2.1 Analysis of raw microarray data
We analyzed sample data downloaded from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. The GSE25099 dataset was divided into two groups: 10 normal oral tissues (GSM616588-GSM616597) and 10 OC patient tissues (GSM616647-GSM616656), which were analyzed to generate lncRNA/mRNA expression profiles. The GSE98463 dataset were divided into two groups: 4 normal oral tissues (GSM2596879-GSM2596882) and 4 OC patient tissues (GSM2596874-GSM2596877), which was analyzed to generate miRNA expression profiles. The criteria for differential expression were P < 0.05, lncRNAs/mRNAs/miRNAs were selected by the criteria of |fold change| ≥ 2, and hierarchical clustering heatmap or volcano plot was constructed. KEGG pathway enrichment analysis was performed using the functional annotation tool of DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/summary.jsp). Binding between lncRNAs and miRNAs was predicted using Starbase (https://starbase.sysu.edu.cn/). miRNA-mRNA binding was predicted using Targetscan 7.2 (https://www.targetscan.org/vert_72/) .
2.2 Cell lines and culture and transfection
Human oral cancer cell lines (CAL-27 and FaDu), and human embryonic kidney 293 cells (HEK 293) were purchased from the American Type Culture Collection (ATCC; VA, USA), human oral epithelial cells (HOECs) was purchased from Procell (Wuhan, China). All cells were maintained in complete medium containing the following components: Dulbecco's modified Eagle's medium (HyClone, UT, USA) with 10% fetal bovine serum (FBS; HyClone). RNA was transfected into cells using Lipofectamine 3000 and used in subsequent experiments 48 hours later. FGF11 overexpression (ov-FGF11), LINC00342 small interfering (si)RNA, and miR-149-5p mimic/inhibitor and their scrambled negative controls (ov-NC, si-NC, mimic NC, and inhibitor NC) were purchased from Sangon Biotech Co., Ltd (Shanghai, China).
2.3 Transient LINC00342 silencing
si-LINC00342-1, si-LINC00342-2, si-LINC00342-3, and scrambled siRNA (si-NC) were designed and synthesized by Sangon Biotech Co., Ltd. The siRNA sequences were as follows: si-LINC00342-1, 5′-TGGCTTGTTTACTCCAAATGATC-3′; si-LINC00342-2, 5′-CTCCACAAACCAGAAGACTTATT-3′; si-LINC00342-3, 5′-TACAGAAGATGCTAACTAGAATA-3′; si-NC, 5′-TTCTCCGAACGTGTCACG-3’.
2.4 Nucleocytoplasmic separation
Cytoplasmic and Nuclear RNA purification kit (Norgen Biotek; Ontario, Canada) was isolated from cells according to the manufacturer's instructions. Briefly, 400 µL cell isolation buffer was added to the cell pellet. After 10 minutes of incubation, the cells were centrifuged at 500 × g for 1 minute to separate the cytoplasm and nucleus. The nuclear and cytoplasmic samples were then thoroughly mixed with Lysis Buffer at 21℃. RNA from the cytoplasm and nucleus was added separately to the pre-warmed eluate and then collected for real-time quantitative polymerase chain reaction (RT-qPCR) analysis.
2.5 RNA pull-down assay
Biotinylated NC, LINC00342 and LINC00342 mut were transfected into FaDu and CAL-27 cells. Cell lysates were incubated (30 min; 25 ℃) with Dynabeads M-280 streptavidin (Invitrogen, MA, USA) and shaken slowly according to the manufacturer's instructions. For quantitative analysis, RT-qPCR was performed.
2.6 RNA isolation and RT-qPCR analysis
Total RNA was extracted from FaDu and CAL-27 cells using TRIzol (Invitrogen, MA, USA) according to the manufacturer's instructions. Real-time mRNA quantification for LINC00342, miR-149-5p, FGF11, U6, and 18sRNA was performed using SYBR Green qPCR SuperMix (Invitrogen) on a 7500 RT-qPCR system (Applied Biosystems, MA, USA). Primers for LINC00342, FGF11, ITGB3, PDGFC, miR-149-5p, 18sRNA (as an internal normalization control for LINC00342, FGF11, ITGB3, PDGFC), and U6 (as an internal normalization control for miRNAs) were as follows: LINC00342 forward, 5'-TCCACAGACACTACCCAAAGC-3'; and reverse, 5'-GCAGTTCACTCTGCTGCTTC-3'; FGF11 forward, 5'-TGTCTCTCTCTCCAGAGCCT-3'; and reverse,5'-CTGTGAAATGCGGCGAACTG-3'. ITGB3 forward, 5'-AGATTGGAGACACGGTGAGC-3'; and reverse, 5'-CGGCATACCCCACACTCAAA-3'. PDGFC forward, 5'-TGGCGGTGGTGAAAGAGACT-3'; and reverse, 5'-GCTGAGGATCTTGTACTCCGTTC-3'. miR-30a-5p forward, 5'-ACACTCCAGCTGGGTGTAAACATCCTCGAC-3'; and reverse, 5'-CTCAACTGGTGTCGTGGA-3'. miR-139-5p forward, 5'-ACACTCCAGCTGGGTCTACAGTGCACGTGTC-3'; and reverse, 5'-CTCAACTGGTGTCGTGGA-3'. miR-149-5p forward, 5'-ACACTCCAGCTGGGTCTGGCTCCGTGTCTTC-3'; and reverse, 5'-CTCAACTGGTGTCGTGGA-3'. 18sRNA forward, 5'-CCTGGATACCGCAGCTAGGA-3'; and reverse, 5'-GCGGCGCAATACGAATGCCCC-3'. U6 forward, 5'-CTCGCTTCGGCAGCACA-3'; and reverse, 5′-AACGCTTCACGAATTTGCGT-3'. Relative expression levels of LINC00342, FGF11, and miR-149-5p were calculated using the 2 − ΔΔCt method [31]. All RT-qPCR experiments were performed in triplicate.
