Inhibition LC3B can increase chemosensitivity of ovarian cancer cells

116 cases of malignant ascites were obtained from the Third Affiliated Hospital of Harbin Medical University (Harbin, China) between January 2014 and May 2018. Of the 116 cases of ovarian cancer ascites, 64 cases were treated without chemotherapy, and 52 cases were treated with chemotherapy. We also acquired 60 samples of fresh ovarian cancerous tissue. Of the 60 cases of ovarian cancer tissue, 14 cases were treated without chemotherapy, and 46 cases were treated with chemotherapy. All cases had complete clinical and pathological data (Additional file 1: Table S1). According to the screening criteria for Ovarian cancer, version 3.2012. published by NCCN [14]. The patients who underwent chemotherapy were further divided into the chemosensitive (sensitive) group and the chemoresistant (resistant) group. A flow diagram describing the study and groups is shown in Additional file 2: Fig. S1 and Additional file 1: Table S2.

The supernatants of ovarian cancer ascites was stored at − 80 °C for subsequent analysis by ELISA. Then, moderate ACK lysis (Leagene Biotechnology, China) buffer was added to the precipitates, which were collected into 1.5-mL centrifuge tubes. Subsequently, RNA and protein analyzes were performed.

Four ovarian cancer cell lines (OVCAR3, SKOV3, A2780 and HO8910) and human embryonic kidney cell line (HEK-293TN) were obtained from the Cell Bank, China Academy of Sciences (Shanghai, China). The cells were maintained in DMEM, MycCoy’ 5A or RIPM1640 complete medium supplemented with 2 mM glutamine and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO₂. Through a cytotoxicity assay (Additional file 3: Fig. S2A), we found that A2780 (IC50 = 30 μM) and HO8910 (IC50 = 37.5 μM) cells were more sensitive to cisplatin and SKOV3 (IC50 = 45 μM) and OVCAR3 (IC50 = 45 μM) cells were less sensitive to cisplatin. Accordingly, for follow-up experiments, we considered A2780 and HO8910 cells to be cisplatin high sensitivity. OVCAR3 and SKOV3 cells to be cisplatin low sensitivity.

Twenty female BALB/c nude mice, 6–8 weeks old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were reared under aseptic conditions. Moreover, 5 × 10⁶ OVCAR3 cells stably expressing a luciferase reporter gene, were injected into the abdominal cavity of female BALB/c nude mice. Tumor formation was observed daily and recorded using the Bruker live imaging system (Bruker, Karlsruhe, Germany). After the tumor formation, 20 BALB/c nude mice with ovarian cancer were randomly divided into four groups: cisplatin group, cisplatin and miR-204 agomir group (miR-204 agomir), cisplatin and 3-methyladenine (3MA) group and cisplatin and miR-negative group with each group containing five mice. Drug injection occurred every 3 days for 3 weeks. Doses of miR-204 agomir and miR-negative (Ribobio, Guangzhou, China) were 5 nmol, 3MA (Selleck, Shanghai, China) was 20 nmol and cisplatin (Selleck, Shanghai, China) was 5 mg/kg. The mice were sacrificed after 3 weeks following the start of treatment conditions (Fig. 5a). The collected tissue was subjected to H&E staining, western blotting, immunofluorescence and other experiments. All animals experiments strictly complied with the various standards found in the “Regulations for the Management of Experimental Animals” of Harbin Medical University. The ethical certification number is HMUIRB20170022.

We utilized data from TCGA (311 samples) and GEO databases (189 samples from GSE17260, GSE32062, and GSE32063), which explored the association between LC3B and MDR1 mRNA expression in ovarian cancer, and access the effect of LC3B on ovarian cancer overall survival (OS) and progression free survival (PFS). All TCGA data were downloaded from the GDSC Broad Institute (http://​firebrowse.​org). GEPIA (http://​gepia.​cancer-pku.​cn) was used to access the effect of LC3B on pancancer OS.

Candidate miRNAs targeting LC3B were predicted using the TargetScan algorithm (http://​www.​targetscan.​org/​) and miRbase (http://​www.​mirbase.​org).

