Cell culture, transfection, and treatment of human ovarian cancer cells were performed as described previously (19). Ovarian cancer cells SKOV3 and A2780 were purchased from Procell Life Science & Technology Co., Ltd. 293T cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. DDP was purchased from Dalian Meilun Biology Technology Co., Ltd. Cells were maintained in Minimum Essential Medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA) in a 37°C incubator containing 5% CO₂. The transfection of miR-576-3p mimic, negative control (NC) mimic, miR-576-3p inhibitor, and NC inhibitor (100 pmol) into cells (at 90% confluence) was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. At 24 h post-transfection, cells were used for subsequent experiments. The sequences of the mimics and inhibitors were as follows: miR-576-3p mimic forward, 5′-AAGAUGUGGAAAAAUUGGAAUC-3′ and reverse, 5′-UUCCAAUUUUUCCACAUCUUUU-3′; NC mimic forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT−3′; miR-576-3p inhibitor, 5′-GAUUCCAAUUUUUCCACAUCUU-3′; NC inhibitor, 5′-UUGUACUACACAAAAGUACUG−3′. The sequences were purchased from JTS Scientific Biological Technology Co., Ltd.

DDP-resistant ovarian cancer cell lines SKOV3/DDP and A2780/DDP were established by culturing SKOV3 and A2780 cells with increasing concentrations of DDP. Briefly, SKOV3 and A2780 cells were resuscitated and cultured in an incubator. At 60% confluence, cells were treated with DDP at concentrations of 1, 2, 4, and 8 µM. Once the cells were incubated for 48 h, the medium was discarded and fresh medium was added. After 12 weeks, stable DDP-resistant SKOV3/DDP and A2780/DDP cells were successfully established and stored in liquid nitrogen.

Cells were seeded into 96-well plates at a cell count of 5×10³ per well. At 24 h after transfection, medium (200 µl) containing different concentrations of DPP (5, 10, 20, 40, and 80 µM) was added to each well (23,24). After the cells were incubated for 48 h at 37°C and 5% CO₂ MTT mixture (0.5 mg/ml) was added into each well, and cells were then incubated at 37°C for 5 h. Subsequently, DMSO solution (150 µl) was added to dissolve the purple formazan. After 10 min of incubation in the dark, the optical density value of cells at a wavelength of 570 nm was measured and the half-maximal inhibitory concentration (IC₅₀) was calculated.

Total RNA was extracted from tissues or cells using a total RNA extraction kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. The RNA concentration in each sample was detected by ultraviolet spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific, Inc.). cDNA was reverse-transcribed from RNA using RNase inhibitor (BioTeke Corporation) and Super M-MLV Reverse Transcriptase (BioTeke Corporation). The following temperature protocol was used for reverse transcription for miR-576-3p: 37°C for 30 min, 42°C for 30 min and 70°C for 10 min. The following temperature protocol was used for reverse transcription for cyclin D1 and PD-L1: 25°C for 10 min, 42°C for 50 min and 80°C for 10 min. Subsequently, RT-qPCR analysis was performed with primers, SYBR-Green (Sigma-Aldrich; Merck KGaA) and 2X Power Taq PCR Master Mix (BioTeke Corporation) to detect miR-576-3p expression level. The following thermocycling conditions were used for qPCR for miR-576-3p: 94°C for 4 min, 40 cycles of 94°C for 15 sec, 60°C for 20 sec and 72°C for 15 sec. The following thermocycling conditions were used for qPCR for cyclin D1 and PD-L1: 94°C for 5 min, 40 cycles of 94°C for 15 sec, 60°C for 25 sec and 72°C for 30 sec. The primers used for qPCR were as follows: miR-576-3p forward, 5′-AAGATGTGGAAAAATTGGAATC-3′ and reverse, 5′-GCAGGGTCCGAGGTATTC-3′; 5S rRNA forward, 5′-GATCTCGGAAGCTAAGCAGG-3′ and reverse, 5′-TGGTGCAGGGTCCGAGGTAT-3′; cyclin D1 forward, 5′-GCGAGGAACAGAAGTGCG−3′ and reverse, 5′-TGGAGTTGTCGGTGTAGATGC−3′; PD-L1 forward, 5′-AACTACCTCTGGCACATC−3′ and reverse, 5′-ATCCATCATTCTCCCTTT-3′; β-actin forward, 5′-GGCACCCAGCACAATGAA−3′ and reverse, 5′-TAGAAGCATTTGCGGTGG-3′. The primers were purchased from GenScript. The expression of miR-576-3p, and cyclin D1 and PD-L1 was calculated and normalized to 5S rRNA and β-actin, respectively. Gene expression was quantified using the 2^−∆∆Cq method (25).

