The present study was approved by the ethics committee of the First Affiliated Hospital of Xinxiang Medical University (Weihui, China). Ovarian cancer tissues (n=58) and their matched adjacent normal tissues were collected from 58 patients with ovarian cancer from the First Affiliated Hospital of Xinxiang Medical University between September 2014 and April 2016. The patients were between 44 and 68 years old, with a mean age of 57.7 years. Written informed consent was obtained from all patients. No patients received radiation therapy or chemotherapy prior to surgical resection. The tissues were immediately snap-frozen in liquid nitrogen following surgical removal and stored until use. The clinical characteristics of patients, as determined using tumor, node, metastasis staging are summarized in Table I (22). Patients were included in the present study if they exhibited primary ovarian cancer and were excluded if they had received radiation therapy or chemotherapy prior to surgical resection. In addition, all patients involved in the present study were categorized into a high miR-142-3p expression group and a low miR-142-3p expression group, based on the mean expression value (1.16) of miR-142-3p.

Normal human ovarian epithelial cell line IOSE386, human ovarian cancer cell lines (SKOV3, HOC1, HO-8910) and the cisplatin-resistant ovarian cancer cell line SKOV3/DDP cells were purchased from American Type Culture Collection (Manassas, VA, USA). Ovarian cancer OVAC cells were purchased from Cell Bank of Central South University (Changsha, China). The cell lines were cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% CO².

To study the function of miR-142-3p and SIRT1, SKOV3 and SKOV3/DDP cells were transfected with miR-142-3p mimic (cat. no. B01001), scrambled miR control (cat. no. B01001; miR-NC), negative control (NC) inhibitor (cat. no. B03001; anti-NC), miR-142-3p inhibitor (cat. no. B03001; anti-miR-142-3p; all obtained from Shanghai GenePharma Co., Ltd., Shanghai, China), or co-transfected with miR-142-3p mimic and pcDNA3.1-SIRT1 plasmid (Yearthbio, Changsha, China), or co-transfected with miR-142-3p mimic and blank pcDNA3.1 vector using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA from tissues and cell lines was extracted using RNAiso plus (Takara Bio, Inc., Otsu, Japan). RNA was then converted into cDNA on an ABI 7300 plus system (Thermo Fisher Scientific, Inc.) using a PrimeScript^® RT reagent kit (Takara Bio, Inc.), according to the manufacturer's protocol. RT-qPCR was conducted using a Mir-XTM miRNA qPCR SYBR^® kit (Takara Bio, Inc.) and SYBR Premix Ex Taq II (Takara Bio, Inc.) on an ABI 7300 plus system. U6 was used as the internal reference for miR-142-3p expression. GAPDH was used as internal reference for mRNA expression. The SIRT1 primer sequences were: 5′-TAGCCTTGTCAGATAAGGAAGGA-3′ and 5′-ACAGCTTCACAGTCAACTTTGT-3′. The GAPDH primer sequences were: 5′-CTGGGCTACACTGAGCACC-3′ and 5′-AAGTGGTCGTTGAGGGCAATG-3′. The primers for miR-142-3p (cat. no. HmiRQP0186) and U6 (cat. no. HmiRQP9001) were purchased from Guangzhou Fulengen Co., Ltd. (Guangzhou, China); sequences were not supplied. The PCR reaction conditions were: 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. The relative expression was analyzed using the 2^−ΔΔCq method (23).

The drug sensitivity of SKOV3/DDP cells was measured using a Cell Counting kit-8 (CCK)-8 assay. SKOV3/DDP cells (1×10⁵ cells/ml) were seeded in 96-well plates and cultured at 37°C for 24 h. Cells were treated with cisplatin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at various concentrations (2, 4, 8, 16, 32, 64, 128, 256 and 512 µg/ml). Following incubation at 37°C for 48 h, 10 µl CCK-8 reagent (Sigma-Aldrich; Merck KGaA) was added into each well and then cells were cultured at 37°C for 2 h. The absorbance of each sample was measured at 450 nm using a plate reader (TECAN Infinite M200; Tecan Group, Ltd., Männedorf, Switzerland).

