Oral squamous cell carcinoma (CAL27, SCC-4, SCC-1, TU183 and SCC-9) and normal human oral keratinocyte (NHOK) cells were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Cells were routinely incubated in Dulbecco’s modified Eagle medium (DMEM-Hyclone) mixed with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100U/ml penicillin under a 5% CO2 moist atmosphere at 37 °C.
The full-length sequence of hnRNPC or AK4 was subcloned into the pcDNA3.1 plasmid (pcDNA3.1-hnRNPC or pcDNA3.1-AK4; Invitrogen,Carlsbad, CA, USA). Knockdown of LINC00662 (sh-LINC00662#1 or sh-LINC00662#2) or hnRNPC (sh-hnRNPC) was also achieved by Invitrogen. The transfection was conducted using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the instructions of the manufacturer. The sequences of relevant shRNAs are as follows: sh-NC, 5′-GAGCCTGGATGATACATGATGC-3′; sh-LINC00662#1, 5′-CTCTTCTATCGAACCGGCCCGC-3′; sh-LINC00662#2, 5′-AGTACTGAACACGGGTTTCAAA-3′; sh-NC, 5′-AGCGAAGGGTCAGGAGAAAGAT-3′; sh-hnRNPC#1, 5′-CGCTCTCCCCCACACCCTCTCT-3′; sh-NC, 5′-AAGTGTAAGAGATGAAAGTAGA-3′; sh-AK4#1, 5′-GCTCTGCGTCTGGTGTGCAACG-3′.
Quantitative real time PCR (qRT-PCR)
TRIzol (Life Technologies, Carlsbad, CA, USA) was adopted to extract total RNA from OSCC cells and RNA purity was assessed using spectrophotometer (Bio-Rad, Hercules, CA, USA). The Prime-Script miRNA cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) to synthesize cDNA using 1 μg of total RNA according to manufacturer’s protocol. The SYBR® Premix Ex TaqTM reagent (TaKaRa, Dalian, China) was employed for qPCR analysis on an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). GADPH was the internal control. The 2−ΔΔCT method was adopted to quantify the results. The primer sequences were as follows: LINC00662 forward: 5′-CACGCTTCTGAAACTGGTGT-3′, and reverse: 5′-TGTACAGCCTGGTGACAGAG-3′; AK4 forward: 5′-TGGATTCACCCTCCTAGCGGAA-3′, and reverse: 3′-CTGTCTTAGCCTGGCAGCAACT-5′; hnRNPC forward: 5′-TGGGCTGCTCTGTTCATAAGGG-3′, and reverse: 5′-CTCGGTTCACTTTTGGCTCTGC-3′; GAPDH forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, and reverse: 5′-TGGTGAAGACGCCAGTGGA-3′.
Cell counting kit-8 (CCK-8) assay
CCK-8 assay measured the proliferation capacity of cells. Transfected cells were seeded into 96-well culture dishes with a density of 3 × 103 cells/well for 12-h culture with or without 4Gy radiation treatment at the dose rate of 200 cGy/min using 137Cs γ-ray source (Atomic Energy of Canada Ltd, Mississauga, Ontario, Canada). Then, cell counting kit-8 (10 μl/well, Dojindo, Kumamoto, Japan) was added according to manufacturer’s protocol, followed by incubation at 37 °C for another 3 h. Absorbance at 450 nm was detected by the MRX II microplate reader (Dynex, Chantilly, VA, USA).
Colony formation assay
Transfected OSCC cells were inoculated into a 6-well culture dish (500 cells/well) and were exposed to several radiation doses (0, 2, 4 and 8Gy). All cells were cultured for 2 weeks, then washed by PBS, fixed by 10% formaldehyde, and stained by 0.1% Crystal Violet (Sigma, U.S.A.). The number of colonies more than 50 cells was counted, based on which cell survival fraction (plating efficiency (PE); SF = each dose of PE/non-irradiated PE × 100%) was calculated.
Flow cytometry of cell apoptosis and cell cycle
For cell apoptosis analysis, cells were inoculated into six-well plates and transfected with sh-NC or sh-LINC00662#2 for 48 h, and then treated with a 4Gy irradiation for 24 h. OSCC cells were washed twice by PBS, centrifuged to isolate the debris and then re-suspended in binding buffer at a concentration of 1 × 106 cells per ml. Cells (1 × 106). Afterwards, cells were dyed with Annexin V/propidium iodide (PI) reagents (KeyGen Biotech, Nanjing, China) according to the instructions of the manufacturers. After the addition of binding buffer (0.4 ml), cells were examined by a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). As for cell cycle analysis, cells were co-cultured in 6-well plates (3 × 105 cells/well) with propidium iodide (PI) staining buffer (Dojindo Molecular Technologies, Inc) and subjected to flow cytometer (BD Biosciences). The proportion of cells in G0/G1, S, or G2/M phases was analyzed with the Cell Quest Pro acquisition software (BD Biosciences).
