Patients and tissues
OC tissues samples (n = 63) and matched peritumor normal (non-tumor, defined as tissues surrounding the tumor) tissues (n = 63) were harvested from OC patients from May 2015 to September 2017 in the First Affiliated Hospital of Shangqiu Medical College in Henan Province. The samples used in this study were immersed in liquid nitrogen for 10 min and then stored at -80 ℃. This study was approved by the First Affiliated Hospital of Shangqiu Medical College in Henan Province ethics committee (No.2019021). The written informed consent forms were signed and submitted by all of these OC patients. The OC tissues were subsequently divided into LINC01638 low- and high- expressed groups based on the expressions of LINC01638, and the median of LINC01638 expression level was seemed as the cut-off value. The period from surgery to death was described as overall survival and the overall survival of these OC patients was recorded. The relationship between LINC01683 expressions and OC clinical pathology (Ages, FIGO stage, Grade, Distant metastasis and Tumor size) was analyzed.
In situ hybridization (ISH) analysis
A hybridization solution (Sangon Biotech, Shanghai, China) was used to pre-hybridize OC tissues sides at 37℃ for 2 h. A total of 10 picomoles of digoxigenin-labeled detection probes (Boster Bioengineering, Wuhan, China) complementary to LINC01683 were added and cultured at 37℃ overnight. After washing, a mouse monoclonal antibody to digoxigenin (ab420, Abcam, Cambridge, MA, USA) was used to carry out an immunologic reaction, whereafter alkaline phosphatase-conjugated streptavidin dilution (Boster) was added to determine streptavidin dilution probes. After drying, slides were observed and photographed under a microscope (Leica, German).
OC cell lines, including SKOV3, CAOV3, OVCAR4 and OVCAR3, and normal cell line IOSE80 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). The Dulbecco's Modified Eagle Media (DEME) medium, containing 10% fetal bovine serum (FBS, Invitrogen Carlsbad, CA, USA), was applied to culture the cell lines in this study. The cells were then cultured at 37 ℃ in an incubator with 5% CO2.
The LINC01638 knocked down plasmid/control plasmids (pLKO.1-shLINC01638/pLKO.1), LINC01638 overexpressed plasmid/control plasmid (pBabe-puro-LINC01638/pBabe-puro), microRNAs-128-3p (miR-128-3p) mimics/negative control (NC) mimics (miR-128 mimics/NC mimics) and miR-128 inhibitor/NC inhibitor were purchased from GenePharma (Shanghai, China). Human OC cell lines OVCAR3 and SKOV3 were transfected with 30 nM of pLKO.1-shLINC01638/pLKO.1, pBabe-puro-LINC01638/pBabe-puro, miR-128 mimics/NC mimics or miR-128 inhibitor/NC. And HEK 293T cells were transfected pLKO.1-shLINC01638/pLKO.1, pBabe-puro-LINC01638/pBabe-puro or miR-128 mimics/NC mimics in the 12-well culture plates using Lipofectamine 2000 (Invitrogen, USA). After transfected for 48 h, all of the cells were collected and used for the following experiments.
After the OC cell lines OVCAR3 and SKOV3 were transfected with pLKO.1-shLINC01638/pLKO.1, pBabe-puro-LINC01638/pBabe-puro or miR-128 mimics/NC mimics, the cells were treated with cisplatin (2 μg/mL). Dimethyl Sulfoxide (DMSO, Invitrogen) served as the drug of control group.
Stably transfected OC cells in this study were incubated in 96-well plates (5 × 103 cells/well) with DMEM medium (100 μL, containing 10% FBS) and were cultured at 37°C, 5% CO2. After cultured for 1, 2, 3 and 4 days, CCK-8 kit (10 μL, Dojindo, Kumamoto, Japan) was added for 4 h at 37 °C. Plates were subsequently putted in a microplate reader (Biotek, USA) to detect the optical density (OD) value at 450 nm wavelength.