Tissues and all cells lines used in this study were lysed in cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The protein (50 µg per lane) was separated via 12% SDS-PAGE (Pierce; Thermo Fisher Scientific, Inc.) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). Following blocking in 5% non-fat dried milk in PBS at room temperature for 3 h, the PVDF membrane was incubated with rabbit anti-SIRT1 primary antibody (1:100; ab32441; Abcam, Cambridge, MA, USA) or rabbit anti-GAPDH primary antibody (1:100; cat. no. ab9485; Abcam) at room temperature for 3 h. Following washing with PBS with Tween-20 for 10 min, the membrane was incubated with the horseradish peroxidase conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab205718; Abcam) at room temperature for 1 h. Results were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression levels were analyzed with Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). GAPDH was used as the internal reference.

SKOV3 cells (2×10⁴) were seeded in 96-well plates, each well with 100 µl of fresh serum-free medium with 0.5 g/l MTT (Sigma-Aldrich; Merck KGaA). This cell line was selected as it demonstrated that the lowest expression of miR-142-3p. Following incubation at 37°C for 0, 24, 48 and 72 h, the medium was removed and 50 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added. Following incubation at 37°C for 10 min, the absorbance of each sample was measured at 570 nm using a plate reader (TECAN Infinite M200; Tecan Group, Ltd.).

TargetScan version 7.1 (www.targetscan.org) was used to predict the target genes of miR-142-3p, according to the manufacturer's protocol.

The wild type (WT) or mutant type (MT) of SIRT1 3′UTR was inserted into the multiple cloning site of the psiCHECK^™2 vector (Promega Corporation, Madison, WI, USA). SKOV3 cells were co-transfected with 100 ng WT-SIRT1-3′UTR or MT-SIRT1-3′UTR plasmid, and 100 nM miR-142-3p mimic or miR-NC using Lipofectamine^® 2000, according to the manufacturer's protocols. Following transfection for 48 h, the Renilla luciferase activity and firefly luciferase activity were determined using a Dual-Luciferase Reporter Assay system (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

All data in the present study are expressed as the mean ± standard deviation. Statistical analysis was conducted using SPSS 19.0 (IBM Corp., Armonk, NY, USA). The difference between two groups was analyzed using Student's t-test and differences among >2 groups were analyzed using one-way analysis of variance, followed by a post hoc Turkey's post hot test. The association between miR-142-3p expression and clinicopathological characteristics of patients with ovarian cancer was analyzed using the Chi-square test. Pearson correlation analysis was conducted for the correlation between miR-142-3p and SIRT1 mRNA expression in ovarian cancer tissues. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed in triplicate.

Firstly, RT-qPCR data revealed that miR-142-3p expression levels were significantly reduced in ovarian cancer tissues compared with in adjacent tissues (Fig. 1A). To confirm these findings, the expression of miR-142-3p in several common ovarian cancer cell lines was investigated. As demonstrated in Fig. 1B, the expression levels of miR-142-3p were significantly lower in ovarian cancer cell lines compared with in the normal human ovarian epithelial cell line IOSE386 (Fig. 1B). Thus, miR-142-3p is downregulated in ovarian cancer. In addition, patients were categorized into a high miR-142-3p expression group and low miR-142-3p expression group, based on the mean expression value of miR-142-3p. Further investigation revealed that decreased expression levels of miR-142-3p were significantly associated with poor differentiation (Table I).

The effect of miR-142-3p on the proliferation of ovarian cancer cells was investigated. SKOV3 cells were transfected with miR-142-3p mimic to upregulate its expression. Transfection with miR-142-3p mimic revealed a significant increase in miR-142-3p expression levels in SKOV3 cells compared with in the control group. However, transfection with miR-NC did not affect the expression of miR-142-3p in SKOV3 cells (Fig. 2A). As the transfection with miR-NC did not affect miR-142-3p expression when compared with the control group, the proliferation of SKOV3 cells in the control group was not assessed. An MTT assay was then used to analyze cell proliferation, which demonstrated that the proliferation of SKOV3 cells was significantly reduced within the miR-142-3p-transfected group at 72 h compared with in cells transfected with miR-NC (Fig. 2B). Thus, miR-142-3p may serve a suppressive role in ovarian cancer cell proliferation.

The role of miR-142-3p in chemoresistance of ovarian cancer cells was investigated. Cisplatin-resistant SKOV3/DDP cells were transfected with miR-142-3p mimic or miR-NC, respectively. Following transfection, the miR-142-3p levels were significantly increased in the miR-142-3p group compared with in the control group; transfection with miR-NC demonstrated no effect on the expression of miR-142-3p in SKOV3/DDP cells (Fig. 2C). As the transfection with miR-NC did not affect miR-142-3p expression when compared with the control group, the survival rate of SKOV3/DDP cells in the control group was not assessed. A CCK-8 assay was conducted to assess the survival rate of cells treated with various concentrations of cisplatin. Under the same concentration of cisplatin (from 4, 8, 16, 32 and 64 µg/ml), the survival rate of SKOV3/DDP cells was significantly reduced in the miR-142-3p group compared with in the miR-NC group (Fig. 2D). These findings suggested that miR-142-3p may promote the sensitivity of SKOV3/DDP cells to cisplatin.

