LINC00852 promotes the proliferation and invasion of ovarian cancer cells by competitively binding with miR-140-3p to regulate AGTR1 expression

Eighty-five ovarian cancer tissue and adjacent normal tissue samples were collected from ovarian cancer patients who underwent surgical resection at Cancer Hospital of China Medical University. Written informed consent was obtained from all patients. Tumor samples were confirmed by two pathologists independently and immediately frozen in liquid nitrogen. No patients received preoperative chemotherapy or radiotherapy. This study was approved by the Ethics Committee of Cancer Hospital of China Medical University.

Normal human ovarian epithelial cell line IOSE80 and human ovarian cancer cell lines (A2780, SKOV-3, OV-90, and CAOV3) were used in this study. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, CA, USA) or Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, CA, USA), 2% sodium pyruvate (Gibco, CA, USA), 1% penicillin-streptomycin (Gibco, CA, USA) in an incubator containing 5% CO₂ at 37 °C.

The vectors used for LINC00852 knockdown (sh, short hairpin)-LINC00852-1, sh-LINC00852-2, and sh-LINC00852-3), LINC00852 negative control (sh-NC), LINC00852 overexpression (oe-LINC00852 vecter was constructed by LINC00852 inserting into pcDNA3.1) and its negative control (oe-NC), miR-140-3p overexpression (miR-140-3p mimic) and its negative control (mimic NC), miR-140-3p knockdown (miR-140-3p inhibitor) and its negative control (inhibitor NC), and AGTR1 knockdown (sh-AGTR1) were synthesized by RiboBio (Guangdong, China). Transfection experiments were conducted using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions.

SKOV-3 and OV-90 cells with different transfections were seeded into 96-well plates at a concentration of 1.5 × 10⁴/mL. Forty-eight hours later, SKOV-3 and OV-90 cells were added with CCK-8 solution (10 μL; Sigma-Aldrich, MI, USA) and incubated for 2 h. The absorbance was measured at 450 nm by a microplate reader (Bio-Rad, CA, USA) at 24 h, 48 h and 72 h.

SKOV-3 and OV-90 cells with different transfections were seeded into 6-well plates at a concentration of 200 cells per well and cultured for 14 days. Then, SKOV-3 and OV-90 cell colonies were fixed with 80% methanol for 30 min and stained with 0.25% crystal violet at room temperature for 30 min.

The apoptosis of SKOV-3 and OV-90 cells with different transfections was confirmed by Hoechst 33342 staining according to previous report [25]. SKOV-3 and OV-90 cells were seeded in culture dish, added with 10 μL Hoechst 33342 solution (Beyotime Biotechnology, Nantong, China), and cultured for 10 min at 25 °C. A fluorescence microscopy (Olympus, Tokyo, Japan) was used to observe the changes in morphology of SKOV-3 and OV-90 cells (chromatin condensation, fragmentation and cell shrinkage). The apoptotic cancer cells were counted from 400 cells in 12 fields/well, and apoptosis rate (%) = apoptotic cancer cells/total cancer cells× 100.

Mitomycin C (10 g/ml) was added to the cell culture medium to inhibit cell replication, which ruled out the changes in intercellular space caused by cell proliferation or apoptosis [26]. SKOV-3 and OV-90 cells (2 × 10⁵ cells/mL) were added to the upper chambers of Transwell (8-μm-diameter pore membrane; Corning, NY, USA) coated with Matrigel. SKOV-3 and OV-90 cells were allowed to invade for 24 h, and cotton swabs were used to scrub cancer cells that did not penetrated the filters. Then, chambers were fixed with 4% paraformaldehyde (Beyotime Biotechnology, Nantong, China) for 2 min, stained with 0.3% crystal violet (Beyotime Biotechnology, Nantong, China) for 2 min and observed under a light microscope (Olympus, Tokyo, Japan).