The expression level of miR-204 was measured in EOC samples and cells by qRT-PCR to determine the clinical implications of miR-204 expression in ovarian cancer. We also performed qRT-PCR to compare the expression of autophagy, apoptosis and resistance markers, including LC3B, Beclin1, caspase 3, caspase 9 and MDR1, in miR-204 mimic/inhibitor-transfected and negative control miR (miR-negative)-transfected cells. Total RNA was extracted from fresh tissues and cell lines using TRIzol reagent (Invitrogen, USA). Stem-loop qRT-PCR for mature miRNAs was performed using a LC96 Real-Time PCR Detection System. All PCR reactions were performed in triplicate and normalized using the comparative threshold method (2^−∆∆Ct). The sequences of primers used for quantitative PCR, which were synthesized by GENEWIZ, are listed in Additional file 1: Table S3.

A chemically modified RNA-based miRNA precursor, the miR-204 mimic/inhibitor, was purchased from Ribobio (Guangzhou, China). To transfect cells, 60 pmol of the miR-204 mimic/inhibitor was diluted in 250 ml serum-free RPIM-1640 medium with 5 ml Lipofectamine 3000 (Invitrogen, Carlsbad, CA). MiR-negative was used as a control. To validate the efficiency of transfection, miRNA expression was examined by qRT-PCR.

The 3′-untranslated region (UTR) of LC3B was PCR amplified from genomic DNA of HEK-293TN cells. Putative binding sites for miR-204 within LC3B 3′-UTR were identified using the TargetScan algorithm (targetscan.org). Wild-type or mutated binding sites were cloned separately into the Nhel and Xhol sites of the pGL3-control vector (Promega, USA). The pGL3-control (100 ng) and pRL-TK plasmids (5 ng; for normalization) were transfected into HEK-293TN cells seeded in 24-well plates (3 × 10⁴ cells/well). The synthetic miR-204 mimic was added to the above reactions at a concentration of 20–60 nM. Luciferase activity was measured after 48 h using an Infinite 200pro series luminometer (Tecan Group, Switzerland) with the Dual-Luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions. All experiments were performed in triplicate and normalized to Renilla luciferase activity.

Cell viability was assessed after adding different concentrations of cisplatin to cells according to the Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Japan) protocol. Absorbance was measured at 450 nm using a microplate reader.

The abundance of the LC3 protein in ovarian cancer ascites was measured by the ELISA using kits purchased from Elabscience (Wuhan, China).

Total protein from cells and ovarian cancer samples was extracted, and the concentration in the supernatant was measured using the Bradford assay (Sigma, USA). Equal amounts of total protein were separated by 15% sodium dodecy1 sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore Co. Bedford, MA, USA). After blocking with 5% skim milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies (Additional file 1: Table S4). The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibody (1:5000, Novus Biologicals) for 1 h at room temperature. Next, the bands were visualized using an enhanced luminol-based chemiluminescence detection kit (Bio-Rad Laboratories, Hercules, CA, USA). The protein levels were normalized to GAPDH as an internal control.

OVCAR3 cells in agarose were treated with 1% OsO4 solution for 1 h at 4 °C; this helped to provide an enhanced contrast for TEM images. Samples were dehydrated in a graded series of acetone, and then embedded in Epon 812. The samples were cut into ultrathin sections (50–70 nm in thickness), double-stained with uranyl acetate and lead citrate and examined with an electron microscope (H-7650, Hitachi, Japan). Images were transferred to Adobe Photoshop software for final processing.

The cells were fixed with 4% paraformaldehyde (PFA) for 30 min. The cells were then incubated overnight with corresponding primary antibodies. Next, the cells were incubated with secondary antibodies for 1 h at 37 °C. The cells were washed 3 times and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nuclei. Images were then obtained using a Nikon E800 Multifunctional Biological Microscope and Image Acquisition Software Nikon ACT 21 version 6.1.

Transwell chambers with 8-μm pores were obtained from Corning (Corning, USA). Transfected cells were harvested, resuspended in RPMI-1640 without FBS at a concentration of 1 × 10⁵ cells in 100 μl and then seeded into the upper chambers of a 24-well plate. The lower chambers were filled with 600 μl RPMI-1640 containing 10% FBS. The cells were incubated for 24 h. At the end of the experiment, cells that had migrated to the reverse side of the Transwell membrane were fixed with methanol, stained with crystal violet, and counted under a light microscope at 100× magnifications. An average of 10 visual fields was examined.

