPI3K, pAkt, JNK1and C-JUN.(3) Detecting the expression of proteins involved in cell cycle by western blot in PDCD4overexpression/interference cells, such as C-MYC, CCND1, P-RB (780s), E2F1,P15,P27and CDK4.(4) Detecting the expression of proteins involved in mitochondrial apoptotic pathways by western blot in PDCD4overexpression/interference cells.(5) Detecting the expression of proteins involved in cell cycle by western blot in SUNE1and5-8F cells transfected with miR-184mimics, such as C-MYC, CCND1, P-RB (780s), E2F1, P15, P27and CDK4. (6) Detecting the expression of proteins involved in mitochondrial apoptotic pathways by western blot in SUNE1and5-8F cells transfected with miR-184mimics.5. PDCD4regulated the expression of miR-184through mediating the binding of C-JUN to the promoter region of miR-184(1) Detecting the expression of miR-184by fluorescence quantitative PCR after C-JUN knockdown by siRNA.(2) Using miRBase, Ensembl and TFSEARCH software to predicate the binding sites of C-JUN in miR-184promoter region.(3) Using chromatin immune co-precipitation to validate whether C-JUN bound to miR-184promoter region.Results1. Expression of PDCD4in NPC cells and tissuesThe results of IHC showed that PDCD4was highly expressed in most non-cancerous nasopharyngeal tissues, and had different degrees of expression in NPC tissues, but mainly was lowly expressed, the difference was statistically significant (χ2=94.635, P<0.001). Western blot showed that PDCD4was highly expressed in3cases of nasopharyngeal tissues, and lowly expressed in5cases of nasopharyngeal carcinomas. QPCR showed that PDCD4expression was different in nasopharyngeal carcinomas and non-cancerous nasopharyngeal tissues (t=-28.433, P=0.001). QPCR showed that the expression of PDCD4was different in immortalized nasopharyngeal epithelial NP69cells and NPC cells5-8F,6-10B, C666-1, CNE1, CNE2, HNE1, HONE1and SUNE1, the difference was statistically significant (F=101.776, P<0.001).5-8F with the lowest PDCD4expression, SUNE1and HONE1with the highest PDCD4expression, was chosen to be the object of PDCD4overexpression or interference, separately. 2. PDCD4inhibited cell proliferation, cell cycle transformation from Gl to S phase and induced cell apoptosis(1) Influence on nasopharyngeal carcinoma cell biology after PDCD4overexpression1) PDCD4overexpression vector pGC-FU-PDCD4-GFP was constructed. Lentivirus was packaged in293T cells and then infected5-8f cells to establish monoclonal cells with stable overexpression of PDCD4through96-wells limited dilution, and the expression of PDCD4was detected by QPCR and western blot. Compared with negative control cells and blank cell line5-8f, the expression of PDCD4in clone1and clone2increased significantly.2) Using MTT to detect cell survival of cells with PDCD4overexpression. Compared with scramble and blank cell line5-8f, clone1and clone2significantly reduced cell survival, difference was statistically significant (F=601.052, P<0.001).3) Detecting the ability of cell proliferation in vitro after PDCD4overexpression by plate clone formation. Compared with scramble and blank cell line5-8f, clonel and clone2inhibited colony-forming ability significantly, difference was statistically significant (F=36.643, P<0.001). This suggested PDCD4inhibited cell proliferation in vitro.4) Detecting the ability of cell proliferation in vivo after PDCD4overexpression by nude mice subcutaneously tumorigenesis. Compared with scramble group, clonel and clone2groups inhibited tumor growth significantly, difference was statistically significant (F=49.268, P<0.001). This indicated PDCD4inhibited cell proliferation in vivo.5) Detecting cell cycle after PDCD4overexpression by flow cytometry analysis. Compared with scramble, the ratio