Total RNAs were extracted from tumor tissues and ovarian cancer cells (SKOV-3 and OV-90 cells) using Trizol Reagent (Invitrogen, CA, USA). Complementary DNA (cDNA) was synthesized using High-Capacity RNA-to-cDNA Kit (Applied Biosystems, CA, USA) according to the manufacturer’s instruction. qRT-PCR reactions were conducted on an ABI StepOne Real-time PCR System (Applied Biosystems, CA, USA) using PowerUp SYBR Green Master Mix with the primers as the following: LINC00852 forward, 5′-CGTTGCCTACAGTCAAGTCAGT-3′, reverse, 5′-GCCATGGTTCCCTTACTGATAC-3′; miR-140-3p forward, 5′-ACACTCCAGCTGGGAGGCGGGGCGCCGCGGGA-3′, reverse, 5′-CTCAACTGGTGTCGTGGA-3′; AGTR1 forward, 5′-CCTCAGATAATGTAAGCTCATCCAC-3′, reverse, 5′-GCTGCAGAGGAATGTTCTCTT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-TGTTCGTCATGGGTGTGAAC-3′, reverse, 5′-ATGGCATGGACTGTGGTCAT-3′. Relative gene expressions of LINC00852, miR-140-3p and AGTR1 were calculated using the2^-ΔΔCt method and normalized by GAPDH or U6.

Proteins were extracted from tumor tissues and ovarian cancer cells (SKOV-3 and OV-90 cells) using RIPA lysis and extraction buffer (ThermoFisher Scientific, CA, USA). The concentration of proteins was measured using BCA protein assay kit (ThermoFisher Scientific, CA, USA). Proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (PVDF; Invitrogen, CA, USA). The blots were blocked with 5% skimmed milk, and incubated with primary antibodies overnight at 4 °C: MMP-2 (1:1000; Abcam, Cambridge, UK), MMP-9 (1:5000; Abcam, Cambridge, UK), Ki67 (1:1000; Abcam, Cambridge, UK), PCNA (1:5000; Abcam, Cambridge, UK), MEK (1:1000; Cell Signaling Technology, MA, USA), p-MEK (1:1000; Cell Signaling Technology, MA, USA), ERK1/2 (1:1000; Abcam, Cambridge, U), p-ERK1/2 (1:1000; Cell Signaling Technology, MA, USA), STAT3 (1:1000; Cell Signaling Technology, MA, USA), p-STAT3 (1:2000; Cell Signaling Technology, MA, USA), GAPDH (1:1000; Cell Signaling Technology, MA, USA). The blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technologies, MA, USA), visualized by an enhanced chemiluminescence kit (ThermoFisher Scientific, CA, USA) and normalized to GAPDH expression. The membranes were firstly cut, and then stained with antibodies. Each complete membrane has GAPDH loading control. The bands were photographed with a ChemiDoc™XRS Imaging System (Bio-Rad, CA, USA) and quantified by Quantity One software (Bio-Rad, CA, USA).

The enzymatic activity of MMP-2 and MMP-9 in SKOV3 and OV-90 cells was conducted with gelatine zymography. Proteins (20 μg) extracted from ovarian cancer cells (SKOV-3 and OV-90 cells) were used for each zymographic assay. Under non-reducing condition, mono-dimensional gelatine zymography was conducted on SDS-PAGE (7.5%) copolymerized with gelatine (0.1%). After electrophoresis, SDS was removed from the gels and zymograms were developed for 18 h at 37 °C. Gels were then stained with Coomassie Brilliant Blue for 30 min at 25 °C and visualized after destaining in methanol/acetic acid/H₂O.

FISH was conducted according to previous report [27]. Digoxin (Sigma-Aldrich, MI, USA)-labeled LINC00852 complementary DNA probe was synthesized in vitro. SKOV-3 cells were grown on the slides. After washed with phosphate buffer solution (PBS) for three times, SKOV-3 cells grown on the slides were fixed with 4% paraformaldehyde (Beyotime Biotechnology, Nantong, China). Then, the slides were treated with protease reagent (Invitrogen, CA, USA) and hybridized with digoxin-labeled LINC00852 probe for 12 h at 40 °C. Images were observed by a confocal microscope (Olympus, Tokyo, Japan) and the magnification of images was 400 × objective.

The luciferase reporter gene assay was conducted according to previous report [28]. The sequences of miR-140-3p or AGTR1 3′-UTR was sub-cloned into pGL3 luciferase reporter vectors (Promega, WI, USA). SKOV-3 cells were seeded into 48-well plates at a concentration of 3 × 10⁴ cells/well, and transfected with wide type miR-140-3p vector (pGL3-miR-140-3p-wt) or mutant miR-140-3p vector (pGL3-miR-140-3p-mut), wide type 3′-UTR of AGTR1 vector (pGL3-AGTR1-wt), mutant 3′-UTR of AGTR1 vector (pGL3-AGTR1-mut) using Lipofectamine 2000 reagent (Invitrogen, CA, USA). Forty-eight hours later, luciferase activity was detected by dual luciferase reporter assay system (Progema, WI, USA).