A wound-healing assay was used to assess cell migration. The two groups of cells transfected with miR-204 mimic/inhibitor or miR-negative were cultured to confluence (> 90%) in 6-well plates. A straight scratch wound was then generated with a 200-ml sterile pipette tip. After 24 h, the cells were photographed, and those migrating from the scratch line were counted. This assay was repeated three times.

Tumor tissues of BALB/c nude mice were routinely processed into paraffin blocks and then sectioned at a thickness of 4 to 6 μm. The sections were then stained with H&E.

All data were obtained based on a minimum of three experiments and were presented as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS software version 21.0. One-way analysis of variance (ANOVA) was used to analyze differences between groups. An independent t-test or u-test for differences in mean values was used for comparison. Comparison between two or multiple rates was performed using the Chi square test. Differences with a p value of < 0.05 were considered significant. Spearman correlations were used to find linear relationships between two variables. Kaplan–Meier analysis performed a survival analysis on the prognosis of ovarian cancer patients in TCGA and GEO databases. Univariate analyses with Cox regression were used to determine the proportional hazards. The difference was statistically significant. GraphPad Prism was used for mapping and curve fitting.

The levels of autophagy and apoptosis markers in 116 cases of ovarian cancer ascites were detected by ELISA. Compared with the non-chemotherapy group and the chemosensitive (sensitive) group, the concentrations of LC3B and Beclin1 in the chemoresistant (resistant) group were increased, and the concentrations of caspase 3 and 9 were decreased (Fig. 1a). The results of qRT-PCR and western blot showed that compared with the sensitive group, the levels of LC3B and Beclin1 in the resistant group were significantly increased, the expression of caspase 3 and 9 was decreased, and the expression of MDR1 in the resistant group was higher than that in the sensitive group (Fig. 1b–e). When expression of autophagy markers increased, the expression of apoptosis protease decreased. Spearman correlation analysis showed LC3B and MDR1 were positively correlated (Fig. 1f, p < 0.0001). Correlation analysis between LC3B and MDR1 was also positively correlated in ovarian cancer samples from TCGA and GEO databases (Additional file 3: Fig. S2B, p < 0.005). These results were suggesting that ovarian cancer resistance may be associated with LC3B.

The level of LC3B expression in the EOC may affect the response of tumor cells to chemotherapeutic drugs. We identify this hypothesis through ovarian tumor cell lines. By knocking down LC3B (LC3B⁻) in ovarian cancer cell lines, western blot showed that compared with the control group, the expression of LC3B, Atg7 and MDR1 was decreased in the LC3B⁻ group (Fig. 2a–c). CCK8 results showed that cisplatin had a higher inhibition rate in the LC3B⁻ group than in the control group, and the sensitivity of the LC3B⁻ group was significantly increased (Fig. 2d). The western blot results showed compared with the control group, the expression of apoptosis proteins caspase 3, 6, and 9 in the LC3B⁻ group was significantly increased (Fig. 2e, f). These results suggested that LC3B expression could affect the sensitivity of tumor cells to cisplatin. The expression of LC3B was decreased. The tumor cells were more sensitive to cisplatin.

The LC3B expression could effect on ovarian cancer resistance, but the causes leading to LC3B expression change in ovarian cancer were unknown. So we searched for computationally predicted candidate miRNAs using a web-based miRNA database (http://​www.​targetscan.​org). Among multiple candidate miRNAs with high potential binding capacity to the 3′-UTR of LC3B, four putative miRNAs (miR-204, miR-211 and miR-182, miR-212) were selected. Four miRNA levels were detected in ovarian cancer samples (ascites and tissues). The expression of miR-204 in the resistant group was significantly decreased in both tumor tissues and ascites cells (Additional file 3: Fig. S2C). It was speculated that LC3B expression may be related to miR-204 deletion in ovarian cancer. At the same time, the expression level of four miRNAs was also detected in ovarian cancer cell lines. The expression of miR-204 in A2780 was significantly higher than that in OVCAR3 (Additional file 3: Fig. S2D). Therefore, it was used miR-204 for subsequent experiments. The results showed that OVCAR3 was less sensitive to cisplatin and A2780 was more sensitive (Additional file 3: Fig. S2A). So subsequent in vitro validation was performed using OVCAR3 and A2780 cell lines.

