MicroRNA-4728 mediated regulation of MAPK oncogenic signaling in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer that accounts for 85% of thyroid cancers. MicroRNAs (miRNAs) have been reported to play important roles in the biological processes in cancer. In this study, we analyzed the biological role of miR-4728 in human PTC process in human PTC cell lines in vitro. MiRNA-4728 was observed to down-regulated in human PTC tissues and PTC cell lines. Additionally, miR-4728 inhibited PTC cell proliferation. Further study demonstrated SOS1 was repressed by miR-4728 and overexpression of miR-4728 down-regulated both the mRNA and protein levels of SOS1. Moreover, miR-4728 overexpression also decreased the MAPK signaling activity. These observations suggested that miR-4728 could inhibit the process of human PTC through regulating MAPK signaling pathway. And, appropriate regulation of miR-4728 might be vital to improve human PTC treatment.

Introduction

Aberrant gene regulation features significantly in Papillary thyroid carcinoma (PTC) like other cancers. MiRNAs were well known as small RNA molecules that regulate gene expression at the post-transcriptional level and drive cells towards transformation (Sotiropoulou et al., 2009). Recently, more than 1400 miRNAs have been verified and many of them are highly conserved in human (Griffiths-Jones et al., 2010). It has been showed that more than 60% of all mRNAs are predicted to be under miRNA control and contribute to many cancers (Wang et al., 2008, Bartel, 2009). Currently, aberrant miRNAs have also been reported to mediate gene expression in PTC (Graham et al., 2015, Ab Mutalib et al., 2016). Usually, these genes regulated by the abnormal expressed miRNAs were oncogenes or tumor suppressor genes (Hodge et al., 2011, Srivastava et al., 2013). So it might be a way to regulate tumor progress by appropriate miRNA regulation.

PTC is counted for 80% of the thyroid cancers and is one of the most quickly increasing cancers in cancer patients (Siegel et al., 2012). The surgical removal, radioactive iodine ablation, chemotherapy and radiotherapy are present clinical treatment of PTC. In order to find better and novel therapeutic strategies to treat PTC, it is becoming more and more important to understand the biological mechanism involved in PTC.

Although much is known about the profiling of miRNAs in many tumors, the mechanistic details of miRNA regulation are still not fully elucidated in PTC. It has been reported that overexpression of miR-375 suppressed PTC cell proliferation and induces cell apoptosis by targeting ERBB2 (Wang et al., 2016). In addition, miR-21 was shown to be over expressed in human PTC and promote the cell proliferation and invasion (Zhang et al., 2014). However, there was no report about the biological effects of miR-4728 on human PTC. In this study, we identified that miR-4728 was down-regulated in human PTC tumors and PTC cells. Additionally, MAPK signaling pathway was found to be repressed after miR-4728 overexpression through SOS1 by in vitro assays. Our studies investigated the precise role of miR-4728 in human PTC and may pave the way to a new therapeutic target for PTC.

Material and methods

Cell culture

Human PTC cell TPC-1, K1 and normal thyroid cells Nthy-ori 3-1 cells were used for this study. The cells were maintained in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) and 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich). The cells were cultured in a humidified incubator supplemented with 5% CO2 at 37 °C.

MiR-4728 transfection

miR-4728 mimics (mimic-miR-4728) or miRNA control mimics (mimic-Ctl) were transfected PTC cells or Nthy-ori 3-1 cells as described previously (Schmitt et al., 2015). Briefly, human PTC cells were transfected with 10 nM mimic-miR-4728 (Invitrogen) or 10 nM mimi-Ctl (Invitrogen) after 24 h culture using lipofectamine 2000 (Invitrogen) in antibiotic-free Opti-MEM medium (Gibco). After 6 h transfection, DMEM complete medium replace the culture medium.

RNA isolation and quantitative real-time PCR

Total RNA of tissues or cells was isolated with TRIzol reagent (Invitrogen) according to the manufacturer. RNAs were reverse transcribed into cDNA with 300 units of M-MLV reverse transcriptase (BRL, Gaithersburg, MD) after removing contaminating DNA by DNA-free DNase (Ambion, Austin, TX). For gene expression, the quantitative real-time PCR (qRT - PCR) assays were performed with gene specific probes (Applied Biosystems, Foster City, CA). GAPDH was used as an internal control.

