MicroRNA-206 attenuates the growth and angiogenesis in non-small cell lung cancer cells by blocking the 14-3-3ζ/STAT3/HIF-1α/VEGF signaling

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Angiogenesis is the major hallmark in NSCLC. So, further elucidation of molecular mechanisms underlying the angiogenesis of NSCLC is urgently needed. Here, we found that microRNA-206 (miR-206) decreased the angiogenic ability in NSCLC via inhibiting the 14-3-3ζ/STAT3/HIF-1α/VEGF pathway. Briefly, 14-3-3ζ bond with phosphorylated-STAT3, and in turn, elevated the expression of HIF-1α. Then, by enhancing the recruitment of HIF-1α to VEGF promoter, 14-3-3ζ increased the angiogenesis. However, miR-206 decreased the angiogenesis by targeting 14-3-3ζ, and inhibiting the STAT3/HIF-1α/VEGF pathway. In NSCLC cell xenograft model, either overexpression of miR-206 or inhibition of 14-3-3ζ inhibited the STAT3/HIF-1α/VEGF pathway and decreased the tumor growth and angiogenesis. Furthermore, there was a negative correlation between miR-206 and 14-3-3ζ in NSCLC specimens. NSCLC patients with low expressions of miR-206 but high expressions of 14-3-3ζ had the worst survival. Collectively, our findings provided the underlying mechanisms of miR-206/14-3-3ζ in tumor growth and angiogenesis, and implicated miR-206 and 14-3-3ζ as potential therapeutic targets for NSCLC.


Growth kinetics
For the determination of growth kinetics, 1×10 5 cells were seeded in six-well plates, followed by conventional cultured for 24, 48, or 72 h, respectively. At each time, the cells were collected and counted in triplicate using a hemocytometer under a microscope. The doubling time was calculated using the formula: Td = Δt × log2/ (log Nt -log N0) as we described previously [1].

Quantitative real-time polymerase chain reaction (qRT-PCR)
Primers used were listed in Supplementary Table  S2. Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's recommendations. Then total RNA (2 μg) was transcribed into cDNA using AMV Reverse Transcriptase (Promega, Madison, USA). qRT-PCR was performed using the Applied Biosystems 7300HT machine (Applied Biosystems, CA, USA) and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas, MA, USA). The PCR reaction was evaluated using melting curve analysis. β-actin was amplified to ensure cDNA integrity and to normalize expression. Fold changes in expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2 -(ΔΔCt) .

Enzyme-linked immunosorbent assay (ELISA)
A total of 1×10 5 cells were seeded in 6-well plates. After treatments, the mediums were collected, cleared by centrifugation. ELISA was performed using the human VEGF, angiopoietin-2, or CXC chemokine ligand 8/IL-8 Quantikine kit (R&D Systems, MN, USA) according to the manufacturer's protocol. Recombinant human VEGF, angiopoietin-2, or IL-8 was used for calibration. The absorbance at 450 nm was measured with a multimode microplate reader (Tecan, Männedorf, Switzerland).

Western blots
Total protein was extracted by lysing cells in RIPA buffer (Beyotime Co. Ltd). Then the protein concentrations were measured with the BCA kit (Beyotime Co. Ltd). Afterwards, proteins (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transferring to polyvinylidene fluoride membranes (Millipore, Billerica, USA). After blocking with 10% non-fat milk in TBST for 1 h at the room temperature, the membranes were incubated with the primary antibody (Supplementary Table S3) at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1: 2000, Beyotime Co. Ltd) for 1 h at the room temperature. The immune complexes were detected by the enhanced chemiluminescence (Cell Signaling Technology, Beverly, MA, USA). Densitometric analysis was conducted via an Image-Pro-Plus 6.0 software (Media Cybernetics, Georgia, USA).

HUVECs recruitment assay
As we described previously [1], HUVECs (5×10 4 ) were seeded in the upper compartments of the chambers, [24-well Boyden chambers with 8-μm pore size polycarbonate membranes (Corning, NY, USA)], and NSCLC cells were placed in the lower compartments. The co-cultured tumor cells were pre-transfected by vector, 14-3-3ζ-Flag, Con-siRNA, 14-3-3ζ-siRNA, NC-mimic, miR-206-mimic, anti-Con, or anti-miR-206, respectively for 12 h and refreshed with DMEM before the recruitment experiments. The chambers were incubated at 37 °C for 24 h. Cells migrating through the filter to the lower surface were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Migrated cells were viewed and photographed under a phase-contrast microscope (Olympus, Tokyo, Japan), and counted by Image-Pro-Plus 6.0 software in five randomly chosen fields.

Co-immunoprecipitation (Co-IP)
Cells were extracted for 30 min with immunoprecipitation lysis buffer (Beyotime Co. Ltd). After centrifugation of the preparations, the supernatants were (total proteins) concentrations were measured with the BCA kit (Beyotime Co. Ltd). Then 100 μg proteins were incubated with 14-3-3ζ-Flag antibody at 4°C overnight. Then the protein-antibody complex were incubated with IgA plus IgG sepharose beads (Beyotime Co. Ltd) at 4°C for another 12 h. After then, the supernatants were removed and the beads were washed for three times, resuspended in the SDS sample buffer (Beyotime Co. Ltd), and boiled to remove protein from the beads. Then, such protein samples were analyzed by Western blots with STAT3 (Ser-727) or STAT3 (Tyr-705) antibody. Densitometric analysis was conducted via an Image-Pro-Plus 6.0 software (Media Cybernetics).