of cells in S phase decreased, and cells in G1and G2increased obviously with significant difference[G1phase:F=88.642, P<0.001; S phase:F=28.469, P=0.001; G2phase:F=18.47, P=0.003)]. The results of cell cycle showed that PDCD4inhibited cell proliferation through suppressing cell transformation from Gl to S phase.6) Detecting cell apoptosis after PDCD4overexpression by flow cytometry analysis. The percentage of apoptotic NPC cells was significantly higher in clone1and clone2than that in scramble with significant difference (F=77.368, P<0.001). This prompted that PDCD4induced cell apoptosis.(2) Influence on nasopharyngeal carcinoma cell biology after PDCD4knockdown1) The lentiviral vectors containing specific anti-PDCD4shRNA and control were transfected into293FT cells to produce lentivirus particles, followed by transfected into HONE1and SUNE1cells. Cells were named as shl-PDCD4, sh2-PDCD4and polyclonal negative control PLVTHM. As the efficiencies of infection (GFP+cells) were more than90%, so we detected the expression of PDCD4by QPCR and western blot. Compared with negative control cells PLVTHM, the expression of PDCD4in cells with PDCD4knockdown was downregulated significantly, especially in sh2-PDCD4polyclonal cells, which were chosen for subsequent experiments.2) Using MTT to detect cell viability in HONE1and SUNE1cells with PDCD4knockdown. Compared with sh-scramble, cells with PDCD4interference significantly induced cell survival[F=1151.482, P<0.001(HONE1); F=2752.914, P<0.001(SUNE1)]3) Detecting the ability of cell proliferation in vitro after PDCD4knockdown by plate clone formation. Compared with sh-scramble, cells with PDCD4interference significantly induced colony-forming ability significantly [t=9.879, P=0.001(HONE1), t=24.012, P<0.001(SUNE1)l. This suggested PDCD4inhibited cell proliferation in vitro.4) Detecting cell cycle after PDCD4knockdown by flow cytometry analysis. Compared with sh-scramble, the ratio of cells in S phase increased and cells in G1and G2decreased obviously with significant difference [G1phase:t=-21.199, P<0.001(HONE1), t=-5.97, P=0.004(SUNE1); S phase:t=6.655, P=0.003(HONE1), t=6.292, P=0.003(SUNE1); G2phase:t=6.94, P=0.002(HONE1), t=3.773, P=0.02(SUNE1)]. The results of cell cycle showed that PDCD4knockdown promoted cell transformation from G1to S phase, thus induced cell proliferation.5) Detected cell apoptosis with PDCD4knockdown by flow cytometry analysis. The percentage of apoptotic NPC cells was significantly lower in shPDCD4than that in sh-scramble, differences were statistically significant [t=-21.250, P<0.001(HONE1), t=-15.985, P<0.001(SUNE1)]. This suggested that PDCD4interference inhibited cell apoptosis.3. Function and target genes of differentially expressed miRNAs detected by miRNA array(1) Data of miRNA expression profile chip showed that the expression of miR-184was significantly low in cells PDCD4knockdown. The expression of miR-184was detected by QPCR, in cells with PDCD4knockdown, miR-184was lowly expressed, and in cells PDCD4overexpression, miR-184was highly expressed compared to their controls, separately. So miR-184was chosen to be the object of the research.(2) miR-184inhibited cell growth and induced apoptosis1) Using MTT to detect cell viability of SUNE1and5-8F cells transfected with miR-184mimics. Compared with negative control, cells transfected with miR-184mimics significantly inhibited cell survival, differences were statistically significant [F=99.542, P<0.001(SUNE1); F=201.9, P<0.001(5-8F)]. 