RIP assay was conducted as previously described [29]. SKOV-3 cells (2 × 10⁷) were collected to perform RIP assay using an AGO2 antibody (Millipore, MA, USA). AGO2 antibody (5 μg) for each RIP was used in RIP assay, and normal rabbit IgG was used as negative control. The co-precipitated RNAs were isolated and detected by qRT-PCR.

SKOV-3 cells were transfected with biotin-labeled wild-type miR-140-3p probe (bio-miR-140-3p-wt), biotin-labeled mutant miR-140-3p probe (bio-miR-140-3p-mut) or negative control probe (bio-probe NC). After 48 h, SKOV-3 cells were collected and lysed using RIPA lysis and extraction buffer (ThermoFisher Scientific, CA, USA). Then, cell lysates were incubated with Streptavidin magnetic beads (Pierce, CA, USA) for 12 h at 4 °C. After elution of Streptavidin magnetic beads, the bound RNAs were detected using qRT-PCR.

Ovarian cancer xenograft mouse model was established by subcutaneously injecting BALB/c nude mice (4–5 weeks, weight 20 ± 0.5 g, female) with 1 × 10⁷ SKOV-3 cells transfected with sh-LINC00852 or sh-NC (sh-LINC00852 and sh-NC groups), with five mice in each group. For control group, BALB/c nude mice were injected with SKOV-3 cells without transfection. BALB/c nude mice were obtained from Charles River (Beijing, China), and kept in individual cages with standard chow and ad libitum access to drinking water under a sterile condition. Tumor volumes were assessed by measuring the length and width of the tumor with calipers (tumor volume [mm³] = 0.5 × length×width²). The length and width were detected between 09.00 am to 10.00 am and testing order was randomized daily by two investigators who are unaware of grouping. Six weeks later, all mice were euthanized by an overdose of 100 mg/kg sodium pentobarbital through intravenous injection. And the tumors were excised, photographed and weighed. The animal experiment was approved by the Animal Care and Use Committee of Cancer Hospital of China Medical University.

Tumor tissue sections were de-paraffinized and rehydrated using descending concentrations of ethanol. Then, tumor tissue sections were added with 3% H₂O₂ for 30 min and blocked with 1% FBS for 1 h. After that, tumor tissue sections were incubated with anti-MMP-2 (1:100; Abcam, Cambridge, UK), anti-MMP-9 (1:1000; Abcam, Cambridge, UK), anti-Ki67 (1:1000; Abcam, Cambridge, UK) and anti-PCNA (1:200; Abcam, Cambridge, UK) overnight at 4 °C. Then, the sections were washed with PBS and incubated with secondary antibodies (1:500; Abcam, Cambridge, UK) for 1 h at room temperature. Finally, tumor tissue sections were stained with 3,3′-diaminobenzidine (DAB) staining solution and observed by a fluorescence microscope (Nikon, Tokyo, Japan).

SKOV-3 cells (1 × 10⁶/0.2 ml) transfected with sh-LINC00852-1 or sh-NC were injected into the tail veins of 15 BALB/c nude mice (4–5 weeks, weight 20 ± 0.5 g, female). BALB/c nude mice were obtained from Charles River (Beijing, China), and kept in individual cages with standard chow and ad libitum access to drinking water under a sterile condition. For control group, BALB/c nude mice were injected with SKOV-3 cells without transfection. There were five mice in each group. The mice were sacrificed 4 weeks after the inoculation and lungs were removed. Then, metastatic nodules were counted macroscopically.

GraphPad Prism 5.0 was used for data analysis. Data were expressed as mean ± standard deviation (SD). Comparisons for two groups were analyzed by Student’s t-test. Comparisons for multiple groups were analyzed by one-way analysis of variance ANOVA followed by Bonferroni post hoc test. P value less than 0.05 was considered statistically significant.

As shown in Fig. 1a, LINC00852 expression was significantly up-regulated in ovarian cancer tissues than adjacent normal tissues. Besides, LINC00852 expression was significantly up-regulated in human ovarian cancer cells (A2780, SKOV-3, OV-90 and CAOV3) than normal human ovarian epithelial cells IOSE80, with highest LINC00852 expression in SKOV-3 cells (Fig. 1b). Importantly, the analysis of LINC00852 expression and clinicopathological characteristics of ovarian cancer showed that LINC00852 expression was correlated with TNM stage and lymph node metastasis (Table 1).

