Abstract:

Objective   To construct the recombinant eukaryotic expression vector for human miR-147a and determine its effects on the proliferation and invasion of lung adenocarcinoma cell A549 due to miR-147a expression. Methods   pri-mir-147a sequence was amplified by RT-PCR using total RNA from A549 cells and was inserted into the pSilencer4.1CMV neo expression vector to construct the recombinant pSilencer4.1-147a vector, which was transfected into A549 cells, followed by qRT-PCR and reporter gene assay to detect the expression of miR-147a. MTT assay was utilized to determine the effect of exogenous miR-147a over-expression on the proliferation of A549 cells. Matrigel invasion assay was performed to detect the effect of exogenous miR-147a over-expression on the invasion of A549 cells. Finally，reporter gene assay was used to observe the effects of miR-147a on common tumor signal pathways. Results   The construction  of pSilencer4.1-147a was confirmed to be correct by restriction enzyme digestion and DNA sequencing. qRT-PCR and reporter gene assay displayed significant ectopic expression of miR-147a after the transfection of pSilencer4.1-147a in A549 cells. MTT assay and Matrigel invasion assay showed that miR-147a significantly inhibited the proliferation and invasion ability of A549 cells. Reporter gene assay showed that miR-147a significantly down-regulated relative luciferase activity of pGL4.23-AP1, pGL4.23-ELK1 and pGL4.23-cMYC. Conclusion   The eukaryotic expression vector of human miR-147a is constructed successfully and expressed in A549 cells effectively. The exogenous miR-147a over-expression shows inhibitive effects on the proliferation and invasion of A549 cells, which maybe relevant to the inhibition of EGFR-RAS-MAPK pathway by miR-147a.

Key words: microRNA; miR-147a; Lung neoplasms; Proliferation; Invasion