After transfection, OC cell lines were treated with 0 nM, 0.25 nM, 1 nM, 4 nM, 16 nM, 64 nM, 256 nM and 1024 μM cisplatin for 24 h. At the end of treatment, the MTT assay was used to detect the cell viability. The OC cells in this study were incubated in 96-well plates (5 × 103 cells/well) with DMEM medium (100 μL, containing 10% FBS) and were cultured at 37°C, 5% CO2. After cultured for 48 h, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltet razolium bromide (MTT kits, Sigma-Aldrich, St. Louis, USA) was added into each well for 4 h at 37 °C. The MTT solution was discarded, and then 150 μL DMSO were added into the plates. After mixing, the OD value of 570nM was detected using microplate reader (Biotek). A GraphPad Prism software 6.0 (San Diego, CA, USA) was performed to calculated the IC50 values.
Colony formation assay
Transfected OC cells (400 per group) from each treatment were seeded in 12-well plates for 14 days. After that, crystal violet was employed to stain the OC cell lines. An invert microscope was used to count the number of stained cells. (Olympics, Tokyo, Japan).
After the OC cell lines transfected for 48 h, the cells were collected. For cell cycle analysis, the cells were fixed and permeabilized using 75% cold ethanol, washed using PBS and resuspended using propidium iodide buffer (Sigma). Cell cycle of all this cells were measured by processing flow cytometry (BD Biosciences, NJ, USA) was processed for detecting cell cycle. For apoptosis assays, after harvesting cells, these human OC cells were stained by fluorescein isothiocyanate-conjugated Annexin V (Nanjing KeyGen Biotech, Nanjing, China) and propidium iodide (Nanjing KeyGen Biotech). Flow cytometry was then operated to assess cell apoptosis.
Xenograft tumor model assay
Female Bal/c nude mice (6-8 weeks; n = 6/group) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China) and raising in sterility environments with approval by the First Affiliated Hospital of Shangqiu Medical College in Henan Province. Human OC cell lines OVCAR3, which were transfected with pLKO.1-shLINC01638 or pLKO.1, were hypodermic injected into mice with a density of 1×107 cells. When the volume of the tumors grew to 200 mm3, cisplatin (10 mg/kg) was injected into these nude mice, vehicle served as control group. The mice were divided into 4 groups: pLKO.1 + vehicle, pLKO.1 + Cisplatin, pLKO.1-shLINC01638 + vehicle and pLKO.1-shLINC01638+Cisplatin. The tumor volume was measured and recorded weekly for 35 days after cisplatin injection, Tumor volume (mm3) = 0.5×L×W2, the length was represented as L and the width was represented as W. The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate with the density of 400 mg/kg, and then euthanized by air embolization after 5 weeks. The tumors were removed for tumor weight examination after death confirmation by cardiac arrest and pupil enlargement. And, tumor tissues were harvested for subsequent experiment. Animal experiments were proceeded in accordance with relevant guidelines and regulations of the First Affiliated Hospital of Shangqiu Medical College in Henan Province.
The paraffin was applied to fix and embed tumor tissues. The tissues were into pieces (5 μm) and placed in glass slides. Then the glass slides were rehydrated and paraffinized via xylene and graded alcohols, respectively. The target retrieval solution was used to immerse the slides for 30 min in a water bath. The endogenous peroxidase was blocked in H2O2 (3%) for 15 min, and the nonspecific bindings was buried in goat serum for 50 min. Then, the slides were stained with Ki-67 primary antibodies were applied to stain the slides, following by secondary antibody polymer HRP was employed to stain the slides. 3,3’-diaminobenzidine (DAB) was subsequently used to stain the slices stained and methyl green was used to counterstained them. Images were taken under a light microscope.
The apoptosis of mice tumor tissues was monitored using an in situ cell death detection kit (Roche, Basel, Switzerland). In brief, the tissue sections were blocked using H2O2 (3%) for 5 min and then defined using TdT labeling reaction mix for 1 h. The experiment was processed at 37°C. The sections were stained by DAB for 15 min and observed under a light microscope.
