Contributed equally
Thyroid cancer is the most common tumour of the endocrine system, and its incidence rate has markedly increased over the past several decades. Aberrantly expressed microRNAs (miRNAs) are reportedly involved in the formation and progression of papillary thyroid carcinoma (PTC) by regulating their target genes. Thus, miRNAs may be potential molecular biomarkers for the prediction and prognosis of PTC, and also as novel therapeutic targets for patients with PTC. miR-139 has recently been reported to be aberrantly expressed in several types of cancer. However, the expression levels, biological functions and the associated molecular mechanism of miR-139 in PTC have not been clearly elucidated. The results of the present study revealed that miR-139 expression was downregulated in PTC tissues and cell lines when compared with adjacent normal tissues and normal human thyroid cells, respectively. The restoration of miR-139 expression suppressed cellular proliferation and invasion in PTC
Thyroid cancer is the most common tumour of the endocrine system, and its incidence rate has dramatically increased over the past several decades (
MicroRNAs (miRNAs) belong to a large family of endogenous, single-strand and short non-coding RNAs with a length of approximately 22 nucleotides (
MiR-139 has been recently reported to be aberrantly expressed in several types of cancer (
This study was approved by the Medical Ethics Committee of The Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine. All patients included in this research were required to offer written informed consent. Forty-three paired PTC tissues and adjacent normal tissues were obtained from patients (age range, 35–67 years; median age, 48; eighteen males and twenty-five females) who underwent surgical resection in The Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine between February 2014 and December 2015. All tissue samples were snap-frozen in liquid nitrogen immediately after surgery and stored at −80°C until RNA extraction.
The human PTC cell lines (TPC-1, HTH83, and BCPAP) were acquired from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal human thyroid cell line (Nthy-ori 3–1) was obtained from European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK). All of the cell lines were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), 100 units of penicillin/ml and 100 ng of streptomycin/ml (Gibco, Grand Island, NY) at 37°C in humidified air with 5% CO2.
MiR-139 mimics and corresponding miRNA negative control (miR-NC) were chemically synthesized and purified by Guangzhou RiboBio Co., Ltd (Guangzhou, China). Overexpression FN1 plasmid (pcDNA3.1-FN1) and blank vector (pcDNA3.1) were obtained from GeneCopoeia (Guangzhou, China). Cells were seeded into 6-well plates at a density of 8×105 cells per well and maintained in DMEM medium without antibiotics. When the cell density reached 60–70%, transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Tranfected cells were incubated at 37°C with 5% CO2 for 6 h and the medium was replaced by DMEM with 10% FBS.
According to the manufacturer's instructions, total RNA was isolated from the tissue specimens or cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and stored at −80°C. To determine miR-139 expression levels, cDNA was generated by reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). Quantification PCR (qPCR) was performed with TaqMan MicroRNA PCR Kit (Applied Biosystems, Carlsbad, CA, USA) on an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). To quantify FN1 mRNA expression, cDNA was synthesized with PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and qPCR was conducted with SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Dalian, China). U6 and GAPDH were used to normalize the level of miR-139 and FN1 mRNA expression, respectively. The data were analyzed using the 2−ΔΔCq method (
Cells were seeded into 96-well plates at 3×103 cells per well. After incubation overnight, cell transfection was performed and then incubated at 37°C in humidified air with 5% CO2. Cell proliferation was examined at 0, 24, 48, and 72 h after transfection. Briefly, 10 µl of CCK-8 reagent (Dojindo, Kumamoto, Japan) was added into each well and incubated at 37°C for another 2 h. Finally, the optical density (OD) was detected at a wavelength of 450 nm using the ELISA plate reader (Model 550; Bio-Rad Laboratories, Hercules, CA, USA). At least three independent experiments were performed.
Transwell invasion assay was performed to assess cell invasion capacity by using Matrigel-coated Transwell chambers (Millipore, Billerica, MA, USA). A total of 1×105 transfected cells in 100 µl of FBS-free DMEM medium were placed in the upper chambers. DMEM with 10% FBS was added into the lower chamber as chemoattractant. After 24 h of incubation, the upper surface of the membrane was wiped with a cotton tip. Subsequently, invasive cells were fixed with methanol, stained with 0.5% crystal violet, washed with PBS and photographed under an inverted microscope at 200× magnification (X71; Olympus, Tokyo, Japan). The number of invasive cells was counted at five randomly selected fields.
To predict the potential targets of miR-139, bioinformatics analysis was performed with TargetScan (
The pMIR-FN1-3′-UTR-wild-type (Wt) and pMIR-FN1-3′-UTR-mutant (Mut) containing the putative binding site of miR-139 were synthesized and confirmed by GenePharma, Co., Ltd. (Shanghai, China). Cells were seeded in 24-well plates and cultured until the cell density reached 80–90% confluence. Subsequently, cells were transfected with either the pMIR-FN1-3′-UTR-Wt or the pMIR-FN1-3′-UTR-Mut reporter vector, together with miR-139 mimics or miR-NC using Lipofectamine 2000. After incubation 48 h, the activities of firefly and Renilla luciferases were determined in transfected cells using the dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's recommendations. Renilla-luciferase activity was assayed for normalization.
