Abstract

   MicroRNAs (miRNAs) comprise a class of short, non-coding RNAs that
   directly target 3′UTR of mRNA, causing subsequent degradation or
   suppression of translation. Here, we verified that miR-199a-5p was
   significantly down-regulated in mouse NSCLC tissues and human patient
   samples. To further study the function of miR-199a-5p, lentivirus
   system was adopted to construct stably over-expressing miR-199a-5p
   A549, SPC-A1 and H1299 cell lines. Then, miR-199a-5p played a tumor
   suppression role via directly targeting MAP3K11 gene in non-small cell
   lung cancer (NSCLC). Elevated miR-199a-5p suppressed cell proliferation
   and arrested cell cycle in G1 phase. We found that MAP3K11 was
   negatively correlated with miR-199a-5p in NSCLC patient tissues and
   mouse xenograft tumors. Our results suggest that miR-199a-5p together
   with its target gene MAP3K11 is a key factor and constitutes a
   complicated regulation network in NSCLC.

   Keywords: miR-199a-5p, MAP3K11, MAPK pathway, NSCLC, tumor suppression

Introduction

   Lung cancer is the leading cause of cancer deaths worldwide [37]^1^,
   [38]^2. Based on their distinct clinicopathological features, lung
   cancers are currently divided into two groups: small cell lung cancer
   (SCLC) and non-small cell lung cancer (NSCLC), which accounts for about
   80% of all [39]^3^-[40]^5. Despite improvements in clinical treatment
   strategies, the 5-year survival rate after treatment is still very
   poor; therefore it is crucial to identify better targets for the
   treatment of human NSCLC [41]^6^-[42]^9.

   MicroRNAs (miRNAs) are short non-coding RNAs of 19-25 nucleotides that
   can modulate the expression of at least 30% of all human genes, with
   marked effects on fundamental biological processes such as cell
   proliferation, apoptosis, differentiation and metabolism
   [43]^10^-[44]^13. MiRNAs can bind to partially complementary sequences
   of mRNA 3' untranslated region (3'UTR), subsequently causing mRNA
   degradation or translation inhibition, thus effectively silencing their
   target genes [45]^14^-[46]^16. Hence, not only protein-encoding genes
   but also non-coding RNAs especially miRNAs, should be considered as key
   factors in signaling cascades [47]^17^-[48]^19. Moreover, the targets
   of many miRNAs are still unknown or to be verified, even though we can
   get the putative targets of miRNAs by TargetScan or other
   bioinformatics tools. Thus, in order to unveil the molecular mechanisms
   associated with NSCLC progression and development, the target
   identification of miRNAs is critical, providing new ways for treating
   lung cancer [49]^20. Recent findings have highlighted a relevant role
   for miR-199a-5p in the regulation of tumorigenesis events [50]^21.
   MiR-199a-5p directly targets HIF1-α, which is a prominent transcription
   factor regulating angiogenesis, predominantly via induction of VEGF
   transcription [51]^22. Furthermore, hypoxia induces down-regulation of
   miR-199a-5p, probably through activation of the AKT pathway [52]^23^,
   [53]^24. On these premises, we investigated the functional role of
   miR-199-5p in NSCLC.

   In the present study, we validated the decrease of miR-199a-5p either
   in clinical lung adenocarcinoma samples or mouse NSCLC models, and
   confirmed miR-199a-5p as a tumor suppressor for NSCLC in vitro (human
   cell lines) and in vivo (xenografted tumor assay). We further dissected
   the regulatory signaling of miR-199a-5p, and gave the evidence that
   miR-199a-5p negatively regulates its downstream target MAP3K11, which
   plays the anti-proliferation roles via MAPK pathway in the lung
   adenocarcinoma.

Materials and Methods

Cell culture

   Human lung cancer cells (A549, SPC-A1, H1299) and Human embryonic
   kidney cell (HEK-293T) were obtained from the Cell Bank, China Academy
   of Sciences (Shanghai, China). A549, SPC-A1 and HEK-293T cell lines
   were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and
   H1299 in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA), all
   supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), 100 U/ml
   penicillin and 100 μg/ml streptomycin. All cells were cultured in a 5%
   CO[2] humidified incubator at 37℃.

Cell proliferation assay

   2.0×10^3 cells per well were seeded in 96-well plates and cell
   viability was measured at 24, 48, 72 and 96 h. Cell viability was
   determined by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan). At
   the indicated time, CCK-8 was added and incubated for 2.5 h at 37℃ in
   5% CO[2] and light absorbance was measured at 450 nm wavelength using a
   microplate reader, and their growth rates were recorded with three
   independent experiments.

Cell cycle analysis

   Cells (1×10^6) were digested with a trypsin solution (0.25%) and then
   fixed in 70% ethanol overnight at -20℃. After washing with cold
   phosphate-buffered solution (PBS), the fixed cells were re-suspended in
   PI/RNase Staining Buffer and incubated at 37°C for 30 min in the dark.
   After staining, samples were analyzed with MoFlo XDP flow cytometry
   (Beckman Coulter, Inc., Brea, CA, USA). Data from flow cytometry was
   analyzed using Flow Jo software (Treestar Inc., USA). The flow
   cytometry analysis was repeated three times.

Colony formation assay

   Cells were plated in 6 cm cell plate at 300-500 cells/well and further
   cultured in complete media for 10-15 d. After removal of the media, the
   cells were rinsed twice with PBS, and colonies were fixed with methanol
   for 15 min, stained with 0.1% crystal violet for 10 min and finally
   photographed using a digital camera (Leica, Germany). Experiments were
   performed three times.

Quantitative real-time PCR (qRT-PCR) analysis

   Total cellular RNA was extracted using TRIzol Regent (Invitrogen,
   Carlsbad, CA, USA) following the manufacturer's instructions. RNAs were
   reverse-transcribed using M-MLV RTase cDNA Synthesis Kit (TaKaRa,
   Dalian, China). A cDNA library of miRNAs was constructed by QuantiMir
   cDNA Kit (TaKaRa, Dalian, China). The level of mRNA or miRNA was
   quantified by qRT-PCR using SYBR Green PCR master mixture (TaKaRa,
   Dalian, China). U6 snRNA and 18S RNA was used as endogenous references