RNA pull down assay
The target miRNA and gene of LINC01638 were predicted using miRDB and Targetscan 7.1, respectively. RNA pull down assay was carried out in human OC cell line OVCAR3 with the silence of LINC01638 or overexpression of LINC01638 to validate whether miR-128-3p could combine with LINC01638. In brief, the biotinylated pLKO.1-shLINC01638/pLKO.1 or pBabe-puro-LINC01638/pBabe-puro probes were inoculated in streptavidin-coupled magnetic beads for 48 h. And qRT-PCR was performed to detect the purified RNA complexes expression. This experiment was processed under 25°C.
Luciferase reporter gene assay
Luciferase reporter vectors, containg PDK1-WT and PDK1-Mut, were constructed. HEK293T cells were transfected with miR-128 mimics/NC, pLKO.1-shLINC01638/pLKO.1 or pBabe-puro-LINC01638/pBabe-puro and then respectively cotransfected with the above luciferase reporter vectors. After 48 h transfection, the double-luciferase assay system (Promega, USA) was proceeded to assess the cells luciferase activity.
RNA extraction and qRT-PCR
LINC01638, miR-128-3p and PDK1 expressions in OC tissues and cells was measured by qRT-PCR. In brief, TRIzol (Sigma-Aldrich) was applied to harvest the total RNA in tissues and cells. The cDNA was synthesized by the reverse transcription reaction of RNA. PrimeScript™ RT reagent kit (Takara, Japan) was used to conduct the reverse transcription reaction according to the instructions. The expression levels of LINC01638, miR-128-3p and PDK1 were then detected by qRT-PCR using SYBR Premix Ex Taq™ kit (Takara). ABI-7900 thermal cycling instrument used in this study was provided by Applied Biosystems, USA. The relative expressions of LINC01638, miR-128-3p and PDK1 were analyzed by 2−ΔΔCT method. GAPDH was served as internal control. The primer sequences were as follows: LINC01638 forward primers: 5′-AATACATCAGCACTGTTGCCTTT-3′, reverse primers: 5′-CTCCATACATACATCTCCAAAAAGT-3′; miR-128-3p forward primers: 5′-GGGTCACAGTGAACCGGT-3′, reverse primers: 5′-TCCTCCTCTCCTCTCCTCTC-3′; PDK1 forward primers: 5′-CCCCTCAGCTTGCAGAT-3′, reverse primers: 5′-TGCTCCACACAGTCATTCA-3′; GAPDH forward primers: 5′-AAGGTCGGAGTCAACGGA-3′, reverse primers: 5′-TTAAAAGCAGCCCTGGTGA-3′.
Briefly, lysis buffer was used to lyse cells used in the present study, and then total proteins in cells were obtained. BCA protein assay kit, provided by BioVision, USA, was applied to measure the protein concentration of each sample. The sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate these proteins, and then these separated proteins were electrically transferred to the PVDF membrane. The PVDF membrane was blocked by 5% skimmed milk powder, and then primary antibodies, provided by Abcam, was added to incubate the membrane overnight at 4°C and the secondary antibody was added for 2 h at room temperature. The primary antibodies used in this study was as follow: anti-P-glycoprotein (anti-p-gp) (1:1000, ab242104), anti-poly (ADP)-ribose polymerases (anti-PARP, 1:1000, ab191217), anti-cleaved-PARP (anti-c-PARP, 1:1000, ab32064), anti-PDK1 (1:1000, ab110025) and anti-β-actin (1:1000, ab8227). The target bands were respectively reflected and evaluated by ECL and Quantity One software (Bio-Rad, Hercules, CA, USA). β-actin was served as internal control.
Data from this study were analyzed by SPSS 22.0 (IBM, USA) and indicated as mean ± SD of at least three independent experiments. To compare the differences among groups, Student’s t-test, one-way ANOVA or two-way ANOVA were employed. Pearson's χ2 tests were performed to analyze the relationship between LINC01638 expression and OC clinical pathology, including Ages, FIGO stage, Grade, Distant metastasis and Tumor size. Kaplan-Meier survival analysis was carried out for analysis of survival rate. The correlations among LINC01638, miR-128-3p and PDK1 expressions in OC tissues were determined using Spearman's correlation analysis. The log-rank test was applied to calculate P-value, and P<0.05 presented statistically significant.