To quantify miRNA expression, the total RNA was reverse transcribed with a miRNA-specific looped RT primer (Applied Biosystems). MiR-4728 was tested with miRNA-specific Taqman minor groove binder probes (Applied Biosystems). U6 was used as an internal control. All Taqman qRT- PCR studies were performed in triplicate on an ABI 7500 system (Applied Biosystems) and the data were presented as mean ± SE.

Cell proliferation assay

CCK-8 assay was used to determine cell proliferation according to the description of the manufacturer. Briefly, cells with transfection of mimic-miR-4728 or mimic-Ctl were seeded into 96-well plates at a density of 5000 cells per well. Cells without transfection were considered as blank control (BC). 1.5 h before the designed time point, CCK8 reagent was added to cells at a ratio of 1:10. After then, these cells were incubated in a humidified incubator supplemented with 5% CO2 at 37 °C for the indicated period. The absorbance at OD450 represented the proliferation of these cells. All the studies were performed in triplicate with independent cell preparations. Data were presented as mean ± SE.

Dual-luciferase assay

MAPK signaling pathway activity was detected by luciferase reporter assay as previous description (Zhang et al., 2015a, Zhang et al., 2015b). Briefly, cells were seeded in 96-well plate at a density of 2 × 104 cells per well. MAPK pathway-luciferase-reporter construct (SABiosciences, Frederick, MD) was co-transfected with mimic-miR-4728 or mimic-Ctl into cells with lipofectatmine 2000. After culture 48 h, the cells were harvested for the Dual Luciferase Reporter Assay (Promega).

For SOS1 target gene verification, SOS1 luciferase assay was performed as previous description as well (Wang et al., 2016). Mutant SOS1 3′-UTR was used as a corresponding control. MiR-4728 inhibitor and mimic-Ctl were purchased from Invitrogen. The wild type (WT) or mutant SOS1 reporter vectors were co-transfected with mimic-miR-4728 or inhibitor into TPC-1 cells by lipofectamine 2000. The cells were harvested for luciferase assay after 48 h culture.

Results are represented by dividing firefly luciferase activity by Renilla luciferase activity. All the studies were performed in triplicate with independent cell preparations. Data were presented as mean ± SE.

Western blotting

Protein extract from PTC cells were separated in a 4–20% gradient SDS-polyacrylamide gel (Invitrogen) and electrophoretically transferred onto a PVDF membrane (GE). After block, membranes were incubated with primary antibodies (Ab) SOS1 and GAPDH (Abcam) overnight. Corresponding HRP-conjugated secondary Abs (Bio-Rad) were added the next day after washing with TBST for 3 times. The proteins were visualized by ECL chemiluminescence and exposed to X-ray film. All the studies were performed in triplicate.

Statistics

The significance of differences between groups was evaluated by student t-test with SPSS 13.0. A probability (P) value less than 0.05 was considered as statistical significance.

Results

MiR-4728 level in PTC tissues and cell lines

Previous studies reported that miR-4728 was abnormally expressed in many types of human cancers (Persson et al., 2011, Newie et al., 2014, 2015), and involved in many biological functions. In this study, we first determined miR-4728 expression in PTC and normal tissues. 18 pairs of PTC and non-cancerous normal tissues were used to determine miR-4728 expression by qRT- PCR assay. U6 snRNA was the internal control. Fig. 1A shows that miR-4726 level in human PTC tissues was significantly down-regulated compared to normal tissues.

Fig. 1

qRT-PCR analysis of miR-4728 in human PTC tissues and cell lines. (A) Analysis of miR-4728 level in huan PTC tissues and non-cancerous normal tissues. (B) Analysis of miR-4728 level in human PTC cells lines (K1 and TPC-1) and normal thyroid gland cell line (Nthy-ori 3-1). Data are normalized U6 small snRNA. Data was represented as mean ± SE *p < 0.05 vs. Non-cancerous normal tissues or Nthy-ori 3-1 (n = 3).

