Patients and sample collection
A total of forty patients with lung squamous cell carcinoma were included in the Third Hospital of Hebei Medical University during February 2019 through September 2019, none of the patients had received radiotherapy or chemotherapy before surgery. Samples were immediately frozen in liquid nitrogen and then stored at -80 °C until further use. The study protocol had been approved by the ethics committee of the Third Hospital of Hebei Medical University. All patients have signed the informed written consents.
RNA extraction and purification
Total RNA was extracted and purified using RNeasy mini kit (Cat. No.74106, QIAGEN, GmBH, Germany) following the manufacturer’s instructions, and checked for an RNA integrity number (RIN) to inspect the RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). RNA samples having a RIN value of 7.0 or 28s/18s≥0.7 indicated that qualified.
RNA amplification and labeling
Total RNA was amplified and labeled by using Low Input Quick Amp Labeling Kit, One-Color (Cat. No. 5190-2305, Agilent technologies, Santa Clara, CA, US) following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat. No. 74106, QIAGEN, GmBH, Germany).
Microarray hybridization analysis
Each slide of labeled cDNA was hybridized with 1.65 μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat. No. 5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat. No. G2545A, Agilent technologies, Santa Clara, CA, US). After 17 hours hybridization, slides were washed in staining dishes (Cat. No. 121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat. No. 5188-5327, Agilent technologies, Santa Clara, CA, US).
Signal collection and data acquisition
Slides were scanned by Agilent Microarray Scanner (Cat. No. G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, PMT 100%, 20bit. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, limma packages in R. All procedures were carried out according to the standard protocols.
Data analysis
Raw data were subjected to quality control, pre-processing and GC-RMA normalization Hierarchical clustering analysis was used to categorize the data into two groups of different expression patterns. Statistical analysis of genes was performed by paired t-test and the calculation of log2-fold change (log FC). DEGs were identified with the cut-off criteria of false discovery rate (FDR)-corrected P value < 0.05 and |log2 fold-change (FC)| > 2 (Benjamini and Hochberg method). Functional groups and biological pathway enrichment analyses for the common DEGs were performed using the Database for Gene Ontology (GO) functional and Kyoto Encyclopedia of Genesand Genomes (KEGG)(14).
IPA functional analysis
DEGs were functionally analyzed based on the Ingenuity Pathway Analysis (IPA) software, which is based on computational algorithms that analyze the functional connectivity of the genes from information obtained within the IPA database. Canonical pathway analysis was performed using IPA. The created genetic networks describe functional relationships among genes or proteins based on known associations from the database. Networks were ranked according to their biological relevance to the gene list provided. The p value calculated by Fisher's exact test was used to determine the probability of the association not due to chance alone.
Quantitative RT-PCR analyses
Verify gene expression changes measured by microarray analysis. Total RNA from pulmonary tumor tissue samples with and without bone metastases were isolated by using TRIZOL reagent (Invitrogen, USA). The quantity and purity of RNA was assessed by absorbance at 260 nm and 280 nm. 1μg of total RNA was used as template for cDNA synthesis using the Reverse Transcription System kit (TaKaRa Bio, Shiga, Japan). Real-time PCR reactions were performed with DNA Master SYBR Green. Average Ct values from triplicate analyses were normalized from average Ct values of GAPDH. All primer sequences were displayed in Table 1.
Table 1.