Total protein was isolated form tissue samples or cells with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce, Rockford, IL, USA). The concentration of total protein was examined by Bradford assay (Biorad Laboratories, Hercules, CA, USA). Equal amounts of protein samples (about 30 µg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris Buffered saline with Tween (TBST), the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study include rabbit anti-human polyclonal FN1 (15613–1-AP; 1:200 dilution; Proteintech, USA) and mouse anti-human monoclonal GAPDH antibody (sc-47724; 1:1,000 dilution; Santa Cruz Biotechnology, CA, USA). The membranes were then washed with TBST and incubated with corresponding HRP-conjugated secondary antibodies (sc-2204 and sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. Band signals were visualized using an enhanced chemiluminescence kit (Pierce, Minneapolis, MN, USA), and analyzed with Quantity One software version 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as an internal control.
The statistical analyses were performed using the SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). All data were presented as mean ± SEM, and differences between groups were analyzed using two-tailed student's t-test or a one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.
RT-qPCR was performed in 43 pairs of PTC tissues and adjacent normal tissues to determine miR-139 expression levels in PTC. Results showed that miR-139 was down-regulated in PTC tissues compared with adjacent normal tissues (
We up-regulated miR-139 expression in TPC-1 and HTH83 cells through miR-139 mimics transfection to determine the functional roles of miR-139 in PTC. RT-qPCR analysis demonstrated that miR-139 evidently increased in the TPC-1 and HTH83 cells transfected with the miR-139 mimics compared with that in the cells transfected with miR-NC and untreated cells (
The potential targets of miR-139 were predicted through bioinformatics analysis to investigate the mechanism by which miR-139 inhibits PTC cell proliferation and invasion. FN1 plays oncogenic roles in PTC (
We subsequently examined the mRNA and protein expression levels of FN1 in PTC tissues and adjacent normal tissues through RT-qPCR and Western blot analyses. Results showed that FN1 expression was significantly up-regulated at both mRNA (
We carried out rescue experiments in TPC-1 and HTH83 cells co-transfected with the miR-139 mimics with or without pcDNA3.1-FN1 to test whether miR-139 exerts tumour-suppressive functions in PTC cells via the down-regulation of FN1. Western blot analysis determined that FN1 expression was restored in the miR-139 mimics combined with the FN1 overexpression plasmid (
Aberrantly expressed miRNAs are involved in PTC formation and progression by regulating their target genes. Thus, miRNAs may be investigated as molecular biomarkers for the prediction and prognosis of PTC and as novel therapeutic targets for PTC patients (
MiR-139 has been observed to be abnormally expressed in various human cancers. For instance, miR-139 was found underexpressed in tumour tissues and cell lines in colorectal cancer (
Accumulated studies reported that miR-139 functions as a regulator in various cancers through its targeting. Shen
FN1, a member of the FN family, is up-regulated in multiple types of human cancer, including anal cancer (
In conclusion, the study is the first to demonstrate that miR-139 expression is down-regulated in PTC tissues and cell lines. In addition, miR-139 inhibits the proliferation and invasion of PTC cells by directly targeting FN1. These results provide novel insights into the molecular mechanism underlying PTC progression and suggest that miR-139 can potentially serve as an anti-tumour agent in PTC treatment.
In thyroid carcinoma, the dysregulation of thyroid hormone is critical symptom and FN1 may be involved in the production of thyroid hormone. In this study, we did not explore the effect of miR-139 on thyroid hormone. In the following experiments, we will examine the effect of miR-139 on thyroid hormone. In addition, we can not analyze the association between miR-139 and clinical parameters of patients with PTC using TCGA, and this issue is a limitation of the present study. In the following experiments, we will collect more PTC tissues and explored the association between miR-139 and clinical parameters of patients with PTC.
The present study was supported by grants from the Key Disciplines Group Construction Project of Pudong Health Burea of Shanghai (grant no. PWZxq2014-12), the Natural Science Foundation of China (grant no. 81571718), the Shanghai Sailing Program (grant no. 16YF1408800) and the Shanghai Science and Technology Committee Foundation (grant no. 14DZ1940605).
MiR-139 expression is down-regulated in PTC tissues and cell lines. (A) The relative expression of miR-139 in 43 pairs of PTC tissues and adjacent normal tissues was measured by RT-qPCR. (B) MiR-139 expression is detected in PTC cell lines (TPC-1, HTH83 and BCPAP) and normal human thyroid cell line (Nthy-ori 3-1) by using RT-qPCR. *P<0.05 compared with respective control.
Ectopic expression of miR-139 inhibits PTC cell proliferation and invasion
FN1 is a direct target of miR-139 in PTC. (A) Complementary pairings of miR-139 with FN1 wild-type and mutant 3′ UTR reporter constructs are shown. (B, C) Luciferase activities were determined in TPC-1 and HTH83 cells 48 h after co-transfection with the miR-139 mimic or miR-NC and pMIR-FN1-3′-UTR-Wt or pMIR-FN1-3′-UTR-Mut reporter vector. (D, E) Expression levels of FN1 were detected in TPC-1 and HTH83 cells transfected with the miR-139 mimics or miR-NC and untreated cells using RT-qPCR and Western blot analyses. (F, G) mRNA and protein expression levels of FN1 increased in PTC tissues compared with adjacent normal tissues as assessed by RT-qPCR and Western blot analyses. (H) Correlation between miR-139 expression levels and FN1 mRNA levels in PTC tissues was determined using Spearman's correlation analysis. *P<0.05 compared with respective control.
Up-regulation of FN1 rescues the tumour-suppressive effects of miR-139 on PTC cells. (A) FN1 protein was determined in TPC-1 and HTH83 cells transfected with the miR-139 mimic with or without pcDNA3.1-FN1. (B, C) CCK-8 assay and transwell invasion assays were performed in TPC-1 and HTH83 cells transfected with the miR-139 mimic with or without pcDNA3.1-FN1 (magnification, ×200). *P<0.05 compared with respective control.