Luciferase assay were performed to whether miR-204 directly targets the LC3B mRNA. The binding sites of miR-204 and LC3B are shown in Fig. 3a. Intact and mutated 3′UTRs of LC3B, were cloned into a luciferase reporter plasmid that was co-transfected with a miR-204 mimic into HEK 293TN cells. miR-204 reduced the luciferase activity of the 3′-UTR reporter of wild type LC3B, while that of the mutated reporter was not significantly affected (Fig. 3b and Additional file 1: Table S5). Transfection with the miR-204 mimic significantly downregulated LC3B (Fig. 3c, p < 0.001) and Atg7 (Fig. 3d, p < 0.001) expression in OVCAR3 cells and A2780 cells. Treatment of ovarian cancer cells with cisplatin showed a significant decrease expression of LC3B in the miR-204 mimic group (Fig. 3e) and increased expression of LC3B in the miR-204 inhibitor group (Fig. 3f). Immunofluorescent staining showed the co-localization of LC3B and LAMP2 in the miR-204 mimic group was significantly reduced, and the co-localization of LC3B and LAMP2 in the miR-204 inhibitor group was increased (Fig. 3g). Electron microscopy showed that OVCAR3 cells have large nuclei and abundant nucleosomes. In the miR-negative group, more lipid droplets were seen in the cytoplasm. In the miR-204 mimic group, there were large numbers of proliferating mitochondria. In the miR-204 inhibitor group, there were large number of autophagic lysosomes were seen in the cytoplasm (Fig. 3h). These data indicated that miR-204 directly targets the 3′-UTR of LC3B. It repressed the expression of the LC3B protein, and miR-204 could inhibit autophagy by targeting LC3B.

The results of ovarian cancer samples (tissues and ascites) showed apoptosis protease was decreased when LC3B expression was increased in the resistant group, while the apoptosis of ovarian cancer cells was increased when LC3B was inhibited. Due to miR-204 was directly targeted at LC3B. We hypothesized that the mechanism of LC3B expression decreased and apoptosis increased was related to miR-204. CCK8 results showed that the A2780 cells were less sensitive to cisplatin and increased cell viability in miR-204 inhibitor group (Fig. 4a and Additional file 4: Fig. S3A); the OVCAR3 cells were more sensitive to cisplatin and cell viability was significantly reduced in the miR-204 mimic group (Fig. 4b and Additional file 4: S3B). Western blot analysis showed that cleaved-caspase 3, caspase 6, caspase 9, p-Bad, Bax, Bak and cleaved-PARP were decreased andBcl-2, p-Akt and MDR1 were increased in miR-204 inhibitor group. Conversely, cleaved-caspase 3, caspase 6, caspase 9, p-Bad, Bax, Bak and cleaved-PARP were significantly increased and Bcl-2, p-Akt and MDR1 were decreased in miR-204 mimic group (Fig. 4c–h). Western gray value statistics are shown in Additional file 4: Fig. S3C-F. RT-PCR results showed that the mRNA level of caspase 3 and caspase 9 was increased in the miR-204 mimic group and decreased in the miR-204 inhibitor group compared with the control group (Additional file 4: Fig. S3G-H). TUNEL assay showed that a decrease in the number of apoptosis cells in the miR-204 inhibitor group compared to the control group (Fig. 4i); whereas the number of apoptosis cells in the miR-204 mimic group increased (Fig. 4j). Above all, miR-204 could inhibit LC3B and promote apoptosis at the same time in ovarian cancer cells.