Since SK-OV3 and OV-90 cells are commonly used in the study of the growth and invasion in ovarian cancer [30‐33] and LINC00852 expression is highly expressed in SKOV-3 and OV-90 cells, we use SKOV-3 and OV-90 cells for the following experiments. After SKOV-3 and OV-90 cells transfected with sh-LINC00852-1, sh-LINC00852-2 and sh-LINC00852-3, LINC00852 expression was significantly down-regulated in SKOV-3 and OV-90 cells (Fig. 2a), and results showed that sh-LINC00852-1 obtained the best transfection efficiency (Fig. 2a). The viability of SKOV-3 and OV-90 cells was significantly inhibited in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2b). The colony formation ability of SKOV-3 and OV-90 cells was suppressed in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2c). The apoptosis rate of SKOV-3 and OV-90 cells was significantly increased in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2d). The invasion rate of SKOV-3 and OV-90 cells was significantly decreased in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2e). Moreover, protein levels of invasion markers MMP-2, MMP-9, proliferation markers Ki67 and PCNA in SKOV-3 and OV-90 cells were significantly down-regulated in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2f and g), whereas apoptosis markers cleaved caspase-3 and cleaved PARP in SKOV-3 and OV-90 cells were significantly up-regulated in sh-LINC00852-1 and sh-LINC00852-2 groups than control and sh-NC groups (Fig. 2f and g). These findings indicated that sh-LINC00852 inhibited the proliferation and invasion of ovarian cancer cells. According to gelatine zymography analyses, enzymatic forms of MMP-2 and MMP-9 were reduced in sh-LINC00852 group than control and sh-NC groups (Fig. 2h).

According to the online bioinformatics software (http://​lncatlas.​crg.​eu), we found that LINC00852 localized mainly in the cytoplasm (Fig. 3a). FISH assay further confirmed the subcellular localization of LINC00852 in the cytoplasm (Fig. 3b). In addition, bioinformatics software (Starbase) predicted the binding sites between LINC00852 and miR-140-3p (Fig. 3c). Dual luciferase reporter gene assay showed that LINC00852 significantly decreased the luciferase activity of miR-140-3p-wt, whereas LINC00852 did not change the luciferase activity of miR-140-3p-Mut (Fig. 3d). In addition, RIP assay showed LINC00852 was enriched in Ago2 group than IgG group, indicating LINC00852 can bind with Ago2 (Fig. 3e). Compared with bio-miR-140-3p-mut or bio-probe NC, LINC00852 can be enriched by bio-miR-140-3p-wt (Fig. 3f).

As shown in Fig. 4a, miR-140-3p expression was significantly down-regulated in ovarian cancer tissues than adjacent normal tissues. Compared with oe-NC + mimic NC group, miR-140-3p expression in SKOV-3 cells was significantly up-regulated in oe-NC + miR-140-3p mimic group, and miR-140-3p expression was further down-regulated in oe-LINC00852 + miR-140-3p mimic group compared with oe-NC + miR-140-3p mimic group (Fig. 4b). Besides, the viability of SKOV-3 cells was significantly inhibited in oe-NC + miR-140-3p mimic group compared with oe-NC + mimic NC group, and the viability of SKOV-3 cells was further promoted in oe-LINC00852 + miR-140-3p mimic group compared with oe-NC + miR-140-3p mimic group (Fig. 4c). The colony formation ability of SKOV-3 cells was significantly inhibited in oe-NC + miR-140-3p mimic group than oe-NC + mimic NC group, and the colony formation ability of SKOV-3 cells was further promoted in oe-LINC00852 + miR-140-3p mimic group compared with oe-NC + miR-140-3p mimic group (Fig. 4d). The apoptosis of SKOV-3 cells was significantly facilitated in oe-NC + miR-140-3p mimic group compared with oe-NC + mimic NC group, and the apoptosis of SKOV-3 cells was further suppressed in oe-LINC00852 + miR-140-3p mimic group compared with oe-NC + miR-140-3p mimic group (Fig. 4e). The invasion of SKOV-3 cells was significantly inhibited in oe-NC + miR-140-3p mimic group compared with oe-NC + mimic NC group, and the invasion of SKOV-3 cells was further promoted in oe-LINC00852 + miR-140-3p mimic group compared with oe-NC + miR-140-3p mimic group (Fig. 4f). In addition, miR-140-3p mimic significantly down-regulated protein levels of invasion markers MMP-2, MMP-9, proliferation markers Ki67 and PCNA, and promoted protein levels of apoptosis markers cleaved caspase-3 and cleaved PARP in SKOV-3 cells, and oe-LINC00852 + miR-140-3p mimic further reversed protein levels of these invasion markers (Fig. 4g and h). According to gelatine zymography analyses, enzymatic forms of MMP-2 and MMP-9 were reduced in oe-NC + miR-140-3p mimic group than oe-NC + mimic NC group, and enzymatic forms of MMP-2 and MMP-9 were increased in oe-LINC00852 + miR-140-3p mimic group than oe-NC + miR-140-3p mimic group (Fig. 4i).

