The Role And Mechanism Of Mir-424-5p Targeting EIF3a In The Regulation Of Ameloblastoma

Objective: Ameloblastoma is an oral benign tumor that mainly arises in the mandible,the incidence is 10% in odontogenic tumors.According to the WHO 2017 head and neck tumors classification,AB was divided into four categories: conventional,unicystic,extraosseous/peripheral,and metastasizing AB.It is benign,but it is locally invasive and has a high risk of recurrence.In recent years,there are many studies on AB,including the proliferation,differentiation,tumor suppressor genes,osteoclast mechanisms,signal molecule expression,and related signaling pathways,but the exact mechanism of it is still not very clear.eIF3 has a molecular weight of 550-700 kDa in mammals and consists of 13 subunits,eIF3a~eIF3m.The largest subunit,eIF3 a,has many biological functions,such as participation in translation initiation,cell cycle regulation,proliferation,and differentiation.It regulates the translation of certain protein mRNAs and plays an important role in the regulation of cell cycle and proliferation.Many studies have confirmed that the expression of eIF3 a is high related to the occurrence and development of tumors.miRNAs regulate nearly 30% of human genes.Some basic cell biology processes,including growth,differentiation,and apoptosis,are inseparable from the development of tumors.miR-424-5p is a member of the miR-15 family that has been found to play a very important role in the development of tumors in many recent studies.In order to study the effects of eIF3 a,miR-424-5p and their mutual regulation on the biological properties of AB,we present the relative studies in three parts in this thesis.Methods: We performed immunohistochemistry to determine the expression of eIF3 a in AB(N=83)and NOM(N=20)tissues.RT-qPCR and Western blotting analyses were conducted with AB(N=30)and NOM tissues(N=6).The correlation between eIF3 a expression and the clinical/pathological features of AB patients was also presented.The functional role of eIF3 a in AM-1 cells was assessed with lentiviral vector-mediated shRNA by MTT,cell clone formation,and flow cytometry assay.The binding between miR-424-5p and eIF3 a was predicted by microchip and verified by the dual luciferase reporter.In addition,RT-qPCR was used to detect and analyze the expression of miR-424-5p in AB(N=30)and NOM(N=6)tissues.AM-1 was transfected with miR-424-5p mimics/NC,then the relevant cell phenotypic changes of AM-1 cells were examined to determine whether the overexpression of the miR-424-5p could affect the biological properties of AB.Finally,RT-qPCR and WB were used to detect and analyze the regulation between miR-424-5p and eIF3 a.Whether the expression of cell cycle-related protein P21 and Cyclin D1 were affected by eIF3 a and miR-424-5p was also presented.Results: Compared with NOM tissue,the expression of eIF3 a was significantly upregulated in AB tissues at both mRNA and protein levels.The AM-1 cell clone formation and MTT assay after lentivirus transfection revealed that eIF3 a knockdown could significantly inhibit the proliferation and cell viability of AM-1 cells.It was observed by flow cytometry that eIF3 a knockdown could significantly promote cell apoptosis.The dual luciferase reporter gene verified that miR-424-5p and eIF3 a have a target gene binding site and miR-424-5p was expressed at a low level in AB tissues.In addition,overexpression of miR-424-5p showed a significant inhibitory effect on the proliferation and migration ability of AM-1.miR-424-5p can negatively regulate the expression of eIF3 a,and both of them have a significant effect on the regulation of AM-1 cell cycle.Conclusion: eIF3 a in AB tissues was upregulated expressed.When eIF3 a knockdown,it could significantly inhibit cell proliferation and viability of AM-1 but promoted apoptosis.There is a target gene binding site between miR-424-5p and eIF3 a.The expression of eIF3 a in AB tissues was at a low level.When miR-424-5p overexpressed,it could inhibit the proliferation and migration of AM-1 cells,negatively regulated the expression of eIF3 a,and both participate in the regulation of AM-1 cell cycle.