The Effect And Mechanisms Of MiR-25 On Neuroblastoma

BackgroundNeuroblastoma(NB)is the most common tumor in children with an incidence rate of 10%,which is second only to leukemia and central nervous system tumors.The prognosis of NB is poor.NB belongs to neuroendocrine tumors,which is originated from the neural crest progenitors in the sympathetic nervous system,and usually occurs in the nerve tissues of the adrenal gland,neck,chest,and abdomen.NB has the clinical features of high malignancy and heterogeneity,prone to metastasis and relapse and poor prognosis.The clinical treatment effect for NB is limited and the patients’5y survival rate was only 40%,except for a few patients who can spontaneously regress or completely resected at early stage.Genetic abnormalities,such as V-Myc Avian Myelocytomatosis Viral Oncogene neuroblastoma derived ho mo log(MYCN)abnormal expression and missing of tumor suppressor gene sequences lp36.1 and lp36.2,are closely related to child NB classification,treatment and prognosis.In addition,some new biomarkers were reported to be associated with the survival of NB.For instance,upregulation of ARID1B gene in NB patients suggests a worse prognosis.The expression level of miR-149 was negatively correlated with the prognosis of NB patients and had potential clinical diagnostic values.However,the molecular mechanisms of the occurrence and development of NB are still unclear.MiRNA is a class of small non-coding RNA molecules with lengths of 18-22 nt and can bind to the 3’-UTR region of mRNA to inhibit its translation and regulate the protein expression.Evidence shows that miRNA is abnormally expressed in NB,and many miRNA molecules are involved in the proliferation,invasion,metastasis and apoptosis of NB,suggesting that miRNAs play important roles in NB.For example,Li et al.found that miR-1303 could promote SH-SY5Y cell proliferation and metastasis by targeting GSK3β and SFRP1 genes.Li et al.indicated that miR-21 could promote NB cell proliferation and metastasis by regulating CHL1 gene.MiR-25 belongs to the miR-106b-25 family and locates on human chromosome 7.It is upregulated in many tumors and plays important regulatory roles in the tumor occurrence.Petroccaet al.found that miR-106b-25 cluster(miR-106b,miR-25 and miR-93)was highly expressed in gastric cancer cells and regulated by E2F1 regulator.Meanwhile,miR-106b and miR-93 can regulate the expression of E2F1,thereby forming a negative feedback path.In Non-Small Cell Lung Cancer(NSCLC),the expression of miR-25 is upregulated,which promotes the proliferation,invasion and metastasis of tumor cells by targeting F-Box and WD Repeat Domain Containing 7(FBXW7)gene,playing the oncogene functions.These researches indicate that miR-25 plays some important roles in a variety of tumors,and closely related to tumor.proliferation,invasion and metastasis.However,the role and mechanism of miR-25 in NB is still unclear.Therefore,on the basis of the above studies,the effect and mechanism of miR-25 in NB was investigated.This article will be divided into three parts to discuss.Firstly,qRT-PCR method was used to verify the level of miR-25 gene expression in neuroblastoma tissue samples and cells.Secondly,the effect of miR-25 on the proliferation,invasion and migration of neuroblastoma cells were verified.Finally,predict the possible mechanism of miR-25 in neuroblastoma through computer software prediction and molecular biology experiments.This study provides the experimental basis for the function and molecular mechanism of miRNA in neuroblastoma.The basic framework and the main research contents of this thesis are shown in the following figure.The detailed research ideas and technical routes of each part will be further elaborated in the dissertation.Methods1.Part Ⅰ:The expression and significance of miR-25 in neuroblastoma tissue samples and cells:34 cases of pediatric neuroblastoma tissues were collected and the relevant data were recorded.The samples were stored at-70 ℃ immediately after liquid nitrogen quick-freezing in the fridge.The purchased neuroblastoma cell line was rapidly resuscitated and cultured in 10%fetal bovine serum.Subsequently,RNA extraction kit was used to extract the tissues and cells total RNA,and subsequent RNA reverse transcriptase was used to synthesize cDNA.QRT-PCR was used to detect the difference of miR-25 gene in neuroblastoma tissues and cells.2.Part Ⅱ:The effect of miR-25 on the proliferation,invasion and migration of neuroblastoma cells:miR-25 mimics,miR-25 inhibitors and negative