Studies Of CDK4Gene On Its Function And Molecular Basis In Nasopharyngeal Carcinoma

Background and ObjectivesNasopharyngeal carcinoma(NPC) is one of the commonest carcinomas with high degree of malignant phenotype and geographic pattern characteristics in Southern China. Clinically, NPC is classified as a specific type of head and neck squamous cell carcinoma with its unique epidemiology, clinical characteristics, etiology, and histopathology. Synergetic effects of Epstein Barr virus (EBV) infections, genetic alterations, and environmental factors are thought to the key factors driving the aberrant activity of a variety of signal pathways, such as the Akt, cell cycle, mitogen-activated protein kinases, Wnt pathway, and epidermal growth factor receptor signaling pathway et al, which caused the pathogenesis of NPC.Therefore, separate efforts were still needed to investigate its underlying molecular mechanism of carcinogenesis.My research team to participate in the use of multiple sets of nasopharyngeal carcinoma tissue microarray containing8000genes compared (nasopharyngeal carcinoma cell content accounts for more than70%of the entire organization), a mixture of nasopharyngeal carcinoma cells and the cancer gene expression profiles between nasopharyngeal mucosa.We get435raised genes and257down-regulated genes by BRB Array tool (BRB Array tool version36[http://linusncinihgov/BRB-ArrayToolshtml]).Furthermore, we have analyzed these differentially expressed genes by KEGG database,and observed the cell cycle there is an obvious exception signaling pathways in the nasopharyngeal carcinoma, which is involved in the expression of cell cycle dependent kinase4(CDK4) exception.CDK4is a member of the cyclin-dependent kinase family and mainly expressed in tumor cell nucleus,but not is a transcription factor.It often periodic protein D (cyclin D) combine to form compounds and increase the E2F1gene expression of transcription factors by the loss of RB genes phosphorylation level.To participate in the regulation of cell cycle G1to S phase, and lead to the occurrence of tumor.Existing research shows that,compared with the tumor CCND1gene,CDK4gene in malignant skin tumors in mice onset process showed higher activity of carcinoma.furthermore,CDK4have shown high expression in a variety of tumors,such as Oral squamous cells, pancreatic cancer and lung cancer, etc.In nasopharyngeal carcinoma, we also found that the abnormal high expression of CDK4.Above studies suggest the CDK4in initial development process played an important role in the tumor.Micrornas (miRNA) is found in recent years, biological endogenous, length of about20-23non-coding small RNA nucleotides, can through the complementary pairing with target mRNA in the transcription level to negative regulation of gene expression, lead to the degradation of mRNA or translation.Many direct evidence that the miRNA by regulating the expression of target genes, involved in tumor cell migration, invasion and metastasis process.For example,Expression of micrornas-21can directly target hit by MARCKS, PDCD4and PTEN protein expression and influence of prostate cancer, glioma, migration, invasion and metastasis of colorectal cancer and liver cancer.Expression of miR-182through direct inhibition of small eye deformity associated with transcription factors M and FOXO3protein expression of melanoma cells migration and survival. Whether CDK4in nasopharyngeal carcinoma by miRNA regulation,regulation which the miRNA expression, which again micrornas in nasopharyngeal carcinoma, in turn, regulate the cell cycle of CDK4mediated signal path way?These are all worthy of our in-depth study of the problem. Contents and methods 1. Detect the CDK4gene expression level in NPC cells and organization:test CDK4expression level by immunohistochemisty and quantitative real-time PCR(qPCR).2. Silence CDK4genes influence biology behavior of NPC cell(1) Construct the interference carrier targeting CDK4by shRNA slow virus, To observe the change of CDK4expression after inhibition in the ability of cells growth, migration and invasion, through MTT, Transwell chamber,to define the function of CDK4genes in NPC cells.(2). Preliminary research of molecular basis mediated by CDK4in NPCTest the change of cell cycle related important gene,such as CDK6、CCND1、 P21and E2Flbefore and after CDK4knock down by western blot.3. Analysis miRNA array expression before and after CDK4knock down and Function, the mechanism of authentication(1) Detect different expression of miRNAs before and afer CDK4knock down by miRNA array and quantitative real-time PCR(qPCR).(2)Select expressed significant difference Let-7c as the research objectUse the mir-mimics the Let-7c/inhibitor, To observe the change of Let-7c expression after transfection cells in the ability of cells growth, migration and invasion, through MTT, Transwell and Boyden chamber,to define the function of Let-7c in NPC cells.(3)Transfection synthetic siRNA into NPC cells with E2F1low expression through lipofection.To observe the change of Let-7c expression after interference by quantitative real-time PCR(qPCR).