To decipher the biological function of miR-4728 in PTC, we next determined miR-4728 level in human PTC cells (K1 and TPC-1) and normal thyroid cells Nthy-ori 3-1 by qRT- PCR. Fig. 1B indicates that miR-4728 level in TPC-1 and K1 cells were significantly decreased than that of Nthy-ori 3-1 cells. These results suggest that down-regulation of miR-4728 may involve in the development and progression of PTC.

Overexpression of miR-4728 suppresses human PTC cell proliferation

The effects of miR-4728 on the cell proliferation of human PTC cells were evaluated by transfecting TPC-1 and K1 cells with a miR-4728 mimic or mimic-Ctl. The cell proliferation was detected by CCK8 assay. Fig. 2 shows that miR-4728 overexpression remarkably suppressed the proliferation of TPC-1 and K1 cells than mock cells after D2. Our results indicated that miR-4728 overexpression may induce cell growth inhibition in TPC-1 and K1 cells.

Fig. 2

Effect of miR-4728 on cell proliferation of human PTC cells. (A) Mock TPC-1 cells or TPC-1 transfected with mimic-Ctl or mimic-miR-4728 were seeded in 96-well plate to determine to cell proliferation. (B) Mock K1 cells and K1 cells transfected with mimic-Ctl or mimic-miR-4728 were seeded in 96-well plate to determine to cell proliferation. Data was shown as mean ± SE, *p < 0.05 vs. D0 (n = 3).

SOS1 is repressed by miR-4728

To deciper the mechanism of miR-4728 in PTC, the bioinformatics tool, Targetscan, was used to predict the potential genes of miR-4728. SOS1 is one of the candidates. In addition, SOS1 has been reported to increase the Ras nucleotide exchange rate of GDP for GTP and lead to up-regulation of Ras in the active GPT-bound form (Tidyman and Rauen, 2009). Furthermore, MAPK signaling pathway is one of several important downstream cascades of Ras. Activated Ras causes Raf activation and results in the activation of MAPK signaling pathway. Therefore, SOS1 was chosen for our further study.

In order to verify whether SOS1 is repressed by miR-4728, TPC-1 cells were con-transfected with 3′-UTR of SOS1 including the binding site of miR-4728 and mimic-4728/mimic-Ctl. Luciferase results showed that miR-4728 overexpression in TPC-1 cells led to reduction of the activity of SOS1 3′-UTR (Fig. 3A). However, there was no decrease of luciferase activity of SOS1 3′-UTR in TCP-1 cells co-transfected with mimic-Ctl (Fig. 3A). The luciferase activity reduction was blocked when co-transfected miR-4728 inhibitor with the wild type of 3′-UTR of SOS1 (Fig. 3A).

Fig. 3

Effect of miR-4728 regulation on SOS1 gene in TPC-1 cells. (A) TPC-1 cells were co-transfected with SOS1 3′-UTR luciferase reporter and mimic-Ctl or mimic-miR-4728. miR-4728 inhibitor was added to cells co-transfected with mimic-miR-4728. Luciferase activity was normalized to Renilla. (B) SOS1 mRNA level was determined by qRT-PCR. Data was normalized to GAPDH. Data was shown as mean ± SE, *p < 0.05 vs. Mock (n = 3); **p < 0.05 vs. mimic-Ctl (n = 3); ***p < 0.05 vs. miR-4728 inhibitor (n = 3). (C) SOS1 protein expression was determined by WB. GAPDH was used as internal control.

To double confirm luciferase results, we also investigated SOS1 mRNA and protein level after transfection of mimic-miR-4728 or mimic-Ctl. Fig. 3B and C showed that both mRNA and protein levels of SOS1 significantly decreased after miR-4728 overexpression compared with mimic-Ctl. These results indicated that SOS1 is directly repressed by miR-4728 in PTC cells.