2) Detecting cell apoptosis after transfected with miR-184mimics by flow cytometry analysis. The percentage of apoptotic NPC cells was significantly higher in cells transfected with miR-184mimics than that in negative control, with significant difference [t=7.965, P=0.001(SUNE1), t=18.817, P<0.001(5-8F)]. This indicated that miR-184induced cell apoptosis.3) Using MTT to detect cell viability in PDCD4-suppressed cells transfected with miR-184mimics. Compared with negative control, cells transfected with miR-184mimics significantly inhibited cell viability [F=956.238, P<0.001(sh-PDCD4-HONE1); F=793.964, P<0.001(sh-PDCD4-HONE1)]. This suggested that miR-184reversed the promotion of tumor growth after PDCD4knockdown.(3)Using RNAhybrid and miRwalk software to predict that BCL2and C-MYC might be target genes of miR-184, and which was further confirmed by dual-luciferase reporter gene analysis and western blot.1) Constructing dual-luciferase report gene carrier psicheck-2/BCL23’UTR and the mutant carrier psicheck-2/BCL2mt3’UTR, which were then cotransfected into SUNE1cell with miR-184mimics or inhibitor, separately. And the activity of firefly and renilla luciferase was detected48h after transfection. Compared with control group, group cotransfected with psiCHECK-2/BCL2wt3’UTR and miR-184mimics reduced the luciferase activity obviously (t=-33.801, P<0.001), and group cotransfected with psiCHECK-2/BCL2wt3’UTR and miR-184inhibitor increased the luciferase activity significantly (t=15.031, P<0.001). No change of luciferase activity was observed in groups cotransfected with mutation plasmid psiCHECK-2/BCL2mt3’UTR and miR-184mimics or inhibitor, respectively (t=-1.763, P=0.092). Results above showed that miR-184bound to the3’UTR of BCL2. 2) Constructing dual-luciferase report gene carrier psicheck-2/C-MYC CDS, and the mutant carrier psicheck-2/C-MYC mt CDS, which were then cotransfected into SUNE1cell with miR-184mimics or inhibitor, separately. And the activity of firefly and renilla luciferase was detected48h after transfection. Compared with control group, group cotransfected with psiCHECK-2/C-MYC wt CDS and miR-184mimics reduced the luciferase activity obviously (t=-7.314, P<0.001), and group cotransfected with psiCHECK-2/C-MYC wt CDS and miR-184inhibitor increased the luciferase activity significantly (t=14.943, P<0.001). No change of luciferase activity was observed in groups cotransfected with mutation plasmid psiCHECK-2/C-MYC mt CDS and miR-184mimics or inhibitors, respectively (t=-1.353, P=0.19). Results above showed that miR-184bound to the CDS of C-MYC.3) Detecting the expression of BCL2and C-MYC in cells transfected with miR-184mimics or inhibitor by western blot. Compared with negative control, BCL2and C-MYC were downregulated in cells transfected with miR-184mimics, however, in cells transfected with miR-184inhibitor BCL2and C-MYC were upregulated.4) Detecting the expression of PDCD4in cells transfected with miR-184mimics by western blot. No change of PDCD4expression was observed in cells transfected with miR-184mimics, compared with negative control. Combining the data of miRNA expression profile chip analysis and the reversion of tumor growth in PDCD4-suppressed cells by miR-184, we kne that PDCD4regulated miR-184, but miR-184did not modulate PDCD4.4. The molecular mechanism of PDCD4and miR-184which inhibited cell proliferation and promoted apoptosis in nasopharyngeal carcinoma (NPC)(1) Detecting the expression of proteins involved in PI3K/Akt signaling pathways by western blot in PDCD4overexpression/interference cells, such as PI3K, pPI3K, pAkt, JNK1and C-JUN. The expression of pPI3K, pAkt, JNK1and C-JUN was low in PDCD4overexpression cells; however, in cells PDCD4knockdown, pPI3K, pAkt, JNK1and C-JUN was upregulated, while no change of PI3K and AKT was observed.(2) Detecting the expression of proteins involved in PI3K/Akt signaling pathways by western blot in SUNE1and5-8f cells transfected with PI3K inhibitors Ly294002, such as PI3K, pPI3K, pAkt, JNK1and C-JUN. The expression of pPI3K, pAkt, JNK1and C-JUN was low in cells transfected with PI3K inhibitors Ly294002, while no change of AKT was observed.