Expression And Significance Of MiRNA-1-3p, LAMP2 And LC3 In Ameloblastoma

Objective:Ameloblastoma is a benign odontogenic tumor of epithelial origin.It is locally aggressive with unlimited growth capacity and has a high potential for recurrence.The possibility of malignant transformation and even metastasis is existed as well,severely affecting the living quality of patients.Autophagy is a phenomenon in which eukaryotic cells process their own aging,damaged or denatured proteins and biomacromolecules such as organelles,which can degrade or reuse these components derived from the cells themselves in lysosomes.This process can providing energy to help cells survive the survival crisis,and play a role in preventing cell aging and death as well as tumorigenesis.Lysosomes play an important role in autophagy.B-cell lymphoma-2-interacting protein-1(Beclin1)encodes a protein that regulates autophagy.The encoded protein is a component of the phosphatidylinositol-3-kinase(PI3K)complex which mediates vesicle-trafficking processes.Beclin1 is a promoter of autophagy.During autophagosome formation,the microtubule-associated protein 1 light chain 3(LC3)molecule is converted from a LC3-I to LC3-II,which can bind to a newly formed autophagosome membrane until autophagosomes and lysosome fuse.LC3-II is often used as a marker for intracellular autophagy.Therefore,detecting the expression levels of LAMP2,Beclin1 and LC3 in ameloblastoma can reflect the level of tumor autophagy.At present,micro RNA(mi RNA)has been widely concerned with the regulation of gene expression at the post-transcriptional level.Many studies have verified that mi RNA can play a regulatory role in different stages of autophagy.Using microarray analysis and bioinformatics prediction,mi RNA-1-3p differentially expressed in ameloblastoma tissues was screened as a potential regulatory molecule of LAMP2.To examine if the expression of mi RNA-1-3p was further correlated with LAMP2 expression,providing a new direction for the diagnosis and treatment of ameloblastoma from non-coding RNA regulation view.Methords:(1)Immunohistochemical staining and quantitative detection with Image J software were used to detect the expression levels of Beclin1,LC3 and LAMP2 in 104 cases of ameloblastic wax blocks and 20 cases of normal oral mucosa.(2)Western Blot(WB)method was used to detect the expression of Beclin1,LC3II/I and LAMP2 in fresh ameloblastoma tissue(AB)and normal oral mucosa(NOM)to reflect the level of autophagy in ameloblastomas.(3)In combination with the results of the detection of Human mi RNA expression profiles in AB and NOM in the early stage,the differentially expressed mi RNAs related to AB were screened.Bioinformatics analysis was performed in down-regulated mi RNA,and mi RNA-1-3p was selected as the research object.(4)Quantitative real-time PCR(q RT-PCR)was used to detect the expression level of LAMP2 and mi RNA-1-3p in 12 pairs of ameloblastoma and normal oral mucosa as well as in human immortalized ameloblastoma cell line AM-1 and human immortalized epidermal cell line Hacat.(5)Spearman correlation analysis was used to analyze the correlation between mi RNA-1-3p and LAMP2 expression levels.Results:(1)Immunohistochemistry results showed that Beclin1 was localized to the cytoplasm and nucleus of ameloblastoma epithelial cells.The positive rate and quantity in AB were lower than that in NOM(P < 0.05).There was no significant difference in the aspects of gender,age,location,pathological type and recurrence.LC3 was localized to the cytoplasm of ameloblastoma epithelial cells.The positive rate and quantity in AB were higher than that in NOM(P < 0.05).There was no significant difference in gender,age and recurrence.The positive rate in AB occurred in the mandible was significantly higher than that in the maxillary and gingival(P < 0.05),and the positive rate in solid/polycystic AB was significantly higher than that in other three types(P < 0.05).LAMP2 localized to the cytoplasm and cell membrane of ameloblastoma epithelial cells,and the positive rate and quantity in AB were higher than that in NOM(P < 0.05).There is no statistically significant difference in gender,age,pathological type and recurrence.The positive rate of AB in the mandible was significantly higher than that in the maxilla and gingiva(P < 0.05).(2)The results of Western Blot showed that the expression levels of LAMP2 and LC3II/I were increased and the expression of Beclin1 was decreased in AB compared with NOM(P < 0.05).(3)In combination with the results of the detection of Human mi RNA expression profiles in AB and NOM in the early stage,it was found that the expression of mi RNA-1-3p associated with autophagy was down-regulated in AB,and the expression level in AB was only 0.06 times that of NOM.Bioinformatics analysis showed that mi RNA-1-3p might be an upstream regulatory molecule of LAMP2,mi RNA-1-3p was selected to be the further research object.(4)The results of q RT-PCR showed that compared with the NOM tissue,the expression of LAMP2 was up-regulated and the expression of mi RNA-1-3p was down-regulated in AB,so as that in AM-1 cells and Hacat cells.The difference was statistically significant(P < 0.05).(5)Spearman correlation analysis result showed that the expression of LAMP2 was negatively correlated with the expression of mi RNA-1-3p(r =-0.888,P < 0.05).Conclusions:(1)The level of autophagy and the increase of lysosomal activity in AB tissues may be related to the biological behavior of AB recurrence and local invasion.(2)LC3 and LAMP2 are highly expressed in AB and they can be used as reference markers for AB diagnosis.(3)mi RNA-1-3p is down-regulated in AB,which may be a potential upstream regulatory molecule of LAMP2,which can affect AB autophagy by targeting LAMP2 and further affect its biological behavior.