Total protein was extracted from tissues or cells using IP cell lysis buffer (Beyotime Institute of Biotechnology) and phenylmethylsulphonyl fluoride protease inhibitor (Beyotime Institute of Biotechnology). Protein concentration was quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Proteins (20 µl/lane) from each sample were separated via SDS-PAGE (8 and 13% separation gel for different sized proteins) and then transferred to a PVDF membrane (EMD Millipore). After blocking in 5% skimmed milk powder for 1 h at room temperature the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies for 2 h at room temperature (1:5,000; cat. nos. A0208 and A0216; Beyotime Institute of Biotechnology). The primary antibodies used were as follows: PD-L1 (1:1,000; cat. no. DF6526; Affinity Biosciences), cyclin D1 (1:1,000; cat. no. AF0931; Affinity Biosciences), efflux pump multidrug resistance protein 1 (MDR1; 1:1,000; cat. no. AF5185; Affinity Biosciences), pro/cleaved caspase-3 (1:1,000; cat. no. DF6879; Affinity Biosciences), pro/cleaved poly (ADP-ribose) polymerase (PARP; 1:1,000; cat. no. #9532; Cell Signaling Technology, Inc.), and β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). Finally, the membranes were visualized with ECL luminescent reagent (Beyotime Institute of Biotechnology) and the optical density values of the target bands were analyzed using Gel-Pro-Analyzer software (version 4; Media Cybernetics, Inc.). β-actin served as an internal control.

Cells were incubated in 6-well plates at a density of 2×10⁵ cells/well. After transfection with miR-576-3p mimic or NC mimic, cells were treated with DDP at 50% of the IC₅₀ dose at 37°C for 24 h. The treated cells were harvested and fixed in 70% ethanol at 4°C for 2 h, followed by incubation with propidium iodide (PI; 25 µl) and RNase A (10 µl) at 37°C for 30 min in the dark. Finally, the cell cycle distribution was detected by flow cytometry using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress software (version 1.2.5; ACEA Biosciences, Inc.).

Both early and late apoptotic cells were assessed. Cells were seeded in 6-well plates at a density of 2×10⁵ cells/well. After transfection, cells were treated with DDP at 50% of the IC₅₀ dose at 37°C for 24 h. Subsequently, cells were treated with 5 µl Annexin V-fluorescein isothiocyanate (FITC) and 10 µl PI for 20 min at room temperature in the dark. Finally, cells stained with V-FITC and PI were detected by flow cytometry using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress (version 1.2.5; ACEA Biosciences, Inc.).

The binding sites between miR-576-3p and PD-L1 and cyclin D1 were determined using TargetScan (version 7.2; http://www.targetscan.org/vert_72/). PD-L1 and cyclin D1 fragments containing the target sequence of miR-576-3p were inserted into pmirGLO vectors (GenScript) to construct wild-type (wt) and mutant (mut) plasmids. Subsequently, the plasmids were co-transfected with miR-576-3p mimic, NC mimic, miR-576-3p inhibitor or NC inhibitor into 293T cells (at 90% confluence) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, luciferase activity was detected using a Dual-Luciferase Detection kit (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase activities.

The present study was approved by the Institutional Animal Care and Use Committee of Affiliated Yantai Yuhuangding Hospital (approval no. 2018137; Shandong, China) and performed according to the Guidelines for the Care and Use of Laboratory Animals (26). The feeding and treatment of mice were performed as described in our previous study (19). Briefly, 12 healthy female BALB/c nude mice (weight, ~20 g) aged 5–6 weeks were given free access to food and water and fed adaptively for 1 week. Mice were randomly divided into two groups: SKOV3/DDP + LV-NC + DDP group and SKOV3/DDP + LV-miR-576-3p + DDP group (n=6/group). The construction of SKOV3/DDP cells was performed as aforementioned. The nude mice were subcutaneously injected (the right side of the back of the neck) with lentivirus (LV) miR-576-3p infected SKOV3/DDP cells (2×10⁶ in PBS) or LV control-infected SKOV3/DDP cells (2×10⁶ in PBS). The tumor was confirmed by a pathologist. When the tumor volume reached 100 mm³, all nude mice were intraperitoneally injected with DDP (10 mg/kg) twice a week for 3 weeks. Tumor size was monitored every 2 days. After 19 days, nude mice were sacrificed by cervical dislocation. Death verification was confirmed by cessation of heartbeat and respiration, and absence of reflexes and metabolism. After the mice stopped breathing, the tumors were resected.

