Cell culture
Human OS cell lines (MG63, KHOS and U2OS) were obtained from the American Type Culture Collection (ATCC, NY, USA) and cultured in DMEM medium containing 15% fetal bovine serum (FBS, Gibco, MA, USA) and 1% penicillin/streptomycin (Invitrogen, MA, USA) at 37 °C in humidified CO2 (5%) incubator. The Dox resistance cell lines (MG63/DXR, KHOS/DXR) were established by serially increased concentration of Dox as described previously [12]. Briefly. the OS cells were continuously cultured in complete medium supplemented with 0.0035 μM Dox. The second (R2) generation was developed by continuous exposure of the corresponding R1 cells to 0.035 μM Dox. The third (R3) generation of Dox-resistant OS cells was generated by culturing the OS R2 cells in the continuous presence of 0.35 μM Dox. A cell line was considered as stable when the growth rate of the treated cells to a given Dox concentration was constant for at least 30 days.
Cell transfection
Indicated cells were seeded into 6-well plate the day before transfection. Transfection was performed with Lipofectamine 3000 (Invitrogen, MA, USA) according to the manufacturer’s recommendation. The oligo sequences used in this study were provided as below:
si-LINC00426–1: 5′-GGCGCTATTTCGGCTCATTAT-3′;
si-LINC00426–2: 5′-GCGCTATTTCGGCTCATTATA-3′;
si-NC: 5′-TTCTCCGAACGTGTCACGT-3′;
miR-4319: 5′-UGCUCCCUGAGGACGUUAUAUGA-3′;
miR-NC: 5′-UGUGCAAAUCUAUGCAAAACUGA-3′.
Real-time PCR
Total RNA was extracted from either cells or tissues with TriZol reagent (Invitrogen, MA, USA). The single-strand cDNA was prepared using the PrimeScript RT Reagent Kit (Takara, Dalian, China). The relative expression of LINC00426 and miR-4319 was determined with SYBR Green MasterMix Kit (Roche, Basel, Switzerland) on ABI PRISM 7900 HT System (Applied Biosystems, CA, USA). GAPDH and U6 were employed as reference control. All primer sequences were listed as follows:
miR-4319 RT prime: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGGCT-3′;
U6 RT prime: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAA-3′;
LINC00426 qRT-PCR Forward: 5′-CAAGAAGACAGGGACAAGC-3′;
LINC00426 qRT-PCR Reverse: 5′-ACTGAGTACCCAGCCAAAG-3′;
GAPDH qRT-PCR Forward: 5′-CATGAGAAGTATGACAACAGCCT-3′;
GAPDH qRT-PCR Reverse: 5′-AGTCCTTCCACGATACCAAAGT-3′;
miR-4319 qRT-PCR Forward: 5′-CACCCAGAGCAAAGCCAC-3′;
miR-4319 qRT-PCR Reverse: 5′-GTGCAGGGTCCGAGGT-3′;
U6 qRT-PCR Forward: 5′-TGCGGGTGCTCGCTTCGGCAGC-3′;
U6 qRT-PCR Reverse: 5′-GTGCAGGGTCCGAGGT-3′.
Cell counting kit (CCK)-8 assay
CCK-8 kit (Dojindo, Kumamoto, Japan) was used to measure cell viability. Briefly, 4000 indicated cells/well were seeded into 96-well plates in triplicated and subjected to specified transfection for 48 h. CCK-8 working solution was added and allowed for reaction for 2 h at 37 °C. Absorption at 450 nm was determined with microplate reader (Biotek, VT, USA) and relative cell viability was calculated accordingly.
Colony formation assay
Cell proliferation was evaluated by colony formation assay. The transfected cells (1000 cells) were prepared into 6-well plate in triplicate and continuously cultured for 2 weeks. The former colonies were fixed with PFA (4% in PBS) first and stained with crystal violet (0.05% in PBS) for 10 min. Colony number was counted under light microscope and representative images were captured and presented.
Caspase-3 activity assay
The indicated cells were collected by trypsin digestion and caspase-3 activity was determined with Cells were treated with different concentrations of MTX, ADM or DDP for 72 h and then collected with trypsin. The caspase-3 activity was measured with Caspase-3 Activity Assay Kit (Merck, MO, USA) according to the manufacturer’s manual. Briefly, cell lysate was prepared in the supplied lysis buffer and substrate was added for 2 h-incubation at 37 °C in the dark. The absorption at 405 nm was recorded with microplate reader and relative caspase-3 activity was calculated accordingly.
Luciferase reporter assay
LINC00426-driven luciferase reporter plasmids were constructed by PCR-Restriction Digestion-Ligation method. The mutant was generated by mutagenesis PCR method. The primer sequences used for PCR were provided as below. Both luciferase plasmids and miR-4319 were co-transfected into MG63/DXR and KHOS/DXR cells in triplicate in 6-well plate (1◊106 cells/well). The cell lysates were collected and dual luciferase activities were determined with Dual-Luciferase Reporter Assay System (Promega, WI, USA).
LINC00426 PCR Forward: 5′-AATTCTCGAG ACTCGGCCATGAAAGTC-3′;
LINC00426 PCR Reverse: 5′-AATTGCGGCCGC TGTCTTCCAGTAAGACTTTA-3′;
LINC00426 mutagenesis PCR Forward: 5′-CACCACTTGTCTCAGAGGAA-3′;
LINC00426 mutagenesis PCR Reverse: 5′-TTCCTCTGAGACAAGTGGTG-3′.
Pull-down assay
RNA was completely removed by RNase A incubation. Biotin-labeled probes were incubated with cell lysates from indicated cells (2.5 × 107) for one hour at room temperature. Pulldown assay was performed with streptavidin-coated agarose beads (Invitrogen, MA, USA) and enriched RNA specimen was reversely transcribed into cDNA as described previously. The relative abundance of target transcript was determined by real-time PCR. The probe sequences used in this study were provided as below:
Sense DNA probe: 5′-Biotin-GTCAGGACACAGCAAATGGGGGATCT-3′;
Antisense DNA probe: 5′- Biotin-AGATCCCCCATTTGCTGTGTCCTGAC-3′;
Bio-miR-NC: 5′-Biotin-UGUGCAAAUCUAUGCAAAACUGA-3′;
Bio-miR-4319: 5′-Biotin-UGCUCCCUGAGGACGUUAUAUGA-3′.
Statistical analysis
Results were presented as the mean values ± SD unless specified and analyzed with two-tailed t-test, one- or two-way ANOVA with a Bonferroni post hoc test. A value of p < 0.05 was considered significantly different.
