Supplementary Materials and Methods

Study subjects

Lung tumor specimens were collected from 124 patients with primary NSCLC at the Department of

Thoracic Surgery, Taichung Veterans General Hospital (Taichung, Taiwan) between 1998 and 2004.

Patients were asked to submit written informed consent; the study was approved by the Institutional Review

Board. The tumor type and stage of each collected specimen were histologically determined in accordance

with the World Health Organization classification system. Cancer relapse data were obtained via chart

review and confirmed by thoracic surgeons. Clinical parameters and OS and RFS data were collected from

chart review and the Taiwan Cancer Registry, Department of Health, Executive Yuan, ROC.

Cell lines

TL-1 and TL-4 cells were kindly provided by Dr. Y. W. Cheng (Graduate Institute of Cancer Biology and

Drug Discovery, Taipei Medical University, Taipei, Taiwan) 1. A549, H1355, H1299, H1650, and H1975

cells were obtained from the Bioresource Collection and Research Center, the Food Industry Research and

Development Institute (Hsinchu, Taiwan). TL-1, TL-4, H1355, H1650, and H1975 cancer cell lines were

maintained in RPMI-1640 (HyClone, Logan, UT). A549 and H1299 cancer cell lines were maintained in

DMEM (HyClone, Logan, UT). The medium contained 10% fetal bovine serum (FBS) supplemented with

penicillin (100 U/mL) and streptomycin (100 mg/mL). These cells were cultured in accordance with the

suppliers’ instructions. Once resuscitated, cell lines were routinely authenticated (once every 6 months; the

cells were last tested in December 2012) by means of cell morphology monitoring, growth curve analysis,

species verification via isoenzymology and karyotyping, identity verification via short tandem repeat

profiling analysis, and contamination checks.

Plasmid construction, transfection, and stable clone selection

The National RNAi Core Facility, Academia Sinica provided CDC25A, PDCD4, c-Myc, and IκBα shRNA.

The target sequences for shRNA are presented in Supplementary Table S1. The expression vector for

PDCD4 was purchased from Addgene (Cambridge, MA). Nonspecific shRNA control of the scramble

sequence was used as the control in the knockdown experiment, and an empty vector expression was used as

the control for the overexpression plasmid. The transfection and stable clone selection procedures have been

described previously 2.

The precursor and inhibitor of MiR-21 and miR-184 transfection

MicroRNA precursor (pre-miR-184 and pre-miR-21, 20-40 nmol/L/well; Ambion, Foster City, CA),

MicroRNA inhibitor (40-80 nmol/L/well; Ambion, Foster City, CA), and negative control (Ambion, Foster

City, CA) were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, Foster City, CA)

according to the manufacturers’ protocols. Transfection efficiency was evaluated by a real-time polymerase

chain reaction (PCR).

Real-time PCR analysis of miRs and mRNA expression levels

DNase I–treated total RNA (10 ng) was subjected to miRNA RT-PCR analysis with the TaqMan miRNA

Reverse Transcription Kit (Applied Biosystems, Foster City, CA), miRNA Assays (Applied Biosystems,

Foster City, CA), and a Real-Time Thermocycler 7500 (Applied Biosystems, Foster City, CA). RNU6B was

used as the microRNA reference housekeeping gene, and GAPDH was used as the mRNA reference

housekeeping gene. The primers used for real-time PCR analysis of mRNA expression are presented in

Supplementary Table S1. The mRNA and microRNA levels in tumours that were higher than the median

value were defined as “high”, whereas levels lower than the median value were defined as “low”.

Luciferase reporter assay

Double-stranded oligonucleotides corresponding to the wild-type or mutant miR-184 binding site in the

coding region of CDC25A were synthesized and ligated between the SpeI and HindIII restriction sites of

pmiR-REPORT miR Expression Reporter Vector (Ambion, Foster City, CA). The oligonucleotides utilized

are listed in Supplementary Table S1. Cells were transfected using an appropriate plasmid and Pre-miR-184.

