WISP1 induces ovarian cancer via the IGF1/αvβ3/Wnt axis

Ethics statement

This study was started with the ratification of the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University and carried out by referring to the Declaration of Helsinki. All subjects provided informed consent form. Animal experiments were ratified by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University.

Bioinformatics analysis

UALCAN and GEPIA websites were employed to analyze the expression of IGF1 and WISP1 in adjacent normal tissues and ovarian cancer tissues, and the relationship between their expression with the prognosis and tumor stage of patients with ovarian cancer. IGF1-related genes and metastasis-related genes in ovarian cancer were analyzed by UALCAN website. The Venn tool was utilized to obtain the intersection genes to plot a Venn map. GO and KEGG enrichment analysis of the above intersection genes was carried out utilizing the ClueGO plug-in unit of Cytoscape software.

Study subjects

Ovarian cancer tissues and adjacent normal tissues (at least 5 cm away from the tumor) were collected from 57 patients with ovarian cancer (aged 34–76 years old with a mean age of 55.07 ± 10.12 years) at The Second Affiliated Hospital of Harbin Medical University from September 2019 to October 2020. These patients had no other clinicopathological features and did not receive preoperative treatment, such as radiotherapy or chemotherapy. The histological diagnosis of ovarian cancer was evaluated in the light of the World Health Organization criteria. All collected tissues were immediately stored at − 80 °C for the following experiments.

Cell culture and transfection

Human normal ovarian cell line IOSE80 (C1390) was purchased from Shanghai Zeye Biotech Co., Ltd. (Shanghai, China). Human ovarian cancer cell lines CAOV4 (HTB-76) and SKOV3 (HTB-77) were procured from COBIOER Company (Nanjing, China). Human ovarian cancer cell line CoC1 was bought from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China). These cells were incubated in 1640 medium replenishing 10% fetal bovine serum (FBS, Gibco Company, Grand Island, NY), 10 μg/mL streptomycin, and 100 U/mL penicillin (Gibco) and placed in an incubator (Thermo Fisher Scientific Inc., Waltham, MA) at 37 °C with 5% CO2.

Some cells were treated with IGF1 (GF306, Sigma-Aldrich, St. Louis, MO) at concentrations of 0.1 nmoL, 1 nmoL and 10 nmoL, respectively. Logarithmically growing cells were trypsinized and seeded in a 6-well plate (1 × 105 cells/well) and cultured for 24 h. Upon75% confluence, the cells were treated with dimethyl sulfoxide, IGF1, negative control (NC) for short hairpin RNA (shRNA) (shNC), shRNA against IGF1 (shIGF1), and shWISP1 with the help of Lipofectamine 2000 (Invitrogen, Carlsbad, California). The plasmids of shNC, shIGF1, and shWISP1 were purchased from Sigma. The sequences of shRNA are shown in Supplementary Table 1.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted utilizing TRIzol reagent (15,596,026, Invitrogen) and reversely transcribed into complementary DNA (cDNA) with the help of a PrimeScript RT reagent Kit (RR047A, Takara, Japan). RT-qPCR was conducted by means of Fast SYBR Green PCR kit (Applied Biosystems, Carlsbad, CA) and ABI PRISM 7300 RT-PCR system (Applied Biosystems). Three replicates were prepared for each well. GAPDH was adopted as the normalizer for mRNA. The 2-ΔΔCt method was employed to quantify relative expression of target genes. The primers are summarized in Supplementary Table 2.

Cell proliferation analysis

Cell Counting Kit-8 (CCK-8) (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) was employed for testing cell viability. The absorbance at the wavelength of 452 nm was tested utilizing Microplate reader.

Scratch test

Cells were added into the 6-well plate (5 × 105 cells/well) and cultured overnight in serum-free medium. The horizontal lines were constructed evenly every 0.5–1 cm on the bottom surface of the 6-well plate using a sterile 10 μL pipette with the help of ruler and marker, at least five lines across each well. The images were gained under microscope and the cell migration was observed.

Transwell assay

Transwell invasion assay was implemented with a Transwell system (Corning, USA) pre-coated with Matrigel (BD Biosciences, San Jose, CA) [15]. The stained invasive cells were counted under an inverted light microscope (Carl Zeiss, Jena, Germany) in at least 5 randomly-selected fields.

