Patients and specimens

Fifty cases of paired NSCLC samples were collected at Nanfang hospital and Cancer Center of Southern medical university (SMUCC). 202 FFPE sections from patients with pathologically confirmed NSCLC who never received pretreatment before radical surgery between 2013 and 2015, were also enrolled in this study. Follow-up information was obtained by telephone or from the outpatient records. The study was approved by Research Ethics Committee of Nanfang Hospital and SMUCC. All samples were used according to the ethical guidelines of the 1975 Declaration of Helsinki and obtained with the patients’ understanding that it might be published.

Cell culture

All cell lines used in this study were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Human lung epithelial cell EBAS-2B was maintained with RPMI 1640 with 10% FBS. Lung cancer cell lines were cultured with DMEM with 10% FBS. RPMI 1640, DMEM and FBS were purchased from Invitrogen.

miRNA transfection, siRNA interference and infection

Cells at confluency of 40–50% were transfected with 5 μg of miRNAs using Lipofectamine 2000 reagent (Invitrogen; Carlsbad, Calif, USA). The miR-661 mimic, a nonspecific miR control, anti-miR-661, a nonspecific anti-miR control, lenti-virus with miR-661 and a correspondent control were purchased from GenePharma (Shanghai, China). SiRNA against RB1 and control siRNA were used as Table 1.

Table 1 Sequences of human RB1 shRNAs Full size table

Real-time PCR

Total RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA)according to the manufacturer’s instructions. 1 μg of total RNA was used to reverse transcribe cDNA with SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-time PCR was performed using ABI 7500HT system. PCR reactions were carried out in a final volume of 10 uL of mixture consisting of SYBR green 2X master mixture (Invitrogen, Carlsbad, CA, USA). The primer sequences were as Table 2, U6 was used as an internal control.

Table 2 Oligo Sequences for Real-time PCR Full size table

Wound healing and transwell assay

Cells at 80–90% confluency were maintained in serum-free medium for 6 days and then plated in six-well plates containing serum-free medium 24 h before experiments. 10 μl pipette tips were used to scratch constant-diameter stripes in the confluent monolayers. The wounds were photographed at 0, 24, 48 h under an inverted phase contrast microscope. Three random fields were marked and measured. Migration index was expressed by ratio of migrating distance of treated cells to those of control cells.

For transwell assay, cells were treated with mitomycin before seeding in the upper chambers of 24-well transwell plates (Corning Incorporated, New York, NY, USA) pre-coated with 50% matrigel (BD Biosciences, Franklin, New Jersey, USA) in phosphate-buffered saline. The lower chambers were filled with culture medium supplemented with 10% FBS. After incubation for 24 h, invaded cells on the bottom were fixed and stained with 0.5% crystal violet and visualized under a bright field microscopy (OLYMPUScx31, TOKYO, Japan).

Dual reporter luciferase assay

MiRwalk database (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) were used to screen for direct targets of miR-661. The 1.8 kb full-length of RB1 3′ untranslated region (3′UTR) was cloned into psiCHECKTM-2 dual luciferase reporter vector (Promega; Madison, Wis, USA) and named wild-type (wt) 3′UTR. Site-directed mutagenesis of the miR-661 binding site in the RB1 3′UTR was carried out using the GeneTailor Site-Directed Mutagenesis System (Invitrogen) and named mutant (mt) 3′UTR. Cells were seeded in 96-well culture plates (Costar) and transfected with wt or mt 3′UTR construct along with miR-661 mimic or inhibitor in triplicate. Firefly and renilla luciferase activities were measured at 48 h after cotransfection with the Dual-Luciferase Reporter Assay kit (Promega, Madison, Wis, USA).

Western blot

Protein expression was assessed by immunoblot analysis of cell lysates (20–60 μg) in RIPA buffer in the presence of rabbit antibodies to E-cadherin, mouse antibodies to β-catenin, fibronectin, vimentin, β-actin (1:500; Santa Cruz, California, USA); and rabbit antibodies to RB1, GAPDH, E2F1, (1:1000; CST, Danvers, MA). Quantifications were carried out using Scion Image software and normalized to β-actin levels.

Co-immunoprecipitation

Cells were lysed in RIPA buffer with PMSF and proteinase inhibitor for 60 min. Supernatants were collected and incubated with anti-RB1 or anti-E2F1 magnetic beads (biotool. China) for overnight at 4 °C, followed by washing with binding buffer and denaturation in elution buffer. Resuspending and elution. Aliquots of the products were subjected to Western blot analysis as described above.

Animal models

For lung metastasis model, 5*106 cells of each group were injected into the caudal vein of 10 BalB/c-nu mice (Laboratory Animal Center of Southern Medical University, Guangzhou, China). Animals were sacrificed 60 days after injection. Lung tissues were fixed in formalin and sectioned for H&E staining.

For subcutaneous model, 1*106 cells of each group were injected into the flank region of 4-week old BalB/c-nu mice. Five weeks later, animals were sacrificed and tumor were collected and sectioned for immunohistochemistry.

The experiments on mice had been approved by the ethics committee at SMUCC (Laboratory Animal Center of Southern Medical University, Guangzhou, China).

Immunohistochemistry

3–4 μm thick tissue sections were subsequently deparaffinized, rehydrated and rinsed before antigen retrieval and endogenous peroxidase blocking. Following non specific binding blocking with goat serum, the slides were incubated with anti-RB1 (1:50; Abcam, Cambridge, UK), E2F1 (1:50; Abcam, Cambridge, UK), vimentin (1:50; Santa Cruz, California, USA) and E-cadherin (1:50; Santa Cruz, California, USA) primary antibody at 4 °C. Standard DAB method (Beyotime, China) was employed to detect the staining. Counter staining was carried out with hematoxylin.

Immunofluorescence

Cells were cultured on coverslips overnight, fixed with 4% paraformaldehyde and treated with 0.25% Triton X-100. After blocking in 10% normal blocking serum, slides were incubated with rabbit antibodies to E-cadherin, mouse antibodies to β-catenin, vimentin (1:500; Santa Cruz, California, USA), and rabbit antibodies to RB1 and E2F1 (1:100; Abcam, Cambridge, UK) antibodies at 4 °C overnight followed by washing with PBS three times. Cover slips were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or mouse and Texas Red (TR)-conjugated anti-mouse or rabbit antibodies (1:120; SantaCruz) and stained with 6-diamidino-2-phenylindole (DAPI; Invitrogen).

Statistical analysis

Statistical analyses were performed using a statistical software package (SPSS19.0, Chicago, IL). The results from real-time PCR, immunoprecipitation, dual reporter luciferase assay, wound healing assay and transwell assay were determined by independent t-test. Chi-square tests were conducted to assess correlations between miR-661 expression and clinicalpathological factors. Overall survivals were evaluated using the Kaplan and Meier survival curves, and compared by the log-rank test. Numbers of metastatic lung nodules in mice were compared with independent t-test. Pearson′s correlation analysis was carried out to determine the relation between miR-661 and RB1 as well as E2F1 expression. P-value less than or equal to 5% were considered statistically significant.