Flow cytometry of transfection efficiency
The indicated cells (5 × 105) were seeded into each well of 24-well plates and cultured with complete DMEM for 24 h prior to transfection. The cells treated alone with pSEB pooled plasmids containing transfection plasmids were named as group I; with pSEB plasmids with ultrasound were named as group II; with the lipid microbubble loaded with transfection plasmids were named as group III; with ultrasound and the lipid microbubble loaded with transfection plasmids were named as group IV; with non-plasmid control were named as group V. The Lipofection group (Lipo) was used for comparing transfection efficiency. Cells of group II and IV were ultra-sonicated with 1 MHz of radiation frequency, pulse wave and 0.5 W/cm2 of sound intensity for 30 s. After that, the transfected cells were harvested after 48 h for flow cytometry to detect transfection efficiency.
Cell migratory ability was evaluated with the use of the Transwell polycarbonate membrane inserts (Millipore, Billerica, MA, USA). The transfected CAL27 and SCC-4 cells (1 × 105) were planted into the upper chamber of the insert. 10% FBS was used to supplement cell-free medium in the low chamber at 37 °C for 1 day. Cells migrating through the membranes were cultured in 4% paraformaldehyde and stained with 0.1% crystal violet. For cell invasion assay, 1 × 105 cells in sterile medium were seeded into the upper chamber with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). Both migrated and invaded cells were observed and counted under an inverted light microscope (Leica Microsystems, Wetzlar, Germany) at a magnification of ×200.
Knockout of LINC00662 or hnRNPC by CRISPR/Cas9
The CRISPR/Cas9 genome editing system was applied for generating the LINC00662 or hnRNPC knockout cells. The two sgRNAs targeting LINC00662 or hnRNPC were individually transfected into cells and Cas9-EGFP vector. The pCRISPR-LvSG03 for sgRNAs and CP-LvC9NU-02 for construction of Cas9-EGFP were all procured from Genecopoeia (Rockville, MD, USA). Cas9-expressing cells were established before co-infection of two sgRNAs. The genomic deletions of isolated single colonies were detected using T7 endonuclease I cleavage and PCR, and determined with sequencing after cloning the relative PCR products to T vector (Takara).
starBase v2.0 (http://starbase.sysu.edu.cn/starbase2/browseClipSeq.php) was employed to predict the RNA-binding proteins of LINC00662 or AK4. In the Protein-RNA column, we chose protein-lncRNA interactions and protein-mRNA interactions parts, respectively. Among the putative LINC00662-binding proteins and AK4-binding proteins, we selected hnRNPC, one of the top ten RNA-binding protein candidates according to starBase v3.0 (http://starbase.sysu.edu.cn/), for further studies.
RNA immunoprecipitation (RIP) assay
For RNA immunoprecipitation (RIP) assay, a Magna RIP Kit (Millipore, Billerica, MA, USA) was adopted according to the manufacturer’s protocols. The whole cell lysate was incubated in RIP buffer where magnetic beads were absorbed by anti-hnPNRC (ab133607; 1:100; Abcam, Cambridge, MA, USA) or anti-IgG (ab218427; 1:100; CST, Boston, MA, USA). IgG was a normalization control. After the digestion of proteinase K, co-precipitated RNAs including LINC00662 and AK4 were harvested, purified and detected by qRT-PCR.
RNA pull-down assay
LINC00662 or AK4 sense was named as LINC00662-WT or AK4-WT, compared with LINC00662 or AK4 antisense (LINC00662-Mut or AK4-Mut). LINC00662-WT, LINC00662-Mut, AK4-WT and AK4-Mut were in vitro transcribed by TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and subsequently biotin‐labelled through the Biotin RNA Labelling Mix (Roche) and T7 RNA polymerase (Roche) as instructed. After the incubation with DNase I (Takara), the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was utilized to purify the transcribed RNAs. Then, 3 μg of biotinylated RNAs were grown for 1 h at 25 °C in mixture with 1 mg whole cell lysates. Then, the complexes were extracted via streptavidin-conjugated Dynabeads (Invitrogen). Eventually, the pull-down materials were confirmed by western blot.
CAL27 and SCC-4 cells were cultured in RIPA buffer with protease inhibitor cocktails (AMRESCO), lysed for 30 min with brief vortexing every 10 min. Then centrifuge the lysates for 15 min at 4 °C and detect protein concentrations using the BCA Protein Assay Reagent (Pierce, Appleton, Wisconsin, USA). Post separation of 40 μg protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the lysate was transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies against cleaved PARP (ab32064; Abcam), PARP (ab74290; Abcam), cleaved caspase-3 (ab2302; Abcam), caspase-3 (ab13847; Abcam), hnRNPC (ab97541; Abcam), AK4 (ab232888; Abcam) or GAPDH (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. After washing, the membrane was cultured with horseradish peroxidase-conjugated (HRP) secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 2 h. Finally, the signals were estimated by enhanced fluorescence as suggested.
All statistical analyses were conducted via SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (GraphPad, Inc., La Jolla, CA, USA). The data were indicated as the mean ± standard deviation. Statistical significance analysis of two or multiple groups was performed using Student’s t-test or ANOVA. P value less than 0.05 was accepted as statistically significant. Each assay was independently carried out in triplicate (Additional file 1).