MicroRNA-9-3p Regulates Proliferation,Migration And Invasion In Ameloblastoma By Targeting RBPJ

Objective:The morbidity of Ameloblastoma(AB)is high,especially in developing countries.AB is an epithelial tumor and in some countries it accounts for 36.3% of odontogenic tumors.Although AB is a benign tumor,it has the characteristics of local invasion and high recurrence rate.It is a borderline tumor,and even can metastasizes distantly.Most of AB occur in the jaw.With the progress of the disease,the jaw gradually expands,resulting in facial deformity and functional impact.The tumor can eventually penetrate the bone and invade the surrounding soft tissue.So far,the proliferation,differentiation,osteoclastic mechanism,signal molecule expression and related signaling pathways of tumor cells was discovered,but still unclear in AB.MicroRNA(miRNA)is one of the common small non-codingRNA with a length of19-24 nucleotides.They can bind to the 3 ’-UTR of messengerRNA(mRNAs),then silence the expression of target gene mRNA.Participate in the regulation of gene expression at the post transcriptional level.It has been reported that miRNA widely exists in various eukaryotic cells and participates in the growth and development,apoptosis,fat metabolism and tumors.MiR-9-3p is a new kind of miRNA,which has been discovered participate in the occurrence and development of other tumors.The purpose of this test is to evaluate the expression of miR-9-3p in AB and proliferation,migration,invasion of miR-9-3p in AM-1.To investigate the possible molecular regulation mechanism,so as to find sensitive diagnostic markers,specific intervention targets and effective treatment for AB.Methods:1.MiRNA microarray technology was used to screen the differentially expressed miRNAs in AB.Bioinformatics technology was used to determine miR-9-3p as the follow-up research object.2.In 15 pairs of AB and NOM,qRT-PCR was used to detect the expression of miR-9-3p.Western blot,qRT-PCR and immunohistochemistry was used to detect the expression of RBPJ.3.MiR-9-3p mimics and si-RBPJ was transfected into AM-1 respectively by transfection technology.Proliferation,apoptosis,migration and invision of AM-1 were observed by CCK8,plate cell cloning,flow cytometry,cell scratch and transwell.4.The target of miR-9-3p was preliminarily predicted and screened by online bioinformatics prediction website.qRT-PCR and Western blot were used to identify the changes of target gene mRNA and protein level.Dual luciferase assay was constructed to verify the changes.Different combinations of miR-9-3p mimics,miR-9-3p inhibitor and si-RBPJ were transfected into AM-1 cells by transfection technology to further clarify the relationship of targeting regulation and its effect on Notch signaling pathway.Results:1.Compared with NOM,MiRNA microarray showed that miRNA expression in AB group more than 2.0 times with statistical difference(P<0.05)was identified as differentially expressed miRNA.The expression of miR-9-3p in AB was 0.348 times of that in the control group(P<0.05).Finally,miR-9-3p was determined as the further research object.2.qRT-PCR showed that the relative expression of miR-9-3p in AB was significantly lower than that in NOM(P<0.05);the relative expression of RBPJ was tested by qRT-PCR and Western blot showed that AB was significantly higher than NOM(P<0.05);Pearson correlation analysis showed that there was a strong negative correlation between the expression of miR-9-3p and RBPJ in AB(R=-0.6943,P<0.05);IHC found that the relative expression of RBPJ in AB was significantly higher than that in NOM(P<0.05),which was closely related to the invasion of AB.3.MiR-9-3p was overexpressed by transfecting miR-9-3p mimics into AM-1.CCK8,plate cell clone and flow cytometry showed that the proliferation of AM-1 was significantly decreased compared with the control group(P<0.05);Cell scratch test showed that the migration of AM-1 was significantly decreased compared with the control group(P<0.05);Transwell showed that the invasion of AM-1 was significantly decreased compared with the control group(P<0.05);Flow cytometry showed that compared with the control group the apoptosis rate of AM cells had no significant change(P>0.05).4.According to bioinformatics analysis,we predicted that RBPJ might be one of the main targets of miR-9-3p.Dual luciferase assay confirmed that miR-9-3p could bind to3’-UTR of RBPJ.Overexpression of miR-9-3p could inhibit the relative luciferase activity of RBPJ 3’-UTR-wt group.5.RBPJ in AM-1 was decreased by transfecting si-RBPJ.CCK8,plate cell clone and flow cytometry showed that the proliferation of AM-1 was significantly decreased compared with the control group(P<0.05);Cell scratch test showed that the migration of AM-1 was significantly decreased compared with the control group(P<0.05);Transwell showed that the invasion of AM-1 was significantly decreased compared with the control group(P<0.05);Flow cytometry showed that compared with the control group the apoptosis rate of AM cells had no significant change(P>0.05).This is consistent with the function of miR-9-3p mimics in AB.6.AM-1 was transfected with miR-9-3p mimics,miR-9-3p inhibitor or si-RBPJ-3,qRT-PCR and Western blot showed that compared with the control group,Notch4 and Jag1 in miR-9-3p mimics group and si-RBPJ-3 group had no difference,but Hey1 was significantly decreased(P<0.05);Notch4 and Jag1 in miR-9-3p inhibitor group had no change,but Hey1 was significantly increased(P<0.05);Notch4 and Jag1 in mimics +si-RBPJ-3 group did not change,while Hey1 decreased most significantly(P<0.01);Notch4,Jag1 and Hey1 in miR-9-3p inhibitor + si-RBPJ-3 group did not change significantly(P>0.05).Conclusions:1.Combined with human miRNA microarray technology,we found that miR-9-3p was lower expressed in AB,RBPJ was higher expressed in AB,and miR-9-3p was negatively correlated with RBPJ.The expression of RBPJ was closely related to the invasion of AB.2.Overexpression of miR-9-3p or down-expression of RBPJ could significantly inhibit the proliferation,migration and invasion of AM-1,but had no significant effect on the apoptosis.3.RBPJ is one of the target gene of miR-9-3p.4.MiR-9-3p regulates Notch signaling pathway by targeting RBPJ.