This study was approved by the Ethics Committee of Daping Hospital (Chongqing, China), in accordance with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants prior to surgery. In total, 47 pairs of PTC tissues and matched adjacent normal tissues were collected from patients who had received surgical resection at Daping Hospital between June 2015 and May 2017. None of the patients had undergone chemotherapy or radiotherapy prior to surgery. All tissues were quickly frozen in liquid nitrogen after being excised and were stored at −80°C until further use.

A normal human thyroid cell line (HT-ori3), two human PTC cell lines (HTH83 and TPC-1) and a thyroid cancer cell line (BCPAP) were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS; both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% v/v penicillin-streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were used to culture the cells. Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂.

miR-766 mimics and a corresponding negative control (miR-NC) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences were as follows: miR-766 mimics, 5'-ACUCCAGCCCCACAGCCUCAGC-3'; miR-NC, 5'-UUCUCCGAACGUGUCACGUTT-3'. Small interfering RNA (siRNA) targeting insulin receptor substrate 2 (IRS2) expression (IRS2 siRNA) and a negative control siRNA (NC siRNA) were acquired from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows: IRS2 siRNA, 5'-AAUAGCUGCAAGAGCGAUGAC-3'; NC siRNA, 5'-UUCUCCGAACGUGUCACGUTT-3'. An IRS2 overexpression plasmid was chemically synthesized using pcDNA3.1(+) basic vectors at the Chinese Academy of Sciences (Changchun, China). HTH83 and TPC-1 cells were seeded into 6-well plates at a density of 8×105 cells/well. Cells were transfected with mimics (100 pmol), siRNAs (100 pmol) or plasmids (4 µg) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 48 h, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), flow cytometric analysis, and cell migration and invasion assays were performed. MTT and colony formation assays were conducted at 24 h post-transfection. Western blot analysis was conducted 72 h post-transfection.

Total RNA was isolated from cultured cells or homogenized tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For the detection of miR-766 expression, RT was performed using a TaqMan MicroRNA RT kit, according to the manufacturer's protocol, followed by qPCR with a TaqMan MicroRNA PCR kit (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions for qPCR were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec, and a final extension step at 4°C for 5 min. U6 small nuclear RNA served as an internal control for miR-766 expression. For IRS2 mRNA quantification, first strand cDNA was synthesized using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. qPCR was then performed using a SYBR Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.). The cycling conditions for qPCR were as follows: 5 min at 95°C, followed by 40 cycles at 95°C for 30 sec and 65°C for 45 sec, and a final extension step at 40°C for 30 sec. GAPDH was used for IRS2 mRNA normalization. The primers were designed as follows: miR-766, forward 5'-TCG AGTACTTGAGATGGAGTTTT-3', reverse 5'-GGCCGCGTTGCAGTGAGCCGAG-3'; U6, forward 5'-GCTTCGGCAGCACATATACTAAAAT-3', reverse 5'-CGCTTCACGAATTTGCGTGTCAT-3'; IRS2, forward, 5'-CACAATTCCAAGCGCCACAA-3', reverse 5'-ATCAAAGCTCCAGGCTGACC-3'; and GAPDH, forward 5'-ACCCACTCCTCCACCTTTG-3' and reverse 5'-CTCTTGTGCTCTTGCTGGG-3'. Relative gene expression was calculated using the 2^−ΔΔCq method (26).

After 24 h of incubation, transfected cells in the exponential growth stage were collected and seeded into 96-well plates at a density of 3,000 cells/well. At 0, 24, 48 and 72 h following inoculation, the MTT assay was performed to determine cell proliferation. Briefly, 20 µl 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA) was added to each well for 4 h at 37°C and 5% CO₂. Subsequently, the culture medium was gently removed and formazan precipitates were dissolved in 100 µl dimethyl sulfoxide (Beyotime Institute of Biotechnology,, Inc., Shanghai, China). The absorbance was detected at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Logarithmically growing transfected cells were harvested after 24 h of incubation and were plated into 6-well plates at a density of 1,000 cells/well. Cells were then maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 2 weeks. Subsequently, cells were washed with PBS (Gibco; Thermo Fisher Scientific, Inc.), fixed with 100% methanol at room temperature for 20 min and stained with methyl violet (Beyotime Institute of Biotechnology) at room temperature for 20 min. The number of colonies (>50 cells/colony) was counted under an inverted light microscope (IX71; Olympus Corporation, Tokyo, Japan).

An Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Biolegend, Inc., San Diego, CA, USA) was used to evaluate the percentage of apoptotic cells. Briefly, transfected cells were seeded into 6-well plates (1×10⁶ cells/well), incubated for 48 h at 37°C and 5% CO₂, harvested, washed three times with PBS, and suspended in 100 µl binding buffer. Subsequently, 5 µl Annexin V-FITC and 5 µl propidium iodide were added and the transfected cells were incubated for 15 min at room temperature in the dark. Finally, flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ, USA) was used to measure the rate of apoptosis. The data were analyzed with CellQuest version 5.1 (BD Biosciences).

The migratory ability of PTC cells was evaluated using Transwell chambers (BD Biosciences) with an 8-µm pore polycarbonate membrane. A total of 48 h post-transfection, 5×10⁴ cells were suspended in FBS-free DMEM and were inoculated into the upper chamber. The lower chambers were filled with 500 µl DMEM supplemented with 20% FBS. After 24 h at 37°C, the non-migrated cells were removed using a cotton swab, whereas the migrated cells were fixed with 100% methanol and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology). Images of the cells were captured and migratory ability was quantified by counting the number of migrated cells in five randomly selected visual fields from each chamber using an inverted light microscope. The cell invasion assay was conducted in a similar manner to the migration assay; however, the Transwell chambers were initially coated with Matrigel (BD Biosciences).

BALB/c nude mice (female; age, 4 weeks; weight, 20 g) were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China) and were divided into two groups (n=4/group), which were subcutaneously injected with TPC-1 cells transfected with miR-766 mimics or miR-NC, respectively. The animals were maintained under specific pathogen-free conditions (25°C, 50% humidity, 10-h light/14-h dark cycle). The mice received free access to normal rodent food and water, which was autoclaved. Tumor length and width were measured every 2 days. BALB/c nude mice were sacrificed 30 days following implantation, and the tumor xenografts were excised and weighed. The tumor volumes were analyzed using the following formula: Tumor volume = 1/2 × tumor length × tumor width. Experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of Daping Hospital.

The putative miR-766 target genes were predicted using TargetScan software (http://www.targetscan.org) and miRDB software (http://mirdb.org/). The wild-type (wt) and mutant (mut) 3'-UTRs of IRS2 were created by Shanghai GenePharma Co., Ltd., and were inserted downstream of the psiCHECK2 luciferase reporter vector (Promega Corporation, Madison, WI, USA), in order to generate psiCHECK2-IRS2-3'-UTR wt and psiCHECK2-IRS2-3'-UTR mut, respectively. Cells were plated into 24-well plates 12 h prior to transfection at a density of 1.5×10⁵ cells/well. Co-transfection with the constructed luciferase reporter plasmids (0.2 µg) and miR-766 mimics (50 pmol) or miR-NC (50 pmol) was performed using Lipofectamine^® 2000, according to the manufacturer's protocol. A total of 48 h post-transfection, luciferase activity was determined using a dual-luciferase reporter assay system (Promega Corporation), according to the manufacturer's protocol. The activity of firefly luciferase was normalized to that of Renilla luciferase.

Total protein was isolated from tissues or cells using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) and was quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology), followed by blocking at room temperature for 1 h with 5% w/v dried skimmed milk diluted in Tris-buffered saline with 0.1% Tween-20 (TBST). The membranes were then incubated overnight at 4°C with primary antibodies against IRS2 (cat. no. ab52606; 1:1,000 dilution; Abcam, Cambridge, UK), phosphorylated (p)-phosphoinositide 3-kinase (PI3K; cat. no. ab182651; 1:1,000 dilution; Abcam), PI3K (cat. no. ab191606; 1:1000 dilution; Abcam), p-protein kinase B (Akt; cat. no. sc-81433; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Akt (cat. no. sc-56878; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.), or GAPDH (cat. no. ab128915; 1:1,000 dilution; Abcam). After three washes with TBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab205719 or ab6721; 1:5,000 dilutions; Abcam) for 2 h at room temperature and were subjected to visualization using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression was semi-quantified using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).

SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Data are presented as the means ± standard deviation from at least three independent experiments, and the differences between groups were compared using Student's t-test or one-way analysis of variance (ANOVA). Student-Newman-Keuls test was used as a post hoc test following ANOVA. The association between miR-766 and clinicopathological characteristics of patients with PTC was analyzed using the χ² test. Spearman correlation analysis was employed to investigate the association between miR-766 and IRS2 mRNA expression in PTC tissues. P<0.05 was considered to indicate a statistically significant difference.