In vivo experiments were performed on a nude mouse model of intraperitoneal tumors. As showed in Fig. 5b, the miR-204 agomir group had a slower tumor growth rate than the other three groups. ROI values result is shown in Additional file 3: Figure S2E. H&E staining results showed that apoptosis necrosis occurred in many tumor cells in miR-204 agomir group and 3MA group. In cisplatin alone and the control group, tumor cells showed less apoptosis and a small amount of vacuoles with inflammation (Fig. 5c). Western Blot assays showed that compared with the control group and the cisplatin group, LC3B and Beclin1 were lower in miR-204 agomir and 3MA groups, cleaved-caspase 3, 6, and 9 were increased, and MMP2 and MMP9 were decreased. Compared with the other three groups, P110 and MDR1 were significantly decreased in the miR-204 agomir group (Fig. 5d). The results of double immunofluorescence staining showed that the 3MA and miR-204 agomir groups had fewer co-localization, and the other two groups had more co-localization, indicating that autophagy was decreased in the 3MA and miR-204 agomir groups (Fig. 5e). TUNEL assay showed that compared with the other two groups, miR-204 agomir and 3MA group had increased apoptosis, and apoptosis in the miR-204 agomir group was more significant (Fig. 5f).

Above results indicate that miR-204 agomir and 3MA can inhibit tumor autophagy. When tumor autophagy was inhibited, apoptosis also changed. Compared with the 3MA group, apoptosis was more obvious in the miR-204 agomir group. It indicated that miR-204 could inhibit autophagy,promote tumor cell apoptosis and increase the sensitivity of tumor cells to cisplatin.

In vivo studies showed that MMP2 and MMP9 were decreased in miR-204 agomir group and 3MA group. MMP2 and MMP9 were members of metal matrix protease family, which was closely related to tumor migration. The results in vivo suggested that inhibition of autophagy could effect on tumor migration. Therefore, the effects of LC3B expression on migration and invasion in ovarian cancer cells were verified by Transwell and wound-healing experiments. Transwell assay showed that the cell migration rate of the miR-204 mimic group was decreased compared with the control group. After the addition treatment of cisplatin, the migration rate of the miR-204 mimic group was significantly decreased. While the migration rate of the miR-204 inhibitor group was increased compared to the control group, and after treatment with cisplatin, the cell migration rate was significantly increased (Fig. 6a, c). The results of wound-healing assays were consistently with transwell assays (Fig. 6b, d). These results indicated miR-204 inhibited LC3B expression could affect migration in ovarian cancer cell.

To identify clinical application value of LC3B, we analyzed the effect of LC3B on overall survival (OS) of ovarian cancer patients based on a set of TCGA data, including ovarian cancer patients (n = 311) receiving platinum treatment. The result indicated that patients with low levels of LC3B exhibit a significantly longer survival time compared with those with high levels of LC3B (Fig. 7a). We also performed univariate analysis using Cox proportional hazard regression. High LC3B expression was associated with a worse OS, and low expression was associated with improved OS (high vs. low by quartile, HR of death = 1.655, 95% CI 1.048–2.614, p = 0.031). At the same time, 189 ovarian cancer samples obtained from 3 GEO databases were analyzed. The effect of LC3B expression on the overall survival of patients was consistent with results of TCGA data (Fig. 7b, high vs. low by median, HR of death = 1.567, 95% CI 1.035–2.373, p = 0.034). We also analyzed the effect of LC3B on PFS of patients based on TCGA data. The results showed that patients with low LC3B expression had longer PFS than those with high LC3B expression (Fig. 7c, p = 0.0257). In addition, LC3B was associated with the consequence of platinum treatment in ovarian cancer patients (Fig. 7d). Compared with the high expression group, low level of LC3B (76.1% VS% 66.9%) had significantly more complete platinum response. These results indicated that LC3B could be used as a prognostic indicator of ovarian cancer, and LC3B could also play such a role in the other cancer. We used GEPIA website (http://​gepia.​cancer-pku.​cn) to analyze multiple cancer samples in TCGA database. The results showed that the prognosis of low LC3B expression group was better in squamous cell lung cancer, colon cancer and esophageal cancer (Additional file 4: Fig. S3I). Analysis of pancancer samples in TCGA (n = 9502) showed that low LC3B expression group had a better prognosis (Fig. 7e). Above all, it was suggested that LC3B could be a biomarker for predicting the prognosis of cancer.