All data are presented as the mean ± SD of three repeats, except for animal experiments (n=6). GraphPad 8.0 (GraphPad Software, Inc.) was used to perform statistical analysis. An unpaired t-test, one-way ANOVA with Tukey's multiple comparisons test or two-way ANOVA with Sidak's multiple comparisons test were used to evaluate statistical significance. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether miR-576-3p, PD-L1 and cyclin D1 are involved in DDP resistance of ovarian cancer cells, DDP-resistant ovarian cancer cell lines SKOV3/DDP and A2780/DDP were used. The results demonstrated that, as the concentration of DDP increased, the viability of ovarian cancer cells SKOV3 and A2780 and DDP-resistant ovarian cancer cells SKOV3/DDP and A2780/DDP significantly decreased. Furthermore, the cell viability of ovarian cancer cells was more affected by DDP compared with that of DDP-resistant cells (Fig. 1A). In addition, the tolerance of SKOV3/DDP and A2780/DDP cells to DDP was significantly higher compared with that of SKOV3 and A2780 cells (Fig. 1B). RT-qPCR analysis was used to detect the mRNA expression of miR-576-3p in DDP-resistant cells, and the results revealed that the mRNA levels of miR-576-3p in SKOV3/DDP and A2780/DDP cells were downregulated compared with those in SKOV3 and A2780 cells (Fig. 1C). Western blotting demonstrated that the protein expression levels of PD-L1 and cyclin D1 in DDP-resistant cells were significantly higher compared with those in non-resistant cells (Fig. 1D and E). These data indicated that miR-576-3p, PD-L1 and cyclin D1 may be involved in DDP resistance of ovarian cancer cells.

The role of miR-576-3p in ovarian cancer cells was investigated via transfection of miR-576-3p mimic, which significantly increased the expression of miR-576-3p in ovarian cancer cells (Fig. S1A). Furthermore, miR-576-3p overexpression decreased ovarian cancer cell viability, and promoted apoptosis and the accumulation of ovarian cancer cells in the G1 phase (Fig. S2). To study the effects of miR-576-3p on DDP sensitivity of ovarian cancer cells, DDP-resistant ovarian cancer cells were transfected with miR-576-3p mimic or NC mimic. The results demonstrated that miR-576-3p overexpression significantly increased miR-576-3p mRNA expression in DDP-resistant cells (Fig. 2A). miR-576-3p upregulation reduced DDP tolerance in DDP-resistant cells (Fig. 2B). Moreover, the viability of DDP-resistant cells decreased with the increase in DDP concentration, and miR-576-3p overexpression increased the DDP sensitivity of SKOV3/DDP and A2780/DDP cells (Fig. 2C). Furthermore, DDP treatment had no significant effect on DDP-resistant cell cycle progression and apoptosis compared with the control group (Fig. 2D and E). However, overexpression of miR-576-3p resulted in the accumulation of DDP-resistant cells in the G1 phase and promotion of cell apoptosis (Fig. 2D and E). Additionally, DDP treatment reduced the protein levels of PD-L1, cyclin D1 and MDR1, but increased the cleaved/pro caspase-3 ratio and cleaved/pro PARP ratio in DDP-treated DDP-resistant cells compared with the control group (Fig. 3). Moreover, miR-576-3p overexpression further enhanced the effects of DDP on PD-L1, cyclin D1, MDR1, cleaved caspase-3 and cleaved PARP protein levels (Fig. 3). Therefore, miR-576-3p overexpression increased DDP sensitivity of DDP-resistant ovarian cancer cells via regulating cell viability and apoptosis.

A dual-luciferase assay was performed to detect the targeting relationship between miR-576-3p and PD-L1 and cyclin D1. The binding sites between miR-576-3p and PD-L1 and cyclin D1 were determined using TargetScan (version 7.2; http://www.targetscan.org/vert_72/). As shown in Fig. 4A and B, the luciferase activity in cells transfected with wt-PD-L1 and wt-cyclin D1 plasmids was significantly decreased by miR-576-3p mimic transfection. The luciferase activity in cells transfected with mut-PD-L1 and mut-cyclin D1 plasmids showed no significant change. The results indicated that miR-576-3p can directly target PD-L1 and cyclin D1. Additionally, the mRNA and protein expression levels of PD-L1 and cyclin D1 were significantly reduced in SKOV3/DDP and A2780/DDP cells following miR-576-3p mimic transfection, but were significantly increased by transfection with a miR-576-3p inhibitor (Figs. 4C-F and S1B). Therefore, miR-576-3p overexpression may regulate cell viability and apoptosis via targeting PD-L1 and cyclin D1.

The effects of miR-576-3p on DDP resistance in ovarian cancer cells were also investigated using a xenograft tumor model. The results demonstrated that DDP treatment inhibited tumor growth in the nude mice injected with miR-576-3p overexpressing-SKOV3/DDP cells compared with the control group (Fig. 5A and B). The mRNA expression level of miR-576-3p was upregulated in miR-576-3p-overexpressing ovarian tumor tissues (Fig. 5C). Western blotting demonstrated that the protein expression levels of PD-L1, cyclin D1 and MDR1 were downregulated, whereas the cleaved/pro caspase-3 ratio and cleaved/pro PARP ratio were upregulated in miR-576-3p-overexpressing tumor tissues treated with DDP (Fig. 5D). Overall, miR-576-3p overexpression enhanced DDP sensitivity of DDP-resistant ovarian cancer cells in vivo.