Specific primers for Quantitative RT-PCR analyses
ID
|
Sequence(5’- 3’)
|
Product Length(bp)
|
For internal reference
|
GAPDH.F
|
TGTTCGTCATGGGTGTGAAC
|
154
|
GAPDH.R
|
ATGGCATGGACTGTGGTCAT
|
For Osteogenic ability detection
|
ALP.F
|
ACTGGGGCCTGAGATACCC
|
185
|
ALP.R
|
TCGTGTTGCACTGGTTAAAGC
|
RUNX2.F
|
TGGTTACTGTCATGGCGGGTA
|
101
|
RUNX2.R
|
TCTCAGATCGTTGAACCTTGCTA
|
For microarray DEGs verification
|
COA3 F
|
CTGGATTCTAAGCGTGGAGAG
|
236
|
COA3 R
|
AGCTCATCTAGGAAACGCTC
|
APPL1 F
|
GACAGCCCGCAGACAAG
|
136
|
APPL1 R
|
GGTGTGTTGCTGCACTTAAT
|
COL6A1 F
|
ACACCGACTGCGCTATCAAG
|
90
|
COL6A1 R
|
CGGTCACCACAATCAGGTACTT
|
TERF1 F
|
ACAGCGCAGAGGCTATTATTC
|
121
|
TERF1 R
|
TCAAACTGTGCATCAAGGGT
|
TST F
|
TATCAGTGCTCAATGGTGGC
|
104
|
TST R
|
GTCCAGTGTGGCTTTGAAGA
|
CD133 F
|
AAACCTGCAACAGCATCAGA
|
150
|
CD133 R
|
GGGATTGATAGCCCTGTTGG
|
RALY F
|
CCCAAGTCCATCAACTCTCG
|
268
|
RALY R
|
CTGCTCTCTTTAGCCCCTTG
|
Prominin2 F
|
CTCCGTGAAGGTGAATGAGG
|
159
|
Prominin2 R
|
TTGTGCTCTGTCTTCACTCG
|
Western blotting
Total protein was extracted from tissue samples by using RIPA buffer. Protein concentration was determined by Bradford protein assay. Total protein was separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) prior to transferring onto a PDVF membrane (Millipore, Bedford, MA, USA). Blocking was performed for 1 h with 4% milk at room temperature for 1.5 h and membranes were subsequently incubated at 4˚C overnight with anti-COL6A1 (1:500 dilution; Abcam, cat. no. ab151422), anti-ALP (1:1000 dilution; Abcam, cat. no. ab83259) and anti-RUNX2 (1:1000 dilution; Abcam, cat. no. ab23981). Goat anti-Mouse IgG H&L (HRP) (1:2000 dilution; Abcam, cat. no. ab6721) was then applied at room temperature for 2h before detection using enhanced chemiluminescence (ECL) substrate (Beyotime Biotechnology, China). GAPDH protein was used as loading control for normalization. The quantitative results were calculated by the ImageJ software (version 1.50b; National Institutes of Health, Bethesda, MD).
Immunohistochemistry (IHC) assay
Surgically pulmonary tumor tissue samples (with and without bone metastasis) were fixed in 4% paraformaldehyde and embedded in paraffin. Sectioned tissue samples were dehydrated with xylene and ethanol, and incubated in sodium sulphate buffer (pH = 6.0) at 100 °C. After washing samples with PBS, anti-COL6A1 (1:100 dilution; Abcam, cat. no. ab151422) was added and incubated at 4 °C overnight. Goat anti-Mouse IgG H&L (HRP) (11000 dilution; Abcam, cat. no. ab6721) was added and incubated for 30 min at 37 °C. After the DAB staining, the slides were then counterstained with hematoxylin for observation under regular microscopy.
Cell culture and osteogenic induction
HOB, hES-MP 002.5 and M-7 cells were obtained from the ATCC, and maintained in DMEM/HIGH medium (Hyclone) with 10% fetal bovine serum (FBS, Hyclone). All cells were incubated at 37˚C and 5% CO2. HOB, hES-MP 002.5 and M-7 cells were induced using osteoblast inducing conditional media (PUHE Biomedical, Cat. no. CTCC Y001) for 14 days.
Alizarin Red Staining
1×103 cells/well HOB, hES-MP 002.5 and M-7 cells were cultured in 96-well paltes, perspectively. After osteogenic induction for 14 days, Alizarin Red Staining was conducted using the kit (PUHE Biomedical, Cat. No. CTCC JD001) according to the manufacturer’s instructions.
ALP staining
1×103 cells/well HOB, hES-MP 002.5 and M-7 cells were cultured in 96-well paltes, perspectively. After osteogenic induction for 14 days, ALP staining was conducted using the kit (PUHE Biomedical, Cat. No. CTCC JD002) according to the manufacturer’s instructions.
Cell adhesion assay
As described in previous study(15), HOB, hES-MP 002.5 and M-7 cells were grown into 6-well paltes. 1×103 HARA-B cells, which were stable expression of COL6A1 (GFP from the COL6A1 overexpressed plasmid or shRNAs)were co-cultured with HOB, hES-MP 002.5 and M-7 cells for 30 min at 37°C, perspectively. And the treated HARA-B cells (1x103 cells/well) were overlaid directly in a dish and incubated for 30 min at 37°C and 5% CO2. The cells were washed in PBS and fixed with 4% paraformaldehyde. The adhered GFP-expressing cells were observed and counted under the fluorescence microscope.
Statistical analysis
Statistics were performed using GraphPad Prism (GraphPad Software Inc, San Diego, CA, USA) and significance of differences was analyzed by Student’s t-test. All results were derived from at least 3 independent experiments. Statistical significance was defined as p < 0.05.