Patients and tissue samples
Between 2013 and 2018, 33 pairs of cervical cancer tissues and adjacent normal tissues were collected from patients who underwent surgery at The Second Xiangya Hospital, Central South University. What’s more, none of the patients in this study received chemotherapy or radiotherapy before surgery. Tissue specimens were frozen in liquid nitrogen at − 80 °C at the time of collection.
Cervical cancer cells (HeLa, SiHa, CaSki and C33a) and normal cervical cell (Ect1-E6E7) were purchased from the Cell Culture Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were all cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific). Cells were cultured with 5% CO2 at 37 °C.
HeLa or SiHa cells were plated in 6-well plates, and then incubated for one day. After that, the specific short hairpin RNAs (shRNAs) of SFN (sh-SFN-1/2) or LINC01128 (sh-LINC01128–1/2) and their negative controls (sh-NCs), along with mimics and inhibitor of miR-383-5p or miR-107-mimics, NC mimics/inhibitor were purchased from Genepharma (Shanghai, China). The above plasmids were separately transfected into HeLa or SiHa cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) after 48 h.
RNA extraction and real-time quantitative PCR (RT-qPCR)
Tissues and cells were lysed via Trizol reagent (Invitrogen), and then total RNA was extracted and reverse transcribed into cDNA via a Reverse Transcription Kit (Invitrogen). Then, RT-qPCR was conducted through SYBR Green RT-PCR Kit (Invitrogen) on an Applied Biosystems 7300 (Thermo Fisher Scientific). Results were calculated via the 2−ΔΔCt method. GAPDH/U6 level was measured as the internal control.
Cell counting kit-8 (CCK-8) and Colony formation assays
The number of HeLa or SiHa cells was 1000, which was seeded in 96-well plates and incubated under the standard conditions. CCK-8 solution (Dojindo, Tokyo, Japan) was added to each well. Then, cells were incubated for another 4 h. The absorbance at 450 nm was evaluated through a microplate reader (Olympus, Tokyo, Japan). For colony formation assay, HeLa or SiHa cells in medium (Thermo Fisher Scientific) were seeded in 6-well plates and incubated for two weeks. Colonies (≥50 cells) were formed and then manually counted.
5-ethynyl-2′-deoxyuridine (EdU) incorporation assay
EdU labeling medium was added to cell culture with EdU labeling kit (RiboBio, Guangzhou, China). HeLa or SiHa cells after transfection were incubated for 2 h, and then fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed at room temperature by PBS (Sigma-Aldrich) dyed with anti-EdU (Acbam, Cambridge, USA) working solution (Invitrogen). Under the same conditions, cells were again washed with Triton X-100 (Solarbio, Shanghai, China) in PBS (Sigma-Aldrich). Cells were observed via a fluorescent microscopy (Leica, Wetzlar, Germany).
This experiment was performed as previously described .
Transfected HeLa or SiHa cells were isolated via RIPA lysis buffer (Invitrogen). Then, the BCA (Invitrogen) was employed to evaluate the concentration of protein. Proteins were separated by SDS-PAGE (Millipore, MA, USA), followed by transferred into PVDF membranes (Millipore). After being blocked with 5% nonfat milk, membranes were incubated overnight with primary antibodies at 4 °C: anti-Bax (ab32503, Abcam), anti-Bcl-2 (ab182858, Abcam), anti-SFN (ab14123, Abcam) anti-E-cadherin (ab194982), anti-N-cadherin (ab202030), anti-Vimentin (ab193555), anti-ZEB1 (ab228986), anti-Slug (ab51772), anti-Twist (ab187008), anti-Snail (ab229701) and anti-GAPDH (ab8245, Abcam), then cultivation at room temperature with secondary antibody for 1 h. GAPDH was employed as the internal parameter. Finally, the ECL Kit (Thermo Fisher Scientific) was selected to visualize protein bands.
Transfected HeLa or SiHa cells were cultivated into 6-well plates. After being washed twice with cold PBS (Sigma-Aldrich) and re-suspended, cells were fixed through ice-cold ethanol (Sigma-Aldrich). Finally, flow cytometer (BD Biosciences, San Diego, CA, USA) was employed to analyze cell apoptosis.
The re-suspended cells in serum-free medium were seeded into the upper chamber, and the basolateral chamber was filled with 10% PBS. Transwell chambers pre-coated with or without Matrigel (Millipore, Massachusetts, USA) were employed to carry out the invasion or migration assay. An inverted microscope (Carl Zeiss, Jena, Germany) was applied to observe the migrated or invaded cells.
Sphere formation assay
HeLa or SiHa cells were harvested, counted and incubated in 6-well ultra-low attachment plates (Corning Incorporated, Corning, NY, USA) in serum-free RPMI 1640 medium (Invitrogen, Shanghai, China) to form sphere. The RPMI 1640 medium is supplemented with 20 ng/ml human FGF (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/ml human EGF (Gibco), 1% N2 supplement (Gibco) and 1% B27 (Gibco). 2 weeks later, the number of spheres was counted by a light microscope (Nikon Corporation) and the number was recorded.
Isolation and purification of cytoplasmic and nuclear RNA were conducted via a Cytoplasmic and Nuclear RNA Purification Kit from Norgen (Ontario, Canada). The expression levels of LINC01128, GAPDH and U6 were separately evaluated via RT-qPCR analysis.
RNA pull-down assay
miR-383-5p biotin probe and miR-383-5p no-biotin probe were treated with M-280 Streptavidin magnetic beads (Invitrogen) to generate probe-coated beads. Subsequently, cells were collected and dissolved, submitted to sonication and cultivation with probe-coated beads overnight at 4 °C. Lastly, RT-qPCR was employed to measure purified RNA complex.
Luciferase reporter assay
Partial DNA sequences of SFN or LINC01128 containing wild-type (WT) or mutant (MUT) miR-383-5p binding sites were amplified by PCR and then cloned into a pmirGLO dual-luciferase plasmid (Promega, Madison, WI, USA) to produce SFN-WT, SFN-MUT, LINC01128-WT, and LINC01128-MUT reporter plasmids, which were separately co-transfected with miR-383-5p mimics or NC mimics into HeLa or SiHa cells. Finally, luciferase activities were evaluated through the dual luciferase reporter assay system (Promega).
Results were presented as mean ± SD of three independent experiments at least. Statistical analyses were imported into SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Student’s t-test or one-way ANOVA was employed for analysis of differences. P < 0.05 had statistically significant.