According to the prediction of bioinformatics software (Starbase), AGTR1 was a target of miR-140-3p (Fig. 5a). Dual luciferase reporter gene assay showed that miR-140-3p mimic significantly decreased the luciferase activity of AGTR1-Wt, whereas miR-140-3p mimic did not change the luciferase activity of AGTR1-mut (Fig. 5b). Different transfection experiments showed that LINC00852 could negatively regulate miR-140-3p expression and positively regulate AGTR1 expression (Fig. 5c). Importantly, loss- and gain-of-function experiments showed that protein levels of phosphorylation MEK (p-MEK), p-ERK1/2 and p-STAT3 in SKOV-3 cells were significantly down-regulated in oe-LINC00852 + miR-140-3p mimic group than oe-LINC00852 + mimic-NC group (Fig. 5d). Protein levels of p-MEK, p-ERK1/2 and p-STAT3 in SKOV-3 cells were significantly up-regulated in sh-LINC00852 + miR-140-3p inhibitor group than sh-LINC00852 + inhibitor NC group (Fig. 5d). Protein levels of p-MEK, p-ERK1/2 and p-STAT3 in SKOV-3 cells were significantly down-regulated in miR-140-3p inhibitor+sh-AGTR1 group than miR-140-3p inhibitor+sh-NC group (Fig. 5d). Moreover, we found AGTR1 expression level was significantly up-regulated in ovarian cancer cells (SKOV-3, OV-90 and CAOV3) than normal human ovarian epithelial cells IOSE80 (Fig. 5e). In in vivo model, tumor volume was significantly decreased in sh-AGTR1 group than sh-NC group (Fig. 5f and g). Besides, tumor weight was significantly reduced in sh-AGTR1 group than sh-NC group (Fig. 5h). These findings indicated that LINC00852 sponged miR-140-3p to promote AGTR1 expression, thereby facilitating the activation of MEK/ERK/STAT3 pathway.

As shown in Fig. 6a and b, tumor volume was significantly decreased in sh-LINC00852-1 group than sh-NC group, whereas there was no significant difference between control and sh-NC groups. Besides, tumor weight was significantly inhibited in sh-LINC00852-1 group than sh-NC group, whereas there was no significant difference between control and sh-NC groups (Fig. 6c). In addition, there was no significant difference in the body weight of mice among control, sh-NC and sh-LINC00852-1 groups (Fig. 6d). LINC00852 expression in tumor tissues was significantly down-regulated in sh-LINC00852-1 group than sh-NC group, whereas there was no significant difference between control and sh-NC groups, and miR-140-3p expression in tumor tissues was significantly up-regulated in sh-LINC00852-1 group than sh-NC group, whereas there was no significant difference between control and sh-NC groups (Fig. 6e). Moreover, protein levels of AGTR1, p-MEK, p-ERK1/2 and p-STAT3 in tumor tissues were significantly down-regulated in sh-LINC00852-1 group than sh-NC group (Fig. 6f). IHC staining showed the expressions of invasion marker MMP-2, MMP-9, proliferation markers Ki67 and PCNA were inhibited in sh-LINC00852-1 group than sh-NC group (Fig. 6g). Lung metastasis assay showed that sh-LINC00852-1 treatment attenuated lung metastasis of SKOV-3 cells compared with sh-NC group, whereas there was no significant difference between control and sh-NC groups (Fig. 6h and i).