Based on the bioinformatics analysis data, SIRT1 was reported to be a putative target gene of miR-142-3p (Fig. 3A). To confirm this prediction, luciferase vectors containing WT or MT SIRT1 3′-UTR were employed (Fig. 3B). Luciferase reporter assay data indicated that luciferase activity was significantly reduced in SKOV3 and SKOV3/DDP cells co-transfected with the WT-SIRT1-3′UTR plasmid and miR-142-3p mimic, but unaltered in SKOV3 cells co-transfected with the MT-SIRT1-3′UTR plasmid and miR-142-3p mimic, when compared with the control group (Fig. 3C and D). Therefore, miR-142-3p may bind to the 3′UTR of SIRT1 mRNA in ovarian cancer SKOV3 and SKOV3/DDP cells.

In the present study, the effect of miR-142-3p on the expression of SIRT1 in ovarian cancer cells was analyzed. As presented in Fig. 4A and B, the mRNA and protein expression levels of SIRT1 in SKOV3 and SKOV3/DDP cells were significantly reduced in the miR-142-3p group compared with in the miR-NC group. To further confirm these data, SKOV3 and SKOV3/DDP cells were transfected with miR-142-3p inhibitor or NC inhibitor. Following transfection, miR-142-3p expression levels were significantly decreased in the miR-142-3p inhibitor group compared with in the control group; however, transfection with NC inhibitor did not markedly affect miR-142-3p expression in SKOV3 and SKOV3/DDP cells (Fig. 4C). Further investigation demonstrated that the mRNA and protein expression levels of SIRT1 were significantly higher in the anti-miR-142-3p group compared with in the anti-NC group (Fig. 4D and E). Collectively, these findings indicated that miR-142-3p may inhibit SIRT1 expression by directly binding to the 3′UTR of SIRT1 mRNA in ovarian cancer cells.

Based on the aforementioned findings, it was suggested that SIRT1 may be involved in miR-142-3p-mediated suppression of ovarian cancer cell proliferation and chemoresistance. To clarify this speculation, miR-142-3p-overexpressing SKOV3 cells were transfected with an SIRT1-expression plasmid to upregulate its expression, or a blank vector as the control. Following transfection, the mRNA and protein levels of SIRT1 were significantly increased in the miR-142-3p + SIRT1 group compared with in the miR-142-3p + blank group (Fig. 5A and B). MTT assay data further revealed that the proliferation of SKOV3 cells were significantly increased in the miR-142-3p + SIRT1 group compared with in the miR-142-3p + blank group at 48 and 72 h (Fig. 5C), indicating that SIRT1 may have rescued the suppressive effect of miR-142-3p on SKOV3 cell proliferation. Subsequently, miR-142-3p-overexpressing SKOV3/DDP cells were also transfected with an SIRT1-expression plasmid, and the mRNA and protein levels of SIRT1 were significantly upregulated following transfection (Fig. 5D and E). A CCK-8 assay was then conducted to assess the survival rates of SKOV3/DDP cells treated with cisplatin at various concentrations. Under the same concentrations of cisplatin, the survival rate was significantly higher at 4, 8, 16, 32 and 64 µg/ml in the miR-142-3p + SIRT1 group compared with in the miR-142-3p + blank group (Fig. 5F). Thus, SIRT1 rescued the miR-142-3p-mediated suppression of chemoresistance of SKOV3/DDP cells.

The expression of SIRT1 in ovarian cancer tissues and cell lines was detected. The results indicated that SIRT1 mRNA expression levels were significantly higher in ovarian cancer tissues compared with in adjacent tissues (Fig. 6A). Additionally, the mRNA and protein expression levels of SIRT1 were significantly upregulated in ovarian cancer cell lines compared with in normal ovarian epithelial cells (Fig. 6B and C). In addition, a negative correlation between SIRT1 and miR-142-3p expression in ovarian cancer tissues was observed (Fig. 6D). These findings suggested that downregulation of miR-142-3p may contribute to the upregulation of SIRT1 in ovarian cancer.