To uncover the expression pattern of miR-766 in PTC, RT-qPCR analysis was performed to detect miR-766 expression in 47 pairs of PTC tissues and matched adjacent normal tissues. PTC tissues exhibited significantly lower miR-766 expression compared with in the matched adjacent normal tissues (Fig. 1A; P<0.05). Subsequently, the clinical significance of miR-766 in PTC was investigated. Briefly, all patients with PTC were divided into miR-766 low (n=24) or miR-766 high (n=23) expression groups, based on the median value of miR-766 expression. Low miR-766 expression was significantly associated with TNM stage (P=0.008) and lymph node metastasis (P=0.039), whereas no obvious association was observed between miR-766 and age, sex or tumor size (all P>0.05; Table I). In addition, miR-766 expression was measured in two PTC cell lines and in a general thyroid cancer cell line. The data obtained from RT-qPCR indicated that miR-766 was downregulated in the two PTC cell lines (HTH83 and TPC-1) and the thyroid cancer cell line (BCPAP) compared with in the normal human thyroid cell line (HT-ori3) (Fig. 1B; P<0.05). These results indicated that downregulation of miR-766 may have a critical role in PTC tumorigenesis and development.

To clarify the roles of miR-766 in PTC progression, HTH83 and TPC-1 cell lines, which exhibited lower miR-766 expression among the three thyroid cancer cell lines, were selected for functional assays and were transfected with miR-766 mimics or miR-NC. Post-transfection, miR-766 was markedly overexpressed in miR-766 mimics-transfected HTH83 and TPC-1 cells compared with in cells transfected with miR-NC (Fig. 2A; P<0.05). The role of miR-766 overexpression in the proliferation of PTC cells was determined by MTT and colony formation assays. Upregulation of miR-766 significantly inhibited the proliferation (Fig. 2B, P<0.05) and colony formation (Fig. 2C, P<0.05) of HTH83 and TPC-1 cells. Since an alteration in cell proliferation is often accompanied with changes in the rate of apoptosis, the effects of miR-766 on cell apoptosis were assessed using flow cytometry. Transfection of miR-766 mimics markedly promoted the apoptosis of HTH83 and TPC-1 cells (Fig. 2D; P<0.05). Cell migration and invasion assays were used to determine whether miR-766 was implicated in the regulation of PTC cell metastasis. Ectopic miR-766 expression markedly restricted the migration (Fig. 2E; P<0.05) and invasion (Fig. 2F; P<0.05) of HTH83 and TPC-1 cells. These findings suggested that miR-766 may exert tumor suppressor activity in PTC cells.

Based on bioinformatics analysis, the present study revealed that the 3'-UTR of IRS2 matched the seed sequence of miR-766 (Fig. 3A). IRS2 was selected for further experimental identification because this gene has previously been implicated in the initiation and progression of PTC (27). A luciferase reporter assay was performed to test this hypothesis. The assay demonstrated that ectopic miR-766 expression significantly reduced the luciferase activity of psiCHECK2-IRS2-3'-UTR wt in HTH83 and TPC-1 cells (Fig. 3B, P<0.05), but not that of psiCHECK2-IRS2-3'-UTR mut. These findings indicated that the 3'-UTR of IRS2 may be directly targeted by miR-766 in PTC cells. To further clarify the association between miR-766 and IRS2 in PTC, IRS2 mRNA expression was detected in 47 pairs of PTC tissues and matched adjacent normal tissues using RT-qPCR. The mRNA expression levels of IRS2 were significantly increased in PTC tissues (Fig. 3C; P<0.05) and were inversely correlated with the expression levels of miR-766 (Fig. 3D; r=-0.5143, P=0.0002), as determined by Spearman correlation analysis. To investigate whether endogenous IRS2 expression could be regulated by miR-766, RT-qPCR and western blot analysis were carried out to detect IRS2 mRNA and protein expression in HTH83 and TPC-1 cells upon miR-766 overexpression. Compared with in the miR-NC group, HTH83 and TPC-1 cells transfected with miR-766 mimics exhibited significantly decreased levels of IRS2 mRNA (Fig. 3E; P<0.05) and protein (Fig. 3F, P<0.05). Taken together, these results supported the hypothesis that IRS2 is a direct target gene of miR-766 in PTC cells.