Luciferase assays were done with the luciferase reporter assay system (Promega, Fitchburg, WI) 48 hr after

transfection. Normalized luciferase activity was reported as luciferase activity/β-galactosidase activity.

Boyden chamber invasion assay

A Boyden chamber with a pore size of 8 μm was used for the in vitro cell invasion assay. Cells (5 × 10 4) in

0.5% serum containing the culture medium were plated in the upper chamber, and 10% FBS was added to

the culture medium in the lower chamber as a chemoattractant. The upper side of the filter was covered with

0.2% Matrigel (Collaborative Research, San Francisco, CA) diluted in RPMI-1640. After 24 h, cells on the

upper side of the filter were removed, and cells that adhered to the underside of the membrane were fixed in

95% ethanol and stained with 10% Giemsa dye. The number of invasive cells was counted. Ten contiguous

fields of each sample were examined to obtain a representative number of cells that invaded across the

membrane. The procedures and methods followed those described previously 3.

Statistical analysis

All statistical analyses were conducted using the SPSS statistical software program as described previously

(version 17.0; SPSS, Inc., Chicago, IL) 2,4. A two-sided analysis of the variance in the statistical tests was

conducted, and P values < 0.050 were considered statistically significant.

References1. Cheng YW, Wu MF, Wang J, et al. Human papillomavirus 16/18 E6 oncoprotein is expressed in lung cancer and related with p53 inactivation. Cancer research. Nov 15 2007;67(22):10686-10693.2. Sung WW, Wang YC, Cheng YW, et al. A polymorphic -844T/C in FasL promoter predicts survival and relapse in non-small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. Sep 2011;17(18):5991-5999.3. Wu DW, Cheng YW, Wang J, Chen CY, Lee H. Paxillin predicts survival and relapse in non-small cell lung cancer by microRNA-218 targeting. Cancer research. Dec 2010;70(24):10392-10401.4. Wu DW, Liu WS, Wang J, Chen CY, Cheng YW, Lee H. Reduced p21(WAF1/CIP1) via alteration of p53-DDX3 pathway is associated with poor relapse-free survival in early-stage human papillomavirus-associated lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. Apr 2011;17(7):1895-1905.

Supplementary TableSupplementary Table S1. List of primer sequences used in the present study.Target gene SequenceReal-time PCRGAPDH Forward 5’-GGAGCCAAAAGGGTCATCATC-3’GAPDH Reverse 5’-GATGGCATGGACTGTGGTCAT-3’CDC25A Forward 5’-GTGAAGGCGCTATTTGGCG-3’CDC25A Reverse 5’-GGTCCATAGTGACGGTCAGGT-3’PDCD4 Forward 5’-ATGGATATAGAAAATGAGCAGAC-3’PDCD4 Reverse 5’-CCAGATCTGGACCGCCTATC-3’c-Myc Forward 5’-AGCGACTCTGAGGAGGAACAAG-3’c-Myc Reverse 5’-CCTGCCTCTTTTCCACAGAAA-3’pmiR-REPORT miR Expression ReporterWild-type Forward 5’-CTAGTGTCCCTGTTAGACGTCCTCCGTCCATATCAGAACTGTGCCACAATGCAGTTCTGA-3’Wild-type Reverse 5’-AGCTTCAGAACTGCATTGTGGCACAGTTCTGATATGGACGGAGGACGTCTAACAGGGACA-3’Mutant Forward 5’-CTAGTGTATCCGACTGACCCAGCATCAACATATCAGAACTGTGCCACAATGCAGTTCTGA-3’Mutant Reverse 5’-AGCTTCAGAACCGCATTGTGGCACAGTTCTGATATGTTGATGCTGGGTCAGTCGGATACA-3’RNAi targetshCDC25A 5’-GACTTCCATGCCTTAAACCTA-3’shPDCD4 5’-GCGGTTTGTAGAAGAATGTTT-3’shc-Myc 5’-CCTGAGACAGATCAGCAACAA-3’shIκBα 5’-ATCACCAACCAGCCAGAAATT-3’