Luciferase reporter assay
The pGL3-14-3-3ζ-3'-UTR (wild type, WT; or mutant, MT, Figure S5)-Luc constructs were synthesized by Shuntian Bio Co. (Shanghai, China). The plasmid phRL-tk containing the Renilla luciferase gene was purchased from Promega. Briefly, cells were plated in 24-well culture dishes. When cells proliferated to 60 to 80% confluence, Con-mimics or miR-206-mimics was cotransfected with the respective reporter construct, by using Lipofecamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. The cells were lysed with passive lysis buffer (Promega), and the lysates were analyzed immediately with a 96-well plate luminometer (Berthold Detection System, Pforzheim, Germany). The amounts of luciferase and Renilla luciferase were measured with the Dual-Luciferase Reporter Assay System Kit (Promega) following the manufacturer's instructions.

Chromatin-Immunoprecipitation (ChIP) assay
NSCLC cells were transfected by Con-siRNA or 14-3-3ζ-siRNA for 12 h. Then, to crosslink proteins to DNA, such cells were treated with 1% formaldehyde in PBS by gentle agitation at room temperature for 10 min. Next, cells were washed, resuspended in lysis buffer, and sonicated on ice for 15 sec × 10 times at a 25% output in an EpiShear probe sonicator (Active Motif, Carlsbad, CA, USA). For the immunoprecipitation of protein-DNA complexes, 5 μl of specific magnetic Dynal bead (Invitrogen)-coupled antibody against HIF-1α or an isotype IgG (an negative control) was added at 4°C overnight, followed by a centrifugation (3000 round/ min × 1 min). Then, the immunoprecipitated DNA and input DNA (total DNA) were cleaned by RNase A (0.2 mg/ml) and proteinase K (2 mg/ml, Beyotime Co. Ltd) before phenol/chloroform-purification. The specific sequences from immunoprecipitated and input DNA were determined by PCR analysis. Amplifications of the VEGF gene sequences among the pull of DNA were performed with specific primers flanking from -1041 to -750 in the VEGF promoter upstream regions (Supplementary Table  S1). The PCR conditions for were 94 °C for 1 min; 35 cycles of 94 °C for 45 sec, 60 °C for 45 sec, and 72 °C for 45 sec; followed by 72 °C for 10 min, and cooling at 4 °C. The PCR products were then analysed by an agarose gel electrophoresis.

Immunohistochemistry (IHC)
Sections mounted on silanized slides were dewaxed in xylene; dehydrated in ethanol; boiled in 0.01 M citrate buffer, pH 6.0, for 20 min in a microwave oven; and then incubated with 3% hydrogen peroxide for 5 min. After washing with PBS, sections were incubated in 10% normal bovine serum albumin for 5 min, followed by incubation with a rabbit-anti Ki-67 (1: 50), or rabbit-anti CD31 (1: 50) antibody at 4°C overnight. Then the slides were incubated with an anti-rabbit horseradish peroxidaseconjugated secondary antibody (1: 300, Beyotime Co. Ltd) at room temperature for another 30 min. The staining was visualized using diaminobenzadine, and the sections were counterstained with hematoxylin, dehydrated, cleared, mounted, and photographed under a pannaramic-scan digital slice scanning system (3DHISTECH Co. Ltd, Budapest, Hungary). The graphs were analyzed via Image-Pro-Plus 6.0 software by two independent researchers who were blinded regarding the experiments' details. The protocol of IHC Quick-score and the inclusion criteria of intratumoral microvessels were according to our previous study [2].

Tunnel staining
Following the manufacturer's instruction (Roche Diagnostics Ltd, Shanghai, China), sections were pretreatment for dewaxing and dehydration as previously descripted in IHC, and then incubated with proteinase K (10 μg/ml, Beyotime Co. Ltd) for 20 min at 37 °C. After washing with PBS, sections were incubated with TUNEL reaction mixture (negative control without terminal transferase) at 37 °C in for 60 min in dark. Then samples were incubated with Converter-POD at 37 °C for 30 min and visualized using diaminobenzadine under microscope after wash in PBS. Finally, the sections were counterstained with hematoxylin, dehydrated, cleared, mounted, and analyzed as mentioned in IHC.

Statistical analysis
Data sets were compared using Graphpad 6.0 (GraphPad Software, Inc, CA, USA) and/or IBM SPSS software v20.0 (IBM Corp, Armonk, USA). Data were presented as the means ± SD. The difference between two groups was analyzed using a two-tailed student's t test. For the repeated measures data, a one-or two-way ANOVA followed by a Sidak's multiple comparisons test was used. The p values <0.05 were considered statistically significant. The expressions of miR-206 and 14-3-3ζ mRNAs in 116 NSCLC tissue samples were determined in triplicate by qRT-PCR. The boundary (cut-off) was defined as the median values as described previously [2].