2.7 Cell proliferation rate
Cell Counting Kit-8 (CCK8) reagent (Solarbio; 10 µL) was added to 96-well plates (containing FaDu and CAL-27 3 × 103 cells) at 0, 24, 48, and 72 hours. Absorbance was measured at 450 nm with an enzyme-labeled instrument (Thermo Fisher Scientific) after 60 minutes of incubation in the dark at 21°C.
2.8 Cell cycle assay
FaDu and CAL-27 cell suspensions were centrifuged at 1000 × g for 5 minutes, washed twice with PBS, and immobilized in 70% ethyl alcohol at 4°C overnight. The samples were washed with PBS and resuspended in 500 µL PBS containing 50 µg/mL PI and 100 µg/mL RNase A (with 0.2% Triton X-100). After 30 minutes of incubation (4°C), flow cytometry (FCM) was used to analyze cell apoptosis.
2.9 Transwell migration and invasion assays
Prior to determining the invasion ability of FaDu and CAL-27 cells, a 24-well transwell insert (BD Biosciences) was coated with Matrigel® (BD Biosciences) for 6 hours at 37°C. Serum-free medium was then added to the upper chamber and complete medium to the lower chamber. After incubation for 24 hours, invasive cells were fixed with anhydrous ethanol for 30 minutes and stained with 0.1% crystal violet (Solarbio; 25°C) for 25 minutes. Cells were then fixed, stained, and counted. The cells were then examined under a light microscope (Olympus Corporation) at ×200 magnification. Matrigel incubation of the insert is not required for the migration assay.
2.10 Dual-luciferase reporter assay
The amplified 3'-UTR fragments of LINC00342 and FGF11 containing miR-149-5p binding sites were cloned into the psiCHECK-2 dual-luciferase reporter vector (Promega Corp., WI, USA). We transfected HEK 293 cells with 0.5 µg reporter construct and 50 nM miR-149-5p mimic per well using Lipofectamine 3000. After 4 hours of transfection, the transfection medium was replaced with complete culture medium. After 48 hours of culture, cells were lysed with passive lysis buffer (Promega), and luciferase activity was measured at 490 nm using the Dual-Luciferase Reporter assay system (Promega Corporation; Wisconsin, USA). The ratio of firefly to Renilla luciferase activity was used to normalize firefly luciferase values.
2.11 Western blotting
FaDu and CAL-27 cells were lysed using Radio Immunoprecipitation Assay lysis buffer (Solarbio) and estimated using a bicinchoninic acid protein assay kit (Solarbio). Denatured proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Solarbio), and protein bands were transferred to a polyvinylidene fluoride membrane, blocked with 5% bovine serum albumin (Solarbio), and incubated overnight (4°C) with FGF11 (1:500; ab89713, abcam, Cambridge, UK), PI3K (1:1000, ab191606, Abcam), AKT (1:500, ab8805, Abcam), phospho (p)-PI3K (1:500, ab182651, Abcam), p-AKT (1:500, ab38449, Abcam) primary antibodies. They were then incubated with goat anti-rabbit antibody (1:20,000, ab205718; Abcam) for 2 hours at 25°C. Anti-GAPDH antibody (1:10000, ab181602; Abcam) was used as a loading control. Proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Massachusetts, USA). Chemiluminescence signals were captured with x-ray film (Kodak, New York, USA).
2.12 Statistical analysis
Statistical analysis was performed using GraphPad Prism software (v8.3.0), and data are presented as mean ± SD. One-way analysis of variance with Bonferroni post hoc test was performed to determine whether there was an overall statistically significant change before Student's t-test to analyze the difference between any two groups. P < 0.05 was considered statistically significant.