Supplementary Table S2. Relationships between miR-21, PDCD4, miR-184, CDC25A, c-Myc and clinical-pathological parameters in NSCLC patients.

miR-21 PDCD4 mRNA miR-184 CDC25A mRNA c-Myc mRNA

Parameters Case no. Low (%) High (%) P Low (%) High (%) P Low (%) High (%) P Low (%) High (%) P Low (%) High (%) P

124 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0) 62 (50.0)

Age

≦65 59 33 (55.9) 26 (44.1) 0.208 30 (50.8) 29 (49.2) 0.857 30 (50.8) 29 (49.2) 0.857 28 (47.5) 31 (52.5) 0.590 27 (45.8) 32 (54.2) 0.369

＞65 65 29 (44.6) 36 (55.3) 32 (49.2) 33 (50.8) 32 (49.2) 33 (50.8) 34 (52.3) 31 (47.7) 35 (53.8) 30 (46.2)

Gender

Female 41 21 (51.2) 20 (48.8) 0.849 25 (61.0) 16 (39.0) 0.086 25 (61.0) 16 (39.0) 0.086 20 (48.8) 21 (51.2) 0.849 22 (53.7) 19 (46.3) 0.567

Male 83 41 (49.4) 42 (50.6) 37 (44.6) 46 (55.4) 37 (44.6) 46 (55.4) 42 (50.6) 41 (49.4) 40 (48.2) 43 (51.8)

Tumor type

AD 102 52(51.0) 50 (49.0) 0.638 56 (54.9) 46 (45.1) 0.019 54 (52.9) 48 (47.1) 0.158 54 (52.9) 48 (47.1) 0.158 52 (51.0) 50 (49.0) 0.638

SQ 22 10(45.5) 12 (54.5) 6 (27.3) 16 (75.7) 8 (36.4) 14 (63.6) 8 (36.4) 14 (63.6) 10 (45.5) 12 (54.5)

Smoking status

Nonsmoking 72 34 (47.2) 38 (52.8) 0.467 35 (48.6) 37 (51.4) 0.716 37 (51.4) 35 (48.6) 0.716 39 (54.2) 33 (45.8) 0.275 39 (54.2) 33 (45.8) 0.275

Smoking 52 28 (53.8) 24 (46.2) 27 (51.9) 25 (48.1) 25 (48.1) 27 (51.9) 23 (44.2) 29 (55.8) 23 (44.2) 29 (55.8)

T

T1+2 98 52(53.1) 46 (46.9) 0.186 53 (54.1) 45 (45.9) 0.078 51 (52.0) 47 (48.0) 0.853 48 (49.0) 50 (51.0) 0.545 52 (53.1) 46 (46.9) 0.043

T3+4 26 10(38.5) 16 (61.5) 9 (34.9) 17 (65.4) 13 (50.0) 13 (50.0) 11 (42.3) 15 (57.7) 8 (30.8) 18 (69.2)

N

N0 63 37(58.7) 18 (41.3) 0.048 35 (55.6) 28 (44.4) 0.209 28 (44.4) 35 (55.6) 0.105

30

(47.6) 33 (52.4) 0.993 35 (55.6) 28 (44.4) 0.105

N1+2 61 25(41.0) 27 (59.0) 27 (44.3) 34 (55.7) 36 (59.0) 25 (41.0) 29 (47.5) 32 (52.5) 25 (41.0) 36 (59.0)

M

M0 121 59(48.8) 62 (51.2) 0.080 62 (51.2) 59 (48.8) 0.080 63 (52.1) 58 (47.9) 0.521 56 (46.3) 65 (53.7) 0.066 58 (47.9) 63 (52.1) 0.521

M1 3 3(100.0) (0.0) 0 (0.0) 3 (100.0) 1 (33.3) 2 (66.7) 3 (100.0) 0 (0.0) 2 (66.7) 1 (33.3)

