Tissues and clinical data
The human ovarian cancer tissue microarray was purchased from Shanghai Outdo Biotech (Sample No. HOvaC154Su01, Shanghai, China). The tissue microarray included 2 cases of benign ovarian tumors and 152 cases of ovarian cancer followed up for 5–9 years. Of these, the serous epithelial ovarian cancer tissues were selected for this study. The selected patients were divided into two groups, namely, the chemosensitive group and the chemoresistant group, according to the NCCN guideline. The study included 63 cases of chemosensitive serous EOC tissues and 32 cases of chemoresistant serous EOC tissues. The clinical features and pathological information are summarized in Table 1. The study was approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University (approval number: 2020-G143) and the ethics committee of Shanghai Outdo Biotech Company (approval number: YB M-05-02).
RNA fluorescence in situ hybridization
The primer for the lnc-SNHG1 fluorescence in situ hybridization (FISH) probe was 5′-GCAGGAAGGGGGTGATAAAATACAGAAATG -3′, while that for the miR-216b-5p probe was 5′- TCACATTTGCCTGCAGAGATTT-3′. The fluorescence probe of lnc-SNHG1 was labeled with Cy3 (red), and that of miR-216b-5p was labeled with FAM (green). One tissue microarray slice was hybridized with Cy3 probes specific for lnc-SNHG1, and the other slice was hybridized with FAM probes specific for miR-216b-5p. The in situ hybridization was performed as follows. (1) The slices were dewaxed to DEPC water followed by boiling in the repair solution for 15 min and cooled naturally. Then, protease K (20 µg/mL) was dripped for digestion for about 25 min. After washing with pure water, PBS was used for washing three times. (2) Prehybridization: The slices were incubated with prehybridization solution at 37°C for 1 h. (3) Hybridization: the prehybridization solution was poured out, the hybrid solution containing the probe of lnc-SNHG1 or miR-216b-5p (concentration 6 ng/UL) was dripped, and the slices were hybridized in the incubator at 37°C overnight. (4) Washing after hybridization: The hybridization solution was washed off with 2× sodium saline citrate (SSC) for 10 min, with 1× SSC for 5 min twice, and then with 0.5× SSC for 10 min at room temperature. (5) The slices were incubated with [4′,6-diamidino-2-phenylindole (DAPI)] dye solution in the dark for 8 min, and then anti-fluorescence quenching sealing agent was added after washing. (6) Fluorescence microscopy and image acquisition: The slices were observed and the images were collected under a Nikon positron fluorescence microscope (Nikon Eclipse Ci, Japan). The nucleus was stained blue with DAPI under UV excitation. The fluorescence using fluorescein indicated positive expression; FAM was stained green, and Cy3 was stained red.
Cell culture
The ovarian cancer paclitaxel-resistant cell line A2780/Taxol and its parental A2780 cell line were purchased from the American Type Culture Collection. Both A2780/Taxol and A2780 cell lines were grown in RPMI 1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies, NY, USA) and 1% penicillin/streptomycin (Hyclone, UT, USA). Moreover, the A2780/Taxol cell line was maintained in the culture medium containing 800 ng/mL paclitaxel (Sigma–Aldrich, MO, USA). Paclitaxel was withdrawn a week before the experiment. The aforementioned cell lines were cultured at 37℃ and in the presence of 5% CO2 and saturated humidity. The cells in the logarithmic growth phase were used for the experiment.
Cell transfection
MiR-216-5p-mimic, miRNA mimic negative control (miRNA mimic NC), MiR-216-5p-inhibitor, miRNA inhibitor negative control (miRNA inhibitor NC), lnc-SNHG1 siRNA, and siRNA NC were chemically synthesized by GeneCreate Biotech (Wuhan, China). MiR-216-5p-mimic and miRNA mimic NC were transfected into cells at a final concentration of 100nM using Lipofectamine 3000 (Invitrogen, CA, USA) following the manufacturer’s protocol. Lnc-SNHG1 siRNA and siRNA NC were transfected at a concentration of 100nM. The cells after transfection were incubated in the presence of 5% CO2 at 37℃ for 48 h. The transfected cells were harvested for the next analysis
CCK-8 assay
After transfection, the cells were seeded in a 96-well plate at a density of 5 × 103 cells/well. The plates were cultured in a 5% CO2 incubator at 37℃. The cells were treated with paclitaxel at a concentration of 0.04μM, 0.2μM, 0.9μM, 5μM, and 23μM for 24 h. The cell viability was assessed using the CCK-8 assay (Dojindo, Kumamoto, Japan). The absorbance of each well at the wavelength of 450 nm was read on a spectrophotometer (XFLUOR4 Version: V 4.51). At least three independent experiments were performed in quadruplicate.
