MicroRNA-138 directly targets TNFAIP8 and acts as a tumor suppressor in osteosarcoma.

MicroRNAs (miRs) have a critical role in the development and malignant progression of osteosarcoma (OS), but the underlying mechanisms have largely remained elusive. The present study aimed to explore the regulatory role of miR-138 in OS growth and metastasis and investigated the associated mechanisms. Reverse-transcription quantitative polymerase chain reaction and western blot analysis were performed to examine the miR-138 and protein expression levels in OS and normal bone tissues and cell lines. An MTT assay and a Transwell assay were used to assess cell proliferation and invasion. Flow cytometry was used to analyze the cell cycle and determine the apoptotic rate. A luciferase reporter assay was used to confirm the targeting association between miR-138 and tumor necrosis factor-α-induced protein 8 (TNFAIP8). It was found that miR-138 was downregulated in OS tissues and cell lines. Overexpression of miR-138 decreased the proliferation, cell cycle progression and invasion of OS cells, while inducing cell apoptosis. TNFAIP8 was then identified as a novel target of miR-138. Similarly to the effects of miR-138 overexpression, inhibition of TNFAIP8 also inhibited OS cell proliferation, cell cycle progression and invasion, and induced cell apoptosis. In addition, miR-138 overexpression as well as downregulation of TNFAIP8 reduced OS cell invasion via inhibition of matrix metalloproteinase-2 and -9 expression. Taken together, the results of the present study demonstrated that miR-138 directly targets TNFAIP8 and acts as a tumor suppressor in OS, suggesting that the miR-138/TNFAIP8 interaction may become a promising therapeutic target for OS.

Introduction

Osteosarcoma (OS), a common malignant bone tumor type, frequently occurs in children and young adolescents (1). Despite improvements in surgical resection combined with chemotherapy and radiotherapy, the survival of patients with advanced or metastatic OS remains poor and their median survival time is only 23 months (2). Therefore, studying the underlying molecular mechanisms of OS growth and metastasis is urgently required for the development of potential diagnostic and therapeutic targets for this disease.

MicroRNAs (miRs) are a class of non-coding small RNAs of 22–25 nucleotides in length, which bind to partially complementary sequences in the 3′-untranslated regions (3′UTRs) of their target mRNAs, causing mRNA degradation and/or translational inhibition (3). Deregulation of various miRs has been implicated in the development and malignant progression of various human cancer types, including OS (4–6). Various miRs have promoting or suppressive effects on OS cell proliferation, cell cycle progression, apoptosis and invasion, such as miR-30a (7), miR-133a (8), miR-137 (4), miR-203 (9), miR-204 (10), miR-205 (11), miR-375 (12) and miR-503 (13).

miR-138 has been demonstrated to be frequently downregulated and act as a tumor suppressor in various human cancers types, including cervical cancer (14), ovarian cancer (15), glioma (16), bladder cancer (17), hepatocellular carcinoma (18), laryngeal carcinoma (19) and non-small cell lung cancer (20). Recently, Zhu et al (21) found that miR-138 has an inhibitory role in OS by targeting EZH2 and enhanced cisplatin-induced apoptosis of OS cells. Furthermore, DEC2 was also found to be a target of miR-138 in OS (22). However, the detailed mechanisms of the effects of miR-138 on OS growth and metastasis have remained largely elusive.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is inducible by transcription factor nuclear factor-κB, has anti-apoptotic and oncogenic functions and is associated with the tumorigenicity of various cancer types (23,24). Li et al (25) found that TNFAIP8 was upregulated in human gastric cancer and promoted tumor cell proliferation, invasion and migration. Recently, Xing and Ren (26) reported that the tumor-suppressive miR-99a inhibited OS cell proliferation via targeting of TNFAIP8. However, whether other miRs also directly target TNFAIP8 in OS has remained elusive.

The present study aimed to explore the regulatory role of miR-138 in OS growth and metastasis, and investigated the underlying mechanisms.

Materials and methods

Clinical tissue collection

The present study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University (Changsha, China). A total of 36 OS patients were recruited and written informed consent was obtained from each of them. Tumor tissues and adjacent non-tumorous tissues were immediately snap-frozen in liquid nitrogen after surgical removal and stored at −80°C prior to use.

Cell culture

The human OS cell lines 143b, LM7, HOS, SaOS-2, U2OS and MG-63 and the normal osteoblastic cell line NHOst were purchased from the Cell Bank of Central South University (Changsha, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a humidified atmosphere containing 5% CO2. The 143b cell line is potentially the same as the HTK cell line (http://web.expasy.org/cellosaurus/CVCL_2270).