(4)Preliminary research of molecular basis overexpression/mediated by Let-7c in NPCTest the change of cell cycle related important gene,such as CDK4、CCND1、 P15、P16、P21and E2F1、c-myc before and after overexpression/mediated Let-7c by western blot.Results1. CDK4gene expression level in nasopharyngeal carcinoma tissue and cell detection Immunohistochemical results show that the CDK4in most of the nasopharyngeal mucosa tissue were lower expression, have different degree of expression in nasopharyngeal carcinoma tissue, but is given priority to with high expression, difference have statistical significance(x2=10.997, P<0.001).From8cases of nasopharyngeal carcinoma tissue and8cases of nasopharyngeal carcinoma tissue by fluorescence quantitative PCR verification CDK4expression in further, results show that the difference is statistically significant(F=3.483, P=0.003). Quantitative real-time PCR shows, CDK4express differences in mRNA level with statistical significance in NPC cells which include5-8F,6-10B, C666-1,CNE1, CNE2, HNE1, HONE1, SUNE1(F=26.745, P＜0.001).6-10B and5-8F wih highest expression can be used as the next interference object.2. Silence CDK4genes influence biology behavior of NPC cell(1) Establish a slow interference carrier of virus and a negative control carrier, packaging5-8F cells after slow virus infection.As the efficiency of infection over at90%, protein expression declined obviously after CDK4interference by western blot, and can be applied directly to the follow-up experiment.Positive interference cloning named Cl-6、D2-4and the negative control cloning namede MOCK.(2) MTT detect that interference of polyclonal cells of Cl-6、D2-4grow slower than the control of5-8F mock proliferation, the difference being statistically significant (F=38.163P＜0.001)(3) In transwell migration assay, the number of Cl-6%、D2-4through polycarbonate film less than mock (F=38.629, P＜0.001) after10h cell culture.(4) Testing the cell cycle by flow cytometry, compared Cl-6、D2-4to control cells(F=6.163,P=0.027),prompt CDK4expression group appeared obvious obstacle from G1to S phase cells, cell block in G1phase.(5)Test the changes of cell cycle related gene expression before and after CDK4knock down by western blot. The results showed that the expression of CCND1、 CDK6、E2F1in C1-6and D2-4compared with control group cells are decrease, while P21expression is increased. 3.Analysis miRNA array expression before and after CDK4knockdown and Function, the mechanism of authentication(1) Interference of CDK4miRNA expression profile chip.Finally received35different miRNAs gene by using BRB software analysis, difference of multiple changes ranging between1.9519.25, and the35miRNAs genes show consistent are on the increase.Fluorescence quantitative PCR showed that mir-Let-7-c expressed significant difference, the selection of mir-Let-7-c as the research object.(2) After targeted silence E2F1, the Let-7c expression decreased obviously (5-8G:t=-36.565, P<0.001;6-10B:t=-34.738, P＜0.001)(3) After use the Let-7c mimics/inhibitor transfection cells,MTT detect that compared with their negative controls, we found that Let-7C mimics or its inhibitor respectively inhibited or promoted cell growth.difference was statistically significant (Mimics F=25.486, P=0.021, Inhibitor F=43.894, P＜0.001).(4) Transwell Chambers migration experiment, cell culture in after12h,Compared to control group, Let-7c mimics cell through the polycarbonate membrane decreased, Let-7c inhibitor group increase in the number of cells through the polycarbonate membrane (Mimics t=7.572, P＜0.001, Inhibitor t=5.494, P＜0.001).Prompt after expressing Let-7c, cell migration ability is abate, interfere with the Let-7c, cell migration ability enhancement.(5) Boyden Chambers invasion experiment, cell culture in after12h,Compared to control group, Let-7c mimics cell through the polycarbonate membrane decreased, Let-7c inhibitor group increase in the number of cells through the polycarbonate membrane (Mimics t=6.147, P＜0.001, Inhibitor t=5.741, P＜0.001).Prompt after expressing Let-7c, cell invasion ability is abate, interfere with the Let-7c, cell invasion ability enhancement.(6)Test the changes of cell cycle related gene expression before and after CDK4knock down by western blot.we found that Let-7C mimics elevated p15and p16expression and decreased the expression of CDK4and E2F1、c-myc in NPC5-8F cells. On the contrary to the specific effect caused by the introduction of Let-7C mimics, we observed that the expression of p15and p16was inhibited and the expression of CDK4and E2F1、c-myc was enhanced by using Let-7C inhibitor in NPC5-8F cells.Conclusions1. After CDK4knocked down, the ability of proliferation,migration have been significantly enhanced in NPC cells, and stop the cells from the G1phase to S phase in cell cycle,suggested that CDK4have the promote cancer genes function.2.After interference CDK4may inhibit the cell cycle signal pathway by inhibiting the CCND1and CDK6and raised the P15, P21gene expression involved in cell cycle regulation.Regulating mir-Let-7-c expression through the transcriptional factor E2F13.Let-7C inhibited cell growth, migration, and invasion in NPC cells. These results hinted Let-7C as a potential tumor suppressor in NPC.we observe that p15and p16, two tumor suppressors as upstream regulator of CDK4(23) were positively modulated by Let-7C, whereas CDK4and E2F1were negatively regulated by Let-7C in NPC cells. These results suggested that Let-7C suppressed cell growth by p15/p16/CDK4/E2F1signal in NPC.