Western blot analysis

Cells were lysed with enhanced radio immunoprecipitation assay lysis appended to protease inhibitor (BOSTER Biological Technology Co., Ltd., Wuhan, Hubei, China). The protein concentration was measured by the Bicinchoninic Acid Assay Kit (BOSTER). Following electrophoresis separation, the protein was transferred onto polyvinylidene difuoride membrane. After blocked with 5% bovine serum albumin for 2 h to inhibit non-specific binding, membranes were incubated with diluted primary antibodies (Supplementary Table 3) overnight at 4 °C. The membranes were then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (1: 2000) (Supplementary Table 3) at room temperature for 1 h. The membrane was developed with ECL working solution (EMD Millipore) for 1 min with the results analyzed with ImageJ software.

Co-immunoprecipitation (co-IP)

For co-IP, a certain amount of cell lysate was incubated with 30 μL Protein A&G Agarose and 1 μg rabbit IgG or IGF1 primary antibody at 4 °C overnight. After incubation, the supernatant was removed through centrifugation. The 0.5 M Nacl lysis buffer was used to rinse the lower layer twice, and then the sample was boiled at 97 °C for 7 min, followed by Western blot analysis. Antibodies used in the experiment are shown in Supplementary Table 3.

Xenograft model

Healthy female nude mice (aged 4–5 weeks) (Vital River Laboratories, Beijing, China) were raised in separate SPF animal laboratory with humidity of 60–65% at 22–25 °C. The mice were acclimated for one week before experiment.

The CAOV4 cell suspension (1 × 105 cells/100 μL) harboring NC for gene overexpression (OE-NC) or overexpression of WISP1 (OE-WISP1) was injected subcutaneously into the nude mice during the feeding process, followed by intraperitoneal injection of IGF1 antibody or phosphate buffered saline (PBS), once every three days. The tumor volume was measured by Vernier calipers on the 7th day, and then once every 7 days. The nude mice were euthanized after 5 weeks to calculate the tumor weight.

In order to monitor the lung metastasis, CAOV4 cell suspension (1 × 106 cells/100 μL) harboring OE-NC or OE-WISP1 was injected into the nude mice via tail vein during the feeding process, followed by intraperitoneal injection of IGF1 antibody or PBS, once every three days. After 4 weeks, lung metastasis was observed.

Hematoxylin and eosin (HE) staining

The HE staining kit (G1120, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was employed for this assay [16]. The changes of tumor morphology were observed under a light microscope.

Immunohistochemistry (IHC) assay

The sections to be tested were heated at 60 °C for 20 min, immersed in xylene for 15 min and rehydrated with 100, 95, 90, 85 and 80% ethanol. Each section was incubated with 3% H2O2 for 15 min at room temperature to remove endogenous peroxidase. The tissues were treated with 0.01 mol/L citrate buffer (pH 6.0) in microwave oven for 10 min for antigen retrieval. After cooling, the sections were sealed with 5% normal goat serum for 15 min at ambient temperature and incubated with antibodies against E-cadherin, ZO-1, N-cadherin and SNAIL at 4 °C overnight, followed by addition of biotinylated goat anti-rabbit IgG and incubation with HRP-streptomycin for 15 min. Subsequently, the sections were treated with diaminobenzidine solution for 3–5 min, counterstained with hematoxylin for 1–3 min, dehydrated, and sealed with neutral balm. The above steps were repeated with 0.1 mol/L PBS (pH 7.4) as NC. Supplementary Table 3 displays the detailed information for the used antibodies.

Statistical analysis

Data analysis was implemented utilizing the SPSS 21.0 software (IBM, Armonk, NY). All measurement data are concluded as mean ± standard deviation. Paired t-test was employed for comparisons between adjacent normal tissues and ovarian cancer tissues. The comparison between two groups was analyzed by independent sample t-test. For multiple independent groups, one-way analysis of variance (ANOVA) with post hoc Tukey’s test was used. Two-way ANOVA was utilized for the comparison of data at different time points (cell viability), and repeated measures ANOVA was employed for tumor volume analysis in combination with post hoc Bonferroni test. Pearson’s correlation analysis was implemented for testing the relationship between indexes. p < 0.05 considered significant.

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