Stage

I 51 30 (58.8) 21 (41.2) 0.100 29 (56.9) 22 (43.1) 0.201 21 (41.2) 30 (58.8) 0.100 29 (56.9) 22 (43.1) 0.206 29 (56.9) 22 (43.1) 0.201

II +III 73 32 (43.8) 41 (56.2) 33 (45.2) 40 (54.8) 41 (56.2) 32 (43.8) 33 (45.2) 40 (54.8) 33 (45.2) 40 (54.8)

miR-21

Low 25 (40.3) 37 (59.7) 0.031 25 (40.3) 37 (59.7) 0.031 40 (64.5) 22 (35.5) 0.001 24 (38.7) 38 (61.3) 0.017

High 37 (59.7) 25 (40.3) 37 (59.7) 25 (40.3) 22 (35.5) 40 (64.5) 38 (61.3) 24 (38.7)

PDCD4 mRNA

Low 40 (64.5) 22 (35.5) 0.001 30 (48.4) 32 (51.6) 0.719 30 (48.4) 32 (51.6) 0.719

High 22 (35.5) 40 (64.5) 32 (51.6) 30 (48.4) 32 (51.6) 30 (48.4)

miR-184

Low 24 (38.7) 38 (61.3) 0.017 40 (64.5) 22 (35.5) 0.001

High 38 (61.3) 24 (38.7) 22 (35.5) 40 (64.5)

Supplementary Figure

Supplementary Figure S1. miR-184 was overexpressed via treatment with a miR-184 precursor in TL-1 cells. In TL-4 cells, miR-184 was reduced by a miR-184 inhibitor. The miR-184 level was determined by real-time PCR. Cell proliferation was evaluated for TL-1 cells subjected to or not subjected to miR-184 precursor treatment, and for TL-4 cells subjected to or not subjected to miR-184 inhibitor treatment for 24h. The invasion ability was evaluated for TL-1 cells subjected to or not subjected to miR-184 precursor treatment, and TL-4 cells subjected to or not subjected to miR-184 inhibitor treatment, and were compared with those of NC controls.

Supplementary Figure S2. TL-1 cells were transfected with miR-184 precursor (40 μmol/L/well; Ambion), 500 ng pMIR-Reporter luciferase vector, including CDS of CDC25A (with WT or Mut miR-184 response element), and β-galactosidase plasmid. TL-4 cells were transfected with miR-184 inhibitor (40 μmol/L/well), 500 ng pMIR-Reporter Luciferase Vector, and β-galactosidase plasmid. In all of the experiments, the relative level in the NC and vector controls was arbitrarily assigned as 1.

Supplementary Figure S3. The alteration of the invasion capability by shIκBα and/or the shRNA combined with BAY or JQ1 was dependent on the expression levels of CDC25A and c-Myc. TL-4 and H1355 cells were treated with miR-21 precursor, NF-κB inhibitor (BAY, 0.5-1μM), and c-Myc inhibitor (JQ1, 1-2μM) for 48h, respectively. Upper and middle: Expression levels of miR-21, miR-184, CDC25A, and c-Myc were evaluated by real-time PCR. Lower: The invasion ability was evaluated by matrix gel boyden chamber assay; the relative level in the NC was arbitrarily assigned as 1.

Supplementary Figure S4. Increase in CDC25A and c-Myc expression by means of a reduction in miR-184 promotes cell invasiveness. H1355 and TL-4 cells were treated with miR-184 inhibitor, shCDC25A, and shc-Myc plasmid, respectively. The invasion ability was evaluated by matrix gel boyden chamber assay; the relative level in the NC was arbitrarily assigned as 1.

Supplementary Figure S5. Kaplan-Meier survival curves of miR-21, PDCD4, miR-184, CDC25A, and c-Myc mRNA expression for overall

survival (OS) and relapse-free survival (RFS) in NSCLC patients.

Supplementary Figure S6. The possible mechanistic route of miR-184 in cell invasiveness.