The human Nthy-ori3-1, and PTC cell lines, TPC-1 and BCPAP were obtained from the American Type Culture Collection (Manassas, VA, USA). Cell lines were authenticated by short-tandem repeat profiling performed by BMR Genomics (Padua, Italy). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare) in a 95% humidified atmosphere with 5% CO₂ at 37°C. The recombinant human Shh N-terminal peptide (rhSHH; R&D system, Minneapolis, MN, USA) was added to the culture medium. Human PTC specimens and their adjacent normal thyroid tissues (30 pairs) were collected from 30 patients (9 males and 21 females; age 27–63 years) who underwent surgery according to an approved human protocol at the Weifang People's Hospital (Weifang, China), between January 2016 and December 2016. All patient materials were obtained with written informed consent.

A LARP7 overexpression plasmid and control vector were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). During cell transfection, the cells (10,000/well) were seeded in 6-well plates and were subsequently cultured until 40–60% confluence. Transfection was performed with a Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The transfection mixture was replaced with a medium containing 10% FBS after 6–8 h. The expression level of LARP7 in the transfected cells was validated via western blot analysis 7 days post-transfection.

Cell viability was examined by Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, Haimen, China). Cells overexpressing LARP7 and control were cultured overnight. Subsequently, cells were trypsinized and seeded at a density of 3,000 cells/well in a 96-well plate with or without ¹³¹I (0.5 ml serum-free DMEM containing 0.1 µCi Na¹³¹I) for 12 h at 37°C. Following culture for the indicated time (0, 24, 48 and 72 h), 10 µl CCK8 was added into each well. The resulting mixtures were incubated at 37°C for 3 h. Subsequently the absorbance of each well was examined using a Multi-skan MK3 spectrophotometer at a wavelength of 450 nm.

Cells transfected with LARP7 and control (200 cells/well) were plated in 6-well plates with or without ¹³¹I. Following 1 week of culture, colonies were fixed with methanol and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 20 min at 37°C, and the images of the stained colonies were captured using a CKX41 light microscope. The number of the colonies that had migrated through the pores was quantified by randomly counting 10 independent visual fields using the images.

Cell apoptosis was assessed using flow cytometry with staining of the cells using an Annexin V/propidium iodide (PI) kit (Nanjing Keygen Biotech, Co, Ltd., Nanjing, Jiangsu, China). Briefly, cells were collected and washed twice in ice cold PBS. The washed cells (2×10⁵) were resuspended in 100 µl binding buffer (included in the kit), and stained with 5 µl Annexin V and 5 µl PI. Following incubation for 15 min in the dark at room temperature, flow cytometry was performed. A flow cytometer (Cytomics FC 500 MPL; Beckman Coulter, Inc., Brea, CA, USA) was utilized to evaluate the apoptotic levels in each sample according to the manufacturer's protocol. Data were analyzed using ModFit LT 3.0 (Verity Software House, Inc., Topsham, ME, USA).

The expression levels of LARP7, sonic hedgehog (SHH), protein patched homolog 1 (PTCH1), glioma-associated protein 1 (GLI1) and sodium-iodide symporter (NIS) proteins were analyzed via western blot assay using the following primary antibodies: Mouse polyclonal LARP7 (1:200; cat. no. sc515209), SHH (1:200; cat. no. sc1194), PTCH1 (1:200; cat. no. sc23929), GLI1 (1:200; cat. no. sc20687) (all from Santa Cruz Biotechnology, Inc.), mouse polyclonal NIS (1:200; cat. no. ab101084; Abcam, Cambridge, MA, USA) overnight at 4°C. Normalization was performed by blotting the same samples with an antibody against mouse monoclonal β-actin (1:800; cat. no. AA128; Beyotime Institute of Biotechnology) overnight at 4°C. Radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) was used to extract total protein from cell lines. Protein concentration of whole cell lysates was assessed using the Pierce™ Bicinchonic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (30 µg) from the lysates of the cells were subjected to electrophoresis via 10% SDS-PAGE (Beyotime Institute of Biotechnology) at 80 V for 30 min then 100 V for 1.5 h, and were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking in 5% skimmed milk for 60 min at room temperature, the membranes were then incubated with the aforementioned diluted primary antibodies. Following incubation with peroxidase-coupled secondary antibody (1:2,000; cat. no. A0216; Beyotime Institute of Biotechnology) at 37°C for 2 h, specific bands were visualized with enhanced chemiluminescence reagent (Nanjing KeyGen Biotech Co., Ltd.) on an autoradiographic film. Images were analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD, USA).