(3) Detecting the expression of proteins involved in cell cycle by western blot in PDCD4overexpression/interference cells, such as C-MYC, CCND1, P-RB(780s), E2F1, P15, P27and CDK4. The expression of C-MYC, CCND1, P-RB(780s) and E2F1was low in PDCD4overexpression cells; however, in PDCD4knockdown cells C-MYC, CCND1, P-RB(780s) and E2F1was upregulated, while no change of P15, P27and CDK4was observed.(4) Detecting the expression of proteins involved in mitochondrial apoptotic pathways by western blot in PDCD4overexpression/interference cells. The expression of cleaved caspase9, cleaved caspase3, cleaved caspase6, cleaved caspase7and cleaved PARP was high and the expression of BCL2was low in PDCD4overexpression cells; however, in PDCD4knockdown cells cleaved caspase9, cleaved caspase3, cleaved caspase6, cleaved caspase7and cleaved PARP was downregulated and the expression of BCL2was high.(5) Detecting the expression of proteins involved in cell cycle by western blot in SUNE1and5-8F cells transfected with miR-184mimics, such as C-MYC, CCND1, P-RB (780s), E2F1, P15, P27and CDK4. The expression of C-MYC, CCND1, P-RB (780s) and E2F1was low in cells transfected with miR-184mimics, while no change of P15, P27and CDK4was observed.(6) Detecting the expression of proteins involved in mitochondrial apoptotic pathways by western blot in SUNE1and5-8F cells transfected with miR-184mimics. The expression of cleaved caspase9, cleaved caspase3, cleaved caspase6, cleaved caspase7and cleaved PARP was high and BCL2was downregulated in cells transfected with miR-184mimics.5. PDCD4regulated the expression of miR-184through mediating the binding of C-JUN to the promoter region of miR-184(1) Detecting the expression of miR-184by fluorescence quantitative PCR after C-JUN knockdown by siRNA. The expression of miR-184was obviously upregulated after C-JUN knockdown by siRNA compared to negative control [t=33.262, P<0.001(SUNE1); t=6.925, P=0.02(5-8F)](2) Using miRBase, Ensembl and TFSEARCH software to predicate the binding sites of C-JUN in miR-184promoter region, and was further validated by chromatin immune coprecipitation. Compared with mock, there were more binding sites of the promoter region of miR-184in cells transfected with pcDNA3.1-C-JUN, difference was statistically significant [t=7.735, P=0.002(PI); t=10.639, P<0.001(P2); t=9.221, P=0.012(P3)]. This indicated that C-JUN regulated the expression of miR-184through binding to the promoter region of miR-184.Conclusions1. PDCD4is highly expressed in nasopharyngeal tissues, and lowly expressed in nasopharyngeal carcinoma.2. PDCD4inhibits cell growth, cell proliferation, cell transformation from G1to S phase, and induces cell apoptosis.3. MiR-184inhibits cell growth and induces cell apoptosis in nasopharyngeal carcinoma. 4. miR-184directly suppressed BCL2and C-MYC in nasopharyngeal carcinoma.5. PDCD4inhibits cell proliferation through PI3K/Akt signaling pathway, and inhibits cell cycle progression through down-regulating C-MYC, CCND1, P-RB (780s) and E2F1. PDCD4activates proteins involved in mitochondrial apoptosis pathway such as cleaved caspase9,3,6,7and PARP, and inhibits the expression of BCL2to induce apoptosis.6. MiR-184inhibits cell growth and induces mitochondrial apoptosis, by regulating of proteins involved in cell cycle and mitochondrial apoptosis pathway through directly suppression of BCL2and C-MYC in nasopharyngeal carcinoma.7. DCD4regulates the expression of miR-184through mediating the binding of C-JUN to the promoter region of miR-184... |
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