Transcriptional Factor MYCN Sustains Cell Proliferation And Tumorigenicity By MINA In Neuroblastoma

Neuroblastoma,developed from the sympathetic nervous system,is a common pediatric extracranial solid outer peripheral nervous system malignant tumor,accounting for childhood cancer rate of 8% ~ 10%,and the children's cancer death 15%.Neuroblastoma is heterogeneous in the process of tumor formation,and its strong ability of proliferation and metastasis are the main factor leading to the death of the patients.Among them,60% children were more than 1 years old at the time of initial diagnosis,and 45% had distant metastasis.The clinical features of neuroblastoma differ from each other.In the later stage of the patients,it commonly has strong resistance to traditional treatments,including surgery,chemotherapy and radiotherapy.Therefore,in recent years,a lot of experiments are devoted to explore new therapeutic methods for neuroblastoma.Approximately 25% to 30% of children with neuroblastoma in patients with MYCN gene amplification,and the higher the amplification factor the worse the prognosis.Studies have shown that MYCN has the role of promoting the proliferation and growth of neuroblastoma cells,inhibiting differentiation and promoting the invasion and metastasis.MINA is MYC-induced nuclear antigen.Structurally,MINA protein contains a conserved jumonji C(Jmj C)domain that belongs to the histone demethylases family.It has been reported that MINA is involved in reducing the trimethylation of histone H3K9(H3K9me3).Although some studies speculated that NMYC and MINA had some relationship in tumor tissue,no molecular characterizations of neuroblastoma were described in thesereports.In this study,we investigate the role of transcriptional factor MYCN in two euroblastoma cells,and our findings demonstrated that MYCN sustains cell proliferation and tumorigenicity by MINA in neuroblastoma.The main results as follows: 1.Analysis the relationship of transcriptional factor MYCN and neuroblastoma prognosisTo investigate the possibility relationship of MYCN and MINA in prognosis of neuroblastoma,the Tumor Neuroblastoma public-Versteeg database,which is available from the online R2: microarray analysis and visualization platform was performed.The Versteeg database contains a cohort of 88 patients with neuroblastoma representative of survival prognosis.12 patients with high MYCN expression showed low survival probability,whereas 76 patients with low MYCN expression showed high survival probability,suggesting that high MYCN expression is prognostic for poor outcome of neuroblastoma.58 patients with high MINA expression showed low survival probability,whereas 30 patients with low MINA expression showed high survival probability,suggesting that high MINA expression is prognostic for poor outcome of neuroblastoma.together,our analysis of the datasets indicated that MYCN and MINA was positively correlated in survival prognosis.Since the elevated expression of MYCN and MINA has been found in various cancers,next we examined MYCN and MINA expression in three neuroblastom cells including SK-N-DZ,BE(2)-C and SHEP1 cells using Western blot.The result shown that MYCN and MINA was expressed at varing levels of protein.Among the neuroblastoma cell lines,high levels of MYCN and MINA was detected in BE(2)-C cells,whereas moderate levels of NMYC and MINA was observed in SK-N-DZ cells and low levels of MYCN and MINA was found in SHEP1 cells.The results showed that the expression of MYCN in neuroblastoma cells was positively correlated with the expression of MINA.2.To explore the effects of MYCN and MINA on neuroblastoma cell growth,proliferation and colony forming abilityT To explore the functional role of MYCN and MINA in regulating cell proliferation and tumorigenesis in neuroblastoma,we investigated the effects of knocked down MYCN in BE(2)-C cells,which had MYCN-high expression.We knocked down NMYC by infection with lentivirus-expressing sh RNA targeting vector control(p LKO.1)or MYCN,we successed to knockdown of endogenous MYCN,the results detected by western blot.Western blot showed that the protein levels of MYCN were decreased in BE(2)-C cells infected with MYCN sh RNA compared with those in control sh RNA cells.Significantly,knockdown of MYCN in BE(2)-C cells by MYCN sh RNA markedly suppressed cell proliferation.In addition,knocking down MYCN in BE(2)-C significantly caused decrease in anchorage-independent growth on soft agar.Then,we knockdown MINA and the same results can be received.We next overexpressed MINA in BE(2)-C cells,which had MYCN knockdown.Western blot showed that the protein levels of MINA were decreased.These findings further confirm the important role of MYCN and MINA in regulating cell proliferation and tumorigenesis in neuroblastoma cells.To further confirm that the effect of MYCN and MINA on cell proliferation,we next investigated the effects of overexpressing MYCN on cell proliferation in a MYCN-low expression cell line(SHEP1).We overexpressed MYCN by lentivirus-mediated infection of SHEP1 cells with vector alone or MYCN.Significantly,overexpression of MYCN in SHEP1 cells caused enhanced cell proliferation compared with vector control cells.The results showed that the expression of MYCN and MINA protein was increased in overexpression of MYCN in SHEP1 cells.To further investigate the effects of MYCN and MINA on the proliferation of neuroblastoma cells,we knocked down MINA in SHEP1 cells,which had overexpressed MYCN,by infection with lentivirus-expressing sh RNA targeting vector control(p LKO.1)or MINA,we successed to knockdown of endogenous MINA,the results detected by western blot.Western blot showed that the protein levels of MINA were significantly decreased in overexpression of MYCN in SHEP1 cells infected with MINA sh RNA compared with those in control sh RNA cells.Significantly,knockdown of MINA in SHEP1 cells by MINA sh RNA markedly suppressed cell proliferation.The results showed that the expression of MYCN and MINA was decreased in knockdown of MINA with overexpression of MYCN in SHEP1 cells.Western blot showed that the protein levels of MINA were decreased in BE(2)-C cells infected with MYCN sh RNA compared with those in control sh RNA cells.And the protein levels of MINA were increased in SHEP1 cells infected with PCDH-MYCN compared with those in GFP cells.These findings further confirm the important role of MYCN and MINA in regulating cell proliferation and tumorigenesis in neuroblastoma cells.3.A preliminary study on the mechanism of MYCN and MINA on the proliferation and tumorigenesis of neuroblastoma cellsIt was found that MYCN was in the promoter region of MINA by chromatin immunoprecipitation assay and the upstream of transcription initiation site region of 593 ~ 472,1043 ~ 839 and 849 ~ 573 were much higher than the other.Therefore,we speculated that MYCN sustains cell proliferation and tumorigenesis by MINA in neuroblastoma.