Total RNA from cells was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Complementary DNA was synthesized from 500 ng of total RNA, reaction mixture (20 µl) containing 1 µg of total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. RT-qPCR was performed using a SYBR Premix Ex Taq (Takara Bio, Inc. Ostu, Japan) according to the manufacturer's protocols. The RT-qPCR conditions were applied for detecting mRNAs: 95°C for 30 sec, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The β-actin was used as internal controls for the detection. The primer sequences used are presented in Table I. The mRNA relative expression levels were calculated using 2^−ΔΔCq and normalized to the reference (8).

Cells were cultured in 24-well plates (10⁵ cells/0.5 ml) and treated with or without 0.5 µg/ml rhSHH (R&D Systems, Inc.) for 24 h, subsequently, radioiodine uptake assays were performed. Cells were washed twice with 0.5 ml PBS and subsequently incubated with 0.5 ml of serum-free DMEM/F-12, which contained µCi carrier-free Na¹³¹I (0.1 m) (Atomic Hi-tech Radiation Co., Ltd., Beijing, China), with or without perchlorate (100 µM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Perchlorate was used as a NIS-competitive inhibitor for radioiodine uptake. Following 1-h incubation at 37°C, the radioiodine-containing medium was removed and cells were rinsed twice with 1 ml PBS. Subsequently, cells in each well were lysed with NaOH and washed twice with PBS. The cell-associated radioactivity of the collected lysed cells was measured using a gamma counter.

All experiments involving animals were approved by the Animal Care and Welfare Committee of Weifang Medical University (Weifang, China). Lv-control or Lv-LARP7 (1×10⁸/ml) (Santa Cruz Biotechnology, Inc.) BCPAP cells (1.0×10⁶) were subcutaneously injected into the right flanks of 4-week-old BALB⁄c athymic nude mice (n=12, female, weight range; 20–25 g) (Laboratory Animal Center of Yangzhou University, Yangzhou, China) and maintained in a specific-pathogen free environment with constant humidity (45–50%) and constant temperature (25–27°C) under a 12 h light/dark cycle with free access to food and water. These nude mice were randomly divided into two groups (6 mice per group) (9). Tumor volume (mm³) was calculated every 3 days over 3 weeks using the following formula: V=0.5 × length × width². Tumors were collected and imaged at 3 weeks following inoculation.

Paraffin-embedded sections of tumor tissue (4 µm thick) were deparaffinized in xylene, rehydrated via graded alcohol solutions, blocked in methanol containing 3% hydrogen peroxide for 10 min at room temperature, and then incubated with mouse anti-human anti-Ki67 antibody (1:200; cat. no. sc-23900; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following rinsing with PBS solution, biotinylated goat anti-rabbit serum IgG (1:2,000; cat. no. ab64256; Abcam) was used as secondary antibodies and streptavidin peroxidase complex reagent were applied for 1 h at room temperature. Finally, the sections were incubated in a 3, 3′-diaminobenzidine solution at room temperature for 10 min and then counterstained with hematoxylin for 3 min at room temperature. Ten randomly selected visual fields per section were examined under a light microscope to evaluate the Ki67 expression.

Statistical analyses were performed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). All experiments were performed in triplicate. Unless otherwise indicated, the data were evaluated as mean ± standard deviation. Differences between two groups were assessed using two-tailed Student's t-test. Data of more than two groups were using one way analysis of vaiance with post hoc test by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

LARP7 expression levels were detected in 30 paired PTC tissues and their corresponding non-neoplastic thyroid tissues by RT-qPCR. The expression level of LARP7 was significantly reduced in PTC tissues when compared with the non-neoplastic thyroid tissues (Fig. 1A). Immunohistochemistry demonstrated that LARP7 was overexpressed in adjacent normal tissues compared with the PTC samples (representative images from 30 pairs; Fig. 1B and C). Furthermore, LARP7 mRNA and protein expression levels were significantly higher in the human thyroid cell line Nthy-ori3-1 cell line when compared with the PTC cell lines using RT-qPCR and western blot analysis (Fig. 1D and E, respectively).