IRS2 was validated as a direct target gene of miR-766 in PTC cells. Therefore, it was hypothesized that the tumor-suppressing roles of miR-766 in PTC cells may be achieved by IRS2 knockdown. To evaluate this hypothesis, HTH83 and TPC-1 cells were transfected with IRS2 siRNA to knockdown endogenous IRS2 expression and examine the function of IRS2 in PTC cells. Western blot analysis indicated that IRS2 protein expression was significantly decreased in IRS2 siRNA-transfected HTH83 and TPC-1 cells (Fig. 4A; P<0.05). Inhibition of IRS2 significantly restricted HTH83 and TPC-1 cell proliferation (Fig. 4B; P<0.05) and colony formation (Fig. 4C; P<0.05), and markedly enhanced cell apoptosis (Fig. 4D; P<0.05). In addition, as shown in Fig. 4E and F, IRS2 knockdown significantly attenuated the migratory (P<0.05) and invasive (P<0.05) abilities of HTH83 and TPC-1 cells compared with in NC siRNA-transfected cells. These results demonstrated that the effects of IRS2 knockdown on PTC cells were similar to the effects of miR-766 overexpression, further suggesting that IRS2 is a functional downstream target of miR-766 in PTC cells.

Rescue experiments were performed to determine whether miR-766 exerts tumor-suppressing roles in PTC cells by inhibiting IRS2 expression. HTH83 and TPC-1 cells were co-transfected with miR-766 mimics and empty pcDNA3.1 plasmid or pcDNA3.1-IRS2 lacking the 3'-UTR. Western blot analysis was used to detect the protein expression levels of IRS2 in the rescue experiment. miR-766 overexpression-induced down-regulation of IRS2 was significantly recovered in HTH83 and TPC-1 cells following co-transfection with miR-766 mimics and pcDNA3.1-IRS2 (Fig. 5A; P<0.05). In addition, functional analyses indicated that restored IRS2 expression could reverse the tumor suppressive roles of miR-766 overexpression on the proliferation (Fig. 5B; P<0.05), colony formation (Fig. 5C; P<0.05), apoptosis (Fig. 5D; P<0.05), migration (Fig. 5E; P<0.05) and invasion (Fig. 5F; P<0.05) of HTH83 and TPC-1 cells. These results collectively indicated that miR-766 may inhibit the development of PTC cells, at least partially, through the downregulation of IRS2 expression.

IRS2 has previously been reported to participate in regulation of the PI3K/Akt pathway (28-30). Therefore, it was hypothesized that miR-766 may target IRS2 to inhibit the activation of PI3K/Akt signaling in PTC cells. To confirm this hypothesis, HTH83 and TPC-1 cells were co-transfected with miR-766 mimics and pcDNA3.1 or pcDNA3.1-IRS2, and western blot analysis was performed 72 h post-transfection to determine the expression of molecules associated with the PI3K/Akt pathway. A marked decrease in p-PI3K and p-Akt was observed in HTH83 and TPC-1 cells upon miR-766 overexpression; however, this did not affect total PI3K and Akt expression (Fig. 6). Furthermore, recovered IRS2 expression rescued the cellular levels of p-PI3K and p-Akt, which were downregulated by miR-766 overexpression. These results suggested that miR-766 may inhibit the PI3K/Akt signaling in PTC cells via regulation of IRS2.

To explore the precise role of miR-766 in PTC in vivo, xenograft experiments were performed by subcutaneously injecting miR-766-overexpressing TPC-1 cells into nude mice. The miR-766-overexpressing tumor xenografts exhibited significant suppression of tumor volume compared with in the miR-NC group (Fig. 7A and B; P<0.05). Subsequently, tumor xenografts were weighed; upregulation of miR-766 decreased tumor weight in vivo (Fig. 7C; P<0.05). In addition, RT-qPCR analysis revealed that miR-766 was markedly upregulated in tumor xenografts that were generated using miR-766 mimics-transfected cells (Fig. 7D; P<0.05). Subsequently, the protein expression levels of IRS2 and molecules associated with the PI3K/Akt pathway were detected in tumor xenografts using western blot analysis. The protein expression levels of IRS2, p-PI3K and p-Akt were downregulated in the tumor xenografts from the miR-766 mimics groups compared with in those from the miR-NC group (Fig. 7E). Taken together, these results suggested that miR-766 may hinder PTC tumor growth in vivo by suppressing IRS2 and activation of the PI3K/Akt pathway.