miRNA-425-5p enhances lung cancer growth via the PTEN/PI3K/AKT signaling axis

Background miRNAs regulate a multitude of cellular processes and their aberrant regulation is linked to human cancer. However, the role of miR-425-5p in lung cancer (LCa) is still largely unclear. Here, we explored the role of miR-425-5p during LCa tumorigenesis. Methods Cell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-425-5p and PTEN. Results We demonstrate that miR-425-5p is overexpressed in LCa tissue and enhances the proliferative and colony formation capacity of the LCa cell lines A549 and NCI-H1299. Through predictive binding assays, PTEN was identified as a direct gene target and its exogenous expression inhibited the pro-cancer effects of miR-425-5p. Through its ability to down-regulate PTEN, miR-425-5p activated the PI3K/AKT axis. Conclusion We conclude that miR-425-5p promotes LCa tumorigenesis through PTEN/PI3K/AKT signaling.


Background
Lung cancer (LCa) is a leading cause of cancer related mortality across the globe. LCa is prevalent in males [1] and asymptomatic during early disease stages. As many as 2 in every 3 cases are at an advanced stage (III or IV) when diagnosed and the 5-year survival rates remain low, particularly for those with metastatic LCa [2]. Improved LCa therapeutics is thus urgently required.

Patient specimens
LCa samples (n = 25) and adjacent healthy tissue (at least 2 cm from the resection margin) were collected from the Fourth Affiliated Hospital, Zhejiang University School of Medicine. The study was fully supported by the Institutional Review Board of the Fourth Affiliated Hospital, Zhejiang University School of Medicine (No.2015-0-09). All participants provided consent for sample analysis and anything about their identities will not be included in the data.
The miR-425-5p mimics and negative control miRNA (NC) were chemically synthesized by Shanghai Gene-Pharma Co., Ltd. (Songjiang, Shanghai, China). Lipofectamine 2000 (Invitrogen, Eugene, OR, USA) was used for transfection according to the manufacturer's protocol. PI3K activity was assayed as previously described [6]. PI3K inhibitor LY294002 was obtained from Abcam. To analyze the effects of miR-425-5p on PI3K/AKT, indicated Lca cells were cultured in the presence or absence of the drugs for 24 h at 37°C, the working concentrations of LY294002 were 30 μM. For experiments with LY294002 treatments, indicated Lca cells were pre-treated with LY294002 for 1 h prior to exposure to proteasome inhibitors.

Cell growth assays
LCa viability was assessed using Cell Counting Kit-8 from Shanghai Haling Biotechnology, Co., Ltd. (Shanghai, China) as per the manufacturer's protocols. Briefly, after incubating the transfected cells for one full day, they were collected after trypsinization and seeded (5 000 cells/well) into 96-well plates. Ten microliters of CCK-8 solution were added per well and kept for 2 h at 37°C. The absorbance of the mixture was estimated in a microplate reader from Bio-Rad Laboratories, Inc. (Hercules, USA) at 450 nm.

Colony formation assay
The colony formation assays were performed as previous [6]. Each group of treated cells (1 × 10 3 per well) was seeded into 10 cm culture dish, and cultured for 2 weeks. Finally, colonies were stained using 1% crystal violet and the number of cell colonies was counted.

Dual luciferase assays
The design and synthesis of PTEN fragments containing binding sites for WT (wild-type) and MUT (mutant) on miR-425-5p was done by Shanghai GenePharma. These were cloned into the Target

Statistical analyses
Data analysis was performed using SPSS 19.0. Treatment groups were compared using a one-way ANOVA. Pvalues < 0.05 were taken as significant. Experiments were performed on at least three occasions. Data represent the mean ± SD.

miR-425-5p is upregulated in LCa
We compared miR-425-5p levels in 25 paired LCa and normal lung tissue samples by qRT-PCR analysis. miR-425-5p was upregulated in LCa specimens (Fig. 1a) and expressed to high levels in A549, NCI-H1299, NCI-H460, and HCC827 cells compared to normal human lung epithelial cell line (BEAS-2B) (Fig. 1b) consistent with previous findings in other cancer types. Previous results indicated that miR-425-5p is up-regulated in LCa.

miR-425-5p enhances proliferation and inhibits apoptosis in LCa cells
To dissect the role of miR-425-5p in LCa, its expression was manipulated using miR-425-5p mimics. Figure 2a-c shows that miR-425-5p upregulation enhanced cell survival, meanwhile enhanced cell colony formation ability ( Fig. 2d and e). Taken together, above results indicated miR-425-5p is thus an oncogene in LCa cells.

Discussion
The poor prognosis of LCa highlights the need for urgent therapeutic strategies. miRNAs are novel targets for  Table 1. Data: mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001  Figure 3. b The PI3K kinase activity was determined in NSCLC cells transfected with LY294002 was significantly lower than that transfected with mimic control, and the PI3K kinase activity in NSCLC transfected with both LY294002 and miR-425-5p mimic was significantly higher than that transfected with LY294002. Data: mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 cancer therapies and their dysregulation occurs in LCa tissue [19][20][21][22]. Duan et al. showed that miR-203 binds to ZEB2 to suppress EMT [23], Yuan et al. showed that miR-30a inhibits EYA2 migration and invasion [24], and Li et al. showed that miR-1304 inhibits LCa cell division through heme oxygenase-1 [25]. The cellular roles of miR-425-5p in LCa are poorly understood. In the present work, we further explore the underlying mechanisms of miR-425-5p-induced LCa cell progression.
In the present study, we confirmed that miR-425-5p is overexpressed in LCa cell lines and tissues implicating a role in LCa tumorigenesis. Upregulating miR-425-5p levels enhanced the cell survival and colony formation ability of LCa cells in vitro, implicating it as a novel LCa oncogene. In the mechanism, using the algorithms Tar-getScan website tools, we identified PTEN as the potential target of miR-425-5p. Furthermore, we performed Luciferase reporter assays and the results showed that miR-425-5p may directly target PTEN-3'UTR. The result of qRT-PCR and western blot also confirmed that overexpression of miR-425-5p could suppress the expression level of PTEN. All the above suggested that PTEN was a potential functional target of miR-425-5p. Moreover, MiR-425-5p also negatively related PTEN mRNA levels in LCa tissue. Rescue experiment indicated that exogenous PTEN expression inhibited the pro-cancer effects of miR-425-5p. PTEN was downregulated in LCa tissue. PTEN is a tumor suppressor with well-characterized phosphatase activity [26]. PI3K/AKT promotes cell cycle progression, inhibits apoptosis, and is known to be overactive in a multitude of human cancers [27,28]. PTEN can suppress PI3K/AKT signaling and thus displays anticancer effects [29]. Upon assessment of the molecular mechanisms of miR-425-5p in LCa cells, its pro-cancer effects were found to be mediated through manipulation of the PTEN/PI3K/AKT signaling axis.

Conclusion
In conclusion, we highlight miR-425-5p as an oncogene in LCa that promotes an oncogene phenotype by inhibiting PTEN. These findings enhance our knowledge of the role of miR-425-5p and reveal new therapeutic strategies for the diagnosis and treatment of LCa. Angiogenesis and metastasis biological experiments will clarify the functions and roles of miR-425-5p in LCa.
Additional file 1: Supplement Figure S1. Uncropped images of blots and gels related to Fig. 3. Supplement Figure S2. Uncropped images of blots and gels related to Fig. 4. Supplement Figure S3. Uncropped images of blots and gels related to Fig. 5. Table S1. The raw data of all colony formation experiments.