In order to investigate the effect of LARP7 in PTC cells, LARP7 was overexpressed in the TPC-1 and BCPAP cells. The expression level of LARP7 was confirmed by western blotting (Fig. 2A and B). The cell viability of the TPC-1 and BCPAP cells was examined using the CCK8 assay 24, 48 and 72 h after incubation. LARP7 overexpression significantly abrogated the viability of both cell lines (Fig. 2C and D). It is of note that LARP7 overexpression significantly increased the inhibitory effect on cell viability, which was reduced following ¹³¹I treatment (Fig. 2C and D). The colony formation assay revealed that LARP7 overexpression inhibited cell proliferation (Fig. 2E and F), and the combination of LARP7 overexpression with ¹³¹I treatment led to a reduced number of colonies compared with LARP7 overexpression or ¹³¹I alone (Fig. 2E and F). In addition, flow cytometry analysis was used to determine the effect of LARP7 overexpression on cell apoptosis. The findings demonstrated that LARP7 overexpression promoted the apoptosis of PTC; however, the combination of LARP7 overexpression with ¹³¹I treatment led to a greater apoptotic rate of PTC compared with LARP7 overexpression or ¹³¹I alone (Fig. 2G and H). The cell iodine uptake ability was investigated further and as demonstrated in Fig. 2I and J, as radioiodine uptake was markedly increased in TPC-1 and BCPAP cell lines.

The present study investigated the association of LARP7 and tumor progression in vivo using subcutaneously transplanted Lv-control or Lv-LARP7 BCPAP cells into BALB⁄c athymic nude mice (n=6/group). Tumors were removed and imaged 3 weeks following cell implantation (Fig. 3A). Results revealed that LARP7 overexpression significantly inhibited tumorigenicity (Fig. 3B and C). Histological analysis of the proliferation index revealed that Lv-LARP7 tumors had a significantly lower number of Ki67 positive cells compared with the Lv-control tumors (Fig. 3D and E). These findings revealed that LARP7 significantly inhibited the tumor growth in vivo.

A recent study revealed that the loss of LARP7 expression contributed to cancer progression and metastasis (5). In cancer cells, one of the primary signaling pathways associated with cancer progression and metastasis is the SHH signaling pathway (10). The present study investigated whether LARP7 is involved in regulating the SHH signaling pathway in PTC cells. Expression levels of the principal components of the SHH signaling pathway in LARP7-overexpressing TPC-1 and BCPAP cells were analyzed. Overexpression of LARP7 inhibited the expression levels of SHH pathway-associated genes, including SHH, PTCH1 and GLI1 in TPC-1 and BCPAP cell lines at the mRNA and protein levels (Fig. 4).

The present study investigated the mechanism through which LARP7 increases the iodine uptake ability of PTC cells, the expression level of NIS in LARP7 overexpressing cells and control TPC-1 and BCPAP cells were also assessed. The mRNA and protein expression levels in LARP7-overexpressing PTC cells were upregulated compared with control cells, as determined by RT-qPCR and western blot analysis (P<0.05; Fig. 5A-D). In order to determine whether LARP7 increased cell iodine uptake ability via the SHH signaling pathway, TPC-1 and BCPAP cells were treated with rhSHH. rhSHH treatment reduced NIS mRNA and protein expression levels in both cell lines (P<0.05; Fig. 5A-D). These findings raveled that the SHH signaling pathway may regulate NIS expression. As presented in Fig. 5E and F, radioiodine uptake was significantly reduced in TPC-1 and BCPAP cells treated with rhSHH. Subsequently, TPC-1 and BCPAP cells overexpressing LARP7 and control cells were treated with rhSHH and RT-qPCR and western blot analysis demonstrated that LAPR overexpression combined with led to rhSHH treatment led to reduced NIS mRNA and protein expression levels (P<0.05; Fig. 5A-D). Radioiodine uptake assay indicated that treatment with rhSHH significantly reduced the radioiodine uptake ability of TPC-1 and BCPAP cell lines, which was previously increased by LARP7 overexpression alone (P<0.05; Fig. 5E and F).