Up-regulated miR-4728 suppresses MAPK signaling pathway in vitro

Since SOS1 is one of the important up-stream regulators of MAPK signaling pathway, our next step was to determine the effect of miR-4728 on MAPK signaling pathway. MAPK dual-luciferase reporter assay was used to monitor the signaling activity. We found that miR-4728 overexpression reduced the activity of MAPK signaling compared with mimic-Ctl and miR-4728 inhibitor blocked the decreased MAPK signaling activity in TPC-1 cells (Fig. 4). The luciferase result indicated that transfection of miR-4728 remarkably decrease MAPK activity in TPC-1 cells.

Fig. 4

Effect of miR-4728 regulation on MAPK signaling in TPC-1 cells. TPC-1 cells were co-transfected MAPK luciferase reporter and mimic-Ctl or mimic-miR-4728. miR-4728 inhibitor was added to cells co-transfected with miR-4728. Luciferase activity was normalized to Renilla. Data was shown as mean ± SE, *p < 0.05 vs. Mock (n = 3); **p < 0.05 vs. mimic-Ctl (n = 3); ***p < 0.05 vs. miR-4728 inhibitor (n = 3).

Discussion

MiRNAs are small non-coding endogenous RNAs and the expression and functions of miRNAs are cell and tissue specific. They are about 22 nucleotides in length and play important roles in the post-transcriptional level of gene expression. It has been reported that about 28,645 miRNAs has been verified in animals, plants and virus (Zhang et al., 2015b). MiRNAs bind to the 3′-UTR of target mRNA to repress protein expression by translational inhibition and mRNA cleavage or decay (Fabian et al., 2010). Currently, miRNAs are attractive in research as oncogenes or tumor suppressor genes in the processes of tumorigenesis (Valencia-Sanchez et al., 2006, Kasinski and Slack, 2011, Van Kouwenhove et al., 2011, Iorio and Croce, 2012). A few dysfunctional miRNAs were found in human PTC related to the progression of PTC (He et al., 2005). However, the roles of miR-4728 in the progression of human PTC are still unclear.

In this present study, miR-4728 was found to be down-regulated in human PTC tissues in comparison with normal tissues by qRT-PCR. MiR-4728 contributes to many cellular regulatory processes in cancers. MiR-4728 was observed to be under-expressed in breast tumors compared with normal tissue and miR-4728 controlled MAPK signaling through targeting MST4 (Newie et al., 2014). In this study, we also found that miR-4728 expression was decreased in human PTC cell lines. Further biological functional study of miR-4728 in vitro indicated that miR-4728 overexpression could significantly suppress the proliferation of human PTC cell lines. These results suggested that induction of miR-4728 in human PTC cells could repress the progression of human PTC.

SOS1 belongs to SOS family. SOS (Son of Sevenless) refers to a set of genes encoding guanine nucleotide exchange factors that act on the Ras sub-family of small GTPases in cell signaling. SOS1 is well-known to be important for EGF-dependent signaling to MAP kinase. It has been reported that EGF receptor uses SOS1 to drive constitutive activation of NF-κB signaling in several cancer cell lines including non-small cell lung cancer (NSCLC) cell lines PC9 and A549 (De et al., 2014). However, it is still not fully understood that whether miRNAs play any role in human PTC through SOS1 and whether MAPK signaling is involved in the regulatory network. In this present study, SOS1 was found to be directly repressed by miR-4728. Moreover, we also observed miR-4728 overexpression suppresses MAPK signaling pathway in vitro. In addition, miR-4728 inhibitor blocked the effect caused by miR-4728 overexpression. Our results indicated that miR-4728 regulated MAPK signaling through SOS1. The future efforts will be directed towards developing in vivo model to verify the in vitro results.

In conclusion, this study indicated that miR-4728 was down-regulated in human PTC tissues and cell lines. MiR-4728 overexpression repressed the PTC cell proliferation. Besides, we also demonstrated that miR-4728 regulate human PTC cell behavior through MAPK signaling via SOS1. These results suggested that appropriate regulation of miR-4728 might possess diagnostic and therapeutic potentials for PTC in the future.

Conflicts of interest

The authors declare no conflict of interest.