control were synthesized and labeled with green fluorescent protein.Transfection of miR-25 miR-25 mimics and miR-25 inhibitors were performed using LipolifectmianTM2000 according to the manufacturer’s instrutions,the transfection efficiency were also determined.The effect of miR-25 mimics and miR-25 inhibitors on proliferation of SH-SY5Y and IMR32 cell lines was observed by Cell Count Kit(CKK-8)assay.The effects of miR-25 mimics and miR-25 inhibitors on the cell cycle activity of neuroblastoma cells were detected by flow cytometry.The invasion and migration of SH-SY5Y and IMR32 cell lines were detected by Transwell assay.This experiment mainly clarified the effect of miR-25 mimics and miR-25 inhibitors on proliferation,migration and invasion of neuroblastoma cells.3.Part Ⅲ:Screening and identification of miR-25 target gene TOB1:Firstly,Bio informatics software and pubmed were used to screen the target gene of miR-25;Secondly,the wild-type and mutant luciferase reporter plasmids of the corresponding target genes were constructed,and the control were co-transfected into HEK293T cells,then the luciferase activity was identified to determine the target gene of miR-25.Finally,miR-25 mimics and miR-25 inhibitors were transfected into cells to detect the target genes mRNA and protein expression level.In this study,we used two-way analysis methods to verify the target gene of miR-25 regulation.Results:1.In this study,we found that the expression of miR-25 in high-risk group increased significantly compared with the low-risk group neuroblastoma tissue,and the expression of miR-25 was positively correlated with the tumor stage(P<0.05).Compared with primary tumor cells,the expression of miR-25 in metastatic cells was significantly increased(P<0.05).2.miR-25 mimics,miR-25 inhibitors and negative control can be successful transfected into the neuroblastoma cells SH-SY5Y and IMR32.The CCK-8 assay showed that miR-25 promotes the proliferation of neuroblastoma cell.Compared with the control group,the growth curve showed that the OD values were significantly increased 48 h-72 h after transfection(P<0.05).Conversely,miR-25 inhibitors transfection in SH-SY5Y cells significantly reduced neuroblastoma cell proliferation(P<0.05).From Transwell migration and invasion assays we found that miR-25 significantly enhance the migration and invasion of neuroblastoma cell lines SH-SY5Y and IMR32.Compared with the control group,we found that the ability of migration and invasion of neuroblastoma cells were significantly enhanced after upregulated miR-25 expressed(P<0.05).In addition,down-regulation the expression of miR-25 showed that the migration and invasion of neuroblastoma cells were significantly inhibited(P<0.05).Cell-cycle analysis demonstrated that miR-25 mimics led to a significant reduction in G1 phase and increase in S phase and G2 phase in neuroblastoma IMR32 cells(P<0.05).In the opposite,down-regulated the miR-25 expression obviously increased the G1 phase cells and reduced the S phase cells(P<0.05).Suggesting that miR-25 plays a role of the period that cell through no-mitosis to mitosis.3.Considering the potential roles of candidate genes in neuorblastoma,TOB1 eas selected as our candidate targets gene.The result of dual luciferase assay further detected the effect of miR-25 on the TOB1 expression by the 3’-UTR of target genes.For example,after co-transfection,the expression of TOB1 was reduced.To further confirm the effect of miR-25 on TOB1 mRNA level,qRT-PCR test showed that miR-25 down-regulated TOB1 mRNA expression(P<0.05).Western blot showed that over-expression of miR-25 substantially reduced TOB1 protein levels(P<0.05),however,miR-25 inhibitors substantially enhanced TOB1 protein levels(P<0.05).Conclusion1.The expression of miR-25 in malignant neuroblastoma tissues and cells is significantly higher than that in low-risk neuroblastoma tissues and cells,so miR-25 can be used as an important indicator for the high and low risk diagnosis and prognosis of neuroblastoma.2.Over-expression of miR-25 substantially enhanced cell proliferation,migration,and invasion of neuroblastoma cells.In the contrary,down-regulated the miR-25 expression can inhibit neuroblastoma cell proliferation,invasion and migration ability.In addition,miR-25 plays an interfering role in the G1-S phase of neuroblastoma cells.MiR-25 plays an ’Oneo-miR’ in the neuroblastoma cells in vitro.3.MiR-25 can be combined with the 3’UTR of TOB1 to control the transcription,providing a new target for the treatment of neuroblastoma.