The Biological Function And Molecular Mechanism Of MLLT3 In Lung Adenocarcinoma

Objective:Lung cancer with high morbidity and mortality is one of the most frequent cancer types around the world.MLLT3,also known as AF9,was first found in acute myeloid leukemia as a fusion partner of the Mixed Lineage Leukemia(MLL)gene.MLL-AF9 fusion gene,formed by chromosome ectopic,is one of the most common 11q23 translocation in acute myeloid leukemia.This topic aimed to explore the elusive functional roles and the underlying molecular mechanisms of MLLT3 in lung adenocarcinoma.Methods:(1)The expression of MLLT3 in 74 pairs of lung adenocarcinoma tissues and their adjacent normal lung tissues was detected by immunohistochemical(IHC).According to the expression intensity of MLLT3,the 74 pairs of tissues were divided into high and low expression group for further analysis between MLLT3 expression and clinical parameters;the expression of MLLT3 in four Lung adenocarcinoma cell lines(PC9,A549,NCI-H1299 and NCI-H1975)and one bronchial epithelial cell line(BEAS-2B)was examined by real-time quantitative PCR and western blot analysis,and high level expression cell lines(A549 and NCI-H1299)were screen out.(2)According to the sequence of MLLT3 gene,two pairs of siRNA were synthesized to knock down the endogenous MLLT3 in A549 and NCI-H1299 cells,The functions of MLLT3 in Lung adenocarcinoma cell lines were examined using CCK8,colony formation,transwell assays,wound-healing assay and flow cytometry in vitro;In the study of mechanism,EGFR-MAPK/ERK signaling pathway,cell cycle and apoptosis related genes and Epithelial-mesenchymal transition(EMT)related molecular markers in the change of protein levels were examined by Western blot.(3)The lentiviral vector containing short hairpin RNA(shRNA)of MLLT3 was further designed to knock down the endogenous MLLT3 in A549 cells,and nude mouse subcutaneous tumor models were used to study lung adenocarcinoma cell growth in vivo.Results:The results showed that human lung adenocarcinoma tissues exhibited increased levels of MLLT3 compared with non-malignant tissues.The expression profiles of MLLT3 was associated with metastasis and advanced TNM stage.Survival analysis showed that high expression level of MLLT3 was negatively correlated with overall survival rate.Expression of MLLT3 in A549 and NCI-H1299 cell lines was higher.Knockdown of MLLT3 repressed lung adenocarcinoma cell proliferation,migration and invasion in vitro,and increased the percentage of lung adenocarcinoma cells in G0/G1 phase and the apoptosis cells,but decreased the percentage of cells in S phase.In the further mechanism research,Western blot confirmed that down regulation of MLLT3 up-regulated the expression levels of cleaved caspase-9,cleaved caspase-3,cleaved PARP,E-cadherin and p21 in A549 and NCI-H1299 cells,Whereas down-regulated the expression levels of P-EGFR,P-ERK,P-MEK,cmyc,CDK4,cyclinD and EMT related molecular markers Vimentin,N-cadherin.Additionally,knocking down MLLT3 significantly reduced the growth rate and weight of subcutaneous tumors in vivo experimental research.Conclusion:The expressions of MLLT3 in lung adenocarcinoma tissues was significantly higher,which was associated with the clinicopathologic characteristics and overall survival of lung adenocarcinoma patients.EGFR-MAPK/ERK pathway might contribute to the effect of MLLT3 in lung adenocarcinoma,which might be a therapeutic target and potential diagnosis for treatment of lung adenocarcinoma.