MicroRNA-1 Inhibits The Proliferation, Migration And Invasion Of Lung Cancer Cell Line A549 Cells By Regulating PIK3CA

Background and objective With nearly two million new cases diagnosed worldwide each year, lung cancer remains one of the most life-threatening tumors. Non-small cell lung cancer (NSCLC), which represents about 75-85% of lung cancers, has a low cure rate because of the lack of early diagnosis and effective treatment methods. The 5-year survival rate of NSCLC patients post-surgery is around 50%, even if they are diagnosed and widely treated with surgical resection in the early stage. Thus, rationally designed and targeted agents that mediate NSCLC progression and that can be used for molecular targeted therapies are urgently required and are of great interest. In our preliminary studies, we found that the high expression of phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) is related with poor prognosis of NSCLC, and PIK3CA expression in NSCLC was negatively correlated with micorRNA-1 (miR-1). miR-1 expression in lung cancer tumor tissues and cells were comparatively low, and significantly lower in patients with metastasis than those that were metastasis-free within 1 year postoperatively, which suggests that the miR-1 expression level is associated with prognosis in lung cancer. PIK3CA has been defined as a driver oncogene responsible for both the initiation and maintenance of NSCLC. Inhibition of PIK3CA may be a new molecular target therapy of NSCLC. We found miR-1 and PIK3CA expression in NSCLC were negatively correlated. Thus, miR-1 has potential therapeutic application against NSCLC. However, the role of miR-1 in NSCLC and the molecular mechanisms by which it exerts its functions remain largely unknown. Based on our previous findings, we investigated the effect of miR-1 transfection on the biological properties of NSCLC cell line A549 and its mechanism is discussed. Identification of novel miR-1 targets would provide new insights into the molecular mechanism underlying miR-1 inhibition of tumorigenic properties and subsequently enable the design of improved therapies.Methods Software and websites of bioinformatics including TargetScan and PicTar were used for target gene prediction. Recombined vector pGL3-promoter was constructed to express PIK3CA 3’UTR and dual-luciferase reporter assay was analyzed. Transfection of miR-1 mimics/inhibitor was performed in human lung adenocarcinoma A549 cells. Quantitative real-time PCR and Western blot were used to detect PIK3CA mRNA and protein expression. Cell proliferation was studied using a cell counting kit-8. Migration and invasion were evaluated by Transwell and Matrigel assays, respectively. Cell cycle and apoptosis were detected by flow cytometry.Results Bioinformatics softwares were used and found that the 3’UTR of PIK3CA mRNA contains a putative site partially complementary to miR-1. The dual-luciferase reporter assay confirmed that miR-1 targeted PIK3CA directly. By western blot, we found PIK3CA protein level increased in which miR-1 is over-expressed and reduction in which miR-1 is down-expressed in A549 cells, compared with the control group. These results further confirmed PIK3CA is a target gene of miR-1. Meanwhile qRT-PCR showed the increase or decrease of miR-1 caused almost no alteration to the expression of PIK3CA mRNA. PIK3CA downregulation by miR-1 mimics led to a significant reduction of phosphorylated Akt and survivin protein, the downstream targets of the PI3K/Akt signaling pathway. In contrast, phosphorylated Akt and survivin protein were significantly increased in the miR-1 inhibitor group as compared with the control group. Overexpression of miR-1 inhibited A549 cell proliferation, migration and invasion. Conversely, transfection of miR-1 inhibitor promoted cell proliferation, migration and invasion. Alteration of miR-1 expression did not affect the cell cycle distribution and apoptosis of A549 cells.Conclusion miR-1 could significant suppress the growth, migration and invasion of lung cancer cells by down-regulating the expression of PIK3CA through the PI3K/Akt pathway. Therapeutic strategies to rescue miR-1 expression may be beneficial to patients with NSCLC in the future.