Cell apoptosis assay
After transfection, the cells were placed in a six-well plate at a density of 2 × 105/2 mL. After 24 h, A2780/Taxol cells were transfected with the miR-216b-5p inhibitor (miRNA inhibitor NC) or siRNA of lnc-SNHG1-2 (siRNA NC). After 48 h of transfection, the cells were cultured in a medium containing 5μM paclitaxel. After 24 h, the cells were collected and washed with PBS twice for apoptosis. The Annexin V-FITC cell apoptosis detection kit (BD, New Jersey, USA) was used for staining, and flow cytometry was used to detect cell apoptosis. The experiment was repeated three times.
Wound healing assay
After transfection, the cells were placed in a six-well plate at a density of 5 × 105/2 mL and cultured to 90% confluence. The wounds were generated via scratching the cell layer with 100-μL sterile plastic pipette tips. The treatment hole was gently washed with 1 mL of precooled sterile PBS three times, the drawn cells were washed out as much as possible, and images were taken under a microscope (Olympus, Tokyo, Japan) at time points of 0 h, 24 h, and 48 h.
Clone formation ability test
The cells after transfection in different treatment groups were plated into 6-well plates at a density of 1000 cells and placed in a 5% CO2 incubator at 37℃ overnight. On the next day, the cells in each group were treated with 5μM paclitaxel for 24 h. After treatment, the cells were cultured in a fresh and complete medium. They were incubated in an incubator for 2 weeks, followed by 0.1% crystal violet staining for 30 min, PBS washing three times, and statistical data cloning. The difference in colony formation was compared.
RNA extraction, cDNA synthesis, quantitative real-time polymerase chain reaction
Total RNA was isolated using TRIzol agent (Invitrogen, CA, USA) following the protocols of the manufacturer and treated with RQ1 DNase (Promega, WI, USA) to remove DNA. Reverse transcription reactions were carried out using a ReverTra Ace quantitative polymerase chain reaction (qPCR) reverse transcription (RT) kit (Toyobo Life Science, Shanghai, China) following the manufacturer’s protocols. The expression levels of miR-216b-5p and lnc-SNHG1 were detected by qRT-PCR. The human actin gene was used as a control. The qRT-PCR was performed on a Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (Toyobo). The relative expression levels of each sample were measured using the 2−ΔΔCt method (Livak and Schmittgen 2001). All reactions were performed in triplicate.
Luciferase activity assay
A2780/Taxol cells were cultured at a density of 2 × 104 cells/well in 96-well culture plates and
transfected with luciferase reporter constructs (400 ng), miR-216b-5p mimics (500nM), and the internal control vector pRL-TK, pRL-SV40, or pRL-CMV (Promega, WI, USA) at a ratio of 20:1 (reporter construct:control vector) using Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. The transfection medium was removed 5 h after transfection and replenished with a medium containing 6μM curcumin (Sigma–Aldrich, MO, USA) solubilized in 100% dimethyl sulfoxide (DMSO) (Sigma–Aldrich, MO, USA). The luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, WI, USA).
RIP
Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, Massachusetts, USA) was applied for the RIP assay which was carried out in A2780/Taxol cells. Cell lysates were mixed with magnetic beads conjugated with IgG or human anti-Ago2 antibody in RIP buffer. Finally, precipitated RNA was purified for qRT-PCR.
Statistical analysis
SPSS version 19.0 (IBM SPSS, IL, USA) was used for statistical analysis. The data were expressed as mean ± standard deviation. The Student t test was used to compare quantitative variables. The chi-square test was used to determine the association of lnc-SNHG1 or miR-216b-5p with clinicopathological variables. The multivariate logistic regression analysis was applied to determine the related factors for chemoresistance. The Cox’s proportional hazard model was applied to identify the independent prognostic factors for overall survival (OS) and disease-free survival (DFS). Spearman’s correlation analysis was used to analyze the correlation between lnc-SNHG1 and miR-216b-5p expression. The OS and DFS curves were analyzed using the Kaplan–Meier test. The log-rank test was used to compare OS and DFS between chemosensitive and chemoresistant groups. A P value less than 0.05 indicated a statistically significant difference.