| Human NDRG2(N-myc downstream regulated gene2) gene was firstly cloned by ourlab. It locates at chromosome14q11.2and encodes a protein of40kD. NDRG2was oneof the N-myc downstream regulated gene family members. In recent years, the functionsof NDRG2have been extensively studied. Several researches indicated that theexpression of NDRG2was decreased in colon cancer, breast cancer, thyroid cancer,glioma tissues or cells. Ectopic expression of NDRG2could promote cell differentiation,inhibit cell proliferation. Although NDRG2was proved to be a tumor suppressor gene invarious kind of tumor, the function that NDRG2exerted in tumors like neuroblastoma has not been elucidated. At the same time, the researches in transcriptional regulationmechanism of NDRG2had proved that NDRG2could be transcriptional regulated byMyc, p53, HIF1, ER, AR and many other transcription factors. Potential binding sites of53transcription factors were found on the promoter of NDRG2by us throughbioinformation analysis prediction. Hence, transcription regulation of NDRG2is muchmore complex beyond our present understanding. At the present study, we will furtherexplore the transcriptional regulation mechanism on NDRG2, and verify the function ofNDRG2on neuroblastoma.In previous reseach, we found that there were several potential binding sites of thetranscription factor---KLF4on NDRG2promoter through bioinformation analysisprediction. KLF4(Krüppel-like factor4) is a transcription factor including zinc fingers,which mainly expressed in intestinal tract and epithelial tissue, and play significant rolesin cell proliferation, cell differentiation and steady state maintenance. The function ofKLF4is relied on the transcription regulation to the downstream target gene. For the pastfew years, several studies indicated that KLF4played a specific tumor suppressor genefunction through inhibiting proliferation or enhancing differentiation of colonic epithelialcell. According to these evidences, we suppose that KLF4might play a role intranscriptional regulation on NDRG2. We explored the excise mechanism in the first part.In order to analyse the mechanism of KLF4regulated on NDRG2, KLF4expressionvector was constructed,. Then pGL3-basic reporter gene vector containing humanNDRG2promoter (-1500bp~+200bp), and a series of truncated and mutant promoterwere constructed respectively. The pGL3-NDRG2vector and KLF4were cotransfectedinto Hela cells. Luciferase reporter gene analysis showed that activity of NDRG2promoter in KLF4expression vector transfected Hela cells was10times higher than thatin parental cells transfected with empty vector. We also analysis the relative activity ofthe series of the truncated and mutant NDRG2promoter, and confirmed the binding sitesof KLF4on NDRG2promoter. It verified distinctly that KLF4could bind with NDRG2promoter through chromatin co-immunoprecipitation (ChIP) and EMSA analysis.Our previous researches confirmed that NDRG2could promote a variety of tumor cell differentiation such as lymphoma cells and colon cancer cells, and it could also inhibitthe proliferation of tumor cells. These results implied that NDRG2might have the samefunction on the human neuroblastoma. Clarifing the molecular mechanism will not onlyprovide theoretical basis for the NDRG2function research, but also provide newstrategies for the clinical diagnosis and treatment of the neuroblastoma. Then weinvestigate the role of the NDRG2in cell proliferation and differentiation in NB in thesecond part.Firstly, we established cell differentiation model of neuroblastoma cells SH-SY5Y andSK-N-SH by using all-trans retinoic acid (ATRA, all-trans retinoid acid). Western blotanalysis showed that NDRG2expression level was increased in differentiated cells, andpositive correlated with cell differentiation level. To further confirm the differentiationfunction of NDRG2, we used lentivirus system to establish the NDRG2over-expressionor down-expression stable clones. We found that NDRG2over-expression stable cloneswere prone to grow neurite, and vice versa.We also found that NDRG2could translocatein the nuclear after cell differentiated by ATRA. Whether the change of intracellularlocalization of NDRG2was related with the role of promoting cell differentiation stillneed further study. The results confirmed that the NDRG2overexpression can promotethe differentiation of neuroblastoma cells, and also suggested that the low expression ofNDRG2in neuroblastoma cells may be associated with tumor development.The methylation of tumor suppressor gene is one of the most important mechanisms oftumorigenesis. Studies have shown that the low expression of NDRG2in breast cancercells or colon cancers was due to promoter methylation. In order to explore the lowexpression of NDRG2in neuroblastoma induced by methylation, we extracted thedifferentiation cell genomic DNA, and designed the methylated PCR primers of NDRG2.After bisulfite treatment, we did PCR amplification and sequencing. Then we analyzedthe methylation status about16CpG sites in the promoter of NDRG2. The sequencingresults showed that the methylation level of the NDRG2promoter was significantly lowerin the differentiation of cells than that in control group cells. After the methylationinhibitors5-aza treatment, we found that the expression levels of NDRG2were significantly higher than control cells by Western blot assay. These results implied thatthe methylation of NDRG2promoter might be the reason of its low expression level.We further detected NDRG2functions on tumor cell proliferation and metastasisability. MTT assay, EdU staining experiments, plate colony-formation assay andmonolayer wound healing assay results had showed that the tumor cell proliferation andmetastasis capabilities are inhibited in NDRG2overexpression cells compared with thecontrol cells. We also detected the cell cycle by flow cytometry, and the results revealedthat the cell cycle arrested in G1/S phase in the NDRG2overexpression cells,accompanied by cell cycle related genes CDK2, cyclinE1expression downregulation,while the expression level of p21and p27was upregulated.In the previous study, we found a NDRG2interacting protein MSP58(58-kDamicrospherule protein) using the yeast two-hybrid method for screening of human braincDNA library. Thus MSP58has become a new clue to explore the function of NDRG2gene further. At present, several studies showed that the expression of MSP58wasclosely correlated with cell cycle, and it could also be associated with a variety of tumorproliferation related proteins. Thus we hypothesized that MSP58played an importantrole in tumor proliferation. In the third part, we explored the MSP58expression level inneuroblastoma and its function in neuroblastoma cell proliferation.The results in the third part showed that MSP58was high expressed in humanneuroblastoma tissues and cell lines. We used SH-SY5Y cells as cell model, andproduced lentivirus by infecting three different pLKO-MSP58vectors andPLKO-scramble control vector, then established the stable cell clones by screening withpuromycin.It was observed that the cell proliferation and migration capabilities weresignificantly inhibited in the MSP58knockdown stable cell clones compared with that inthe control, and the expression of proliferation related genes such as Ki-67and E2F1wasdecreased either. The cell cycle arrested in S phase, moreover the expression of cell cyclerelated molecules CDK2and CyclinA has downregulated.In summary, we studied the NDRG2transcriptional regulatory mechanisms, and itsfunction in proliferation and differentiation of human neuroblastoma cells. The results confirmed that the transcription factor KLF4could transcriptional regulated on NDRG2;the NDRG2could promote the differentiation of NB cells, inhibit cell proliferation, anddiscussed the mechanism of low expression of the tumor suppressor gene NDRG2inneuroblastoma. The results were the important complement of NDRG2transcriptionalregulatory mechanisms, and also for understanding important theoretical basis on clinicalneuroblastoma pathogenesis and providing a new therapeutic target on neuroblastoma. |
PDF Full Text Request