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from tissues and cells using the RNeasy Plus mini kit (cat. no. 74134; Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA was then reversely transcribed into complementary DNA using the PrimeScript reverse transcription-PCR kit (cat. no. RR014A; Takara, Dalian, China), according to the manufacturer's instructions. For quantification, PCR was performed using a miRNA Q-PCR Detection kit (cat. no. R0101S; GeneCopoeia, Rockville, MD, USA) on an ABI 7500 thermocycler (Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. U6 was used as an internal reference. Primers were purchased from Fulengen (Guangzhou, China; miR-138 cat. no. HmiRQP017; U6 cat. no. HmiRQP9001) and sequenced were not provided. Each sample was analyzed using three independent experiments. The relative expression of mRNA were determined using the 2−∆∆Cq method (27).

Transfection

miR-138 mimics, scrambled control miR (miR-NC), TNFAIP8 small interfering (si)RNA or non-specific siRNA (all Yearthbio, Changsha, China) were transfected into Saos-2 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After incubation for 48 h, cells were used for further analysis.

MTT assay

Following transfection, cells (5,000 cells/well) were seeded in a 96-well plate and then incubated for 0, 24, 48 or 72 h. Subsequently, 10 µl MTT solution (5 mg/ml; Beyotime Institute of Biotechnology, Haimen, China) was added to each well, followed by incubation for another 4 h. After the supernatant was removed, 100 µl dimethyl sulfoxide was added to each well. The absorbance was detected at 570 nm with a microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Transwell assay

A Cell Invasion Assay kit (cat. no. ECM550; Chemicon International, Temecula, CA, USA) was used to perform the Transwell assay. Cells were seeded in the upper compartment of the chambers, and DMEM containing 10% FBS was added to the lower chambers. After incubation at 37°C for 24 h, cells on the upper face of the membrane were scraped using a cotton swab. Cells on the lower face were stained with crystal violet at room temperature for 5 min and counted under a light microscope.

Cell cycle distribution analysis

Subsequent to transfection, cells were fixed in 75% ethanol at 4°C for 16 h, washed with cold PBS twice and re-suspended in propidium iodide (PI) staining solution at 4°C for 30 min. The cell cycle distribution was then examined using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Apoptosis analysis

Flow cytometry was used to determine the cell apoptosis with an Annexin V-FITC Apoptosis Detection kit (cat. no. 556547; BD Biosciences), according to the manufacturer's instructions. In brief, cells were washed with cold PBS twice, re-suspended in 200 µl binding buffer containing 10 µl Annexin-V-fluorescein isothiocyanate and 5 µl PI, and incubated in the dark for 30 min. Apoptotic cells were examined using a BD FACSCalibur flow cytometer.

Luciferase reporter assay

Targetscan online software (www.targetscan.org) was used to predict the potential target gene of miR-138. A wild-type (WT) fragment of the 3′-UTR of TNFAIP8 containing the miR-138 binding site and its mutated (MUT) sequence were cloned into the pMirGLO reporter vector (Origene Technologies Inc., Rockville, MD, USA), generating WT-TNFAIP8-3′UTR and MUT-TNFAIP8-3′UTR vector, respectively. WT and MUT sequences were obtained from Yearthbio. Cells were co-transfected with WT-TNFAIP8-3′UTR or MT-TNFAIP8-3′UTR, miR-138 mimics or miR-NC using Lipofectamine® 2000. After transfection for 48 h, the dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA) was used to detect the luciferase activity according to the manufacturer's instructions.

Western blot analysis

The cells were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quanitified using a bicinchonininc acid assay kit. Total protein (50 µg/lane) was separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane was subsequently incubated with rabbit anti-human TNFAIP8 antibody (cat. no. ab64988; 1:100), rabbit anti-human MMP2 (cat. no. ab3715; 1:100), rabbit anti-human MMP9 antibody (cat. no. ab38898; 1:100), rabbit anti-human GAPDH antibody (cat. no ab9485; 1:100; all Abcam, Cambridge, UK) at room temperature for 3 h, and then with a goat anti-rabbit secondary antibody (cat. no. ab7090; 1:10,000; Abcam) at room temperature for 1 h, and then visualized using an enhanced chemilluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

Statistical analysis

All experiments were performed three times. Values are expressed as the mean ± standard deviation. Significant differences among the groups were determined by one-way analysis of variance using GraphPad Prism 5 (GraphPad software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

miR-138 is downregulated in OS

The present study firstly examined the expression of miR-138 in 36 OS and adjacent non-tumorous tissues. As presented in Fig. 1A, miR-138 was significantly downregulated in OS tissues compared with that in adjacent tissues. Subsequently, the expression of miR-138 in OS cell lines, including 143b, LM7, HOS, Saos-2, U2OS and MG-63, as well as in the NHOst normal osteoblastic cell line, was determined. The levels of miR-138 in OS cell lines were lower compared with those in NHOst cells (Fig. 1B). Taken together, the results indicated that miR-138 was downregulated in OS.

Figure 1.

Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the expression of miR-138 in (A) 36 OS and adjacent non-tumorous tissues and (B) in the OS cell lines 143b, LM7, HOS, Saos-2, U2OS and MG-63, as well as in the normal osteoblastic cell line NHOst. **P<0.01 vs. Adjacent or NHOst. OS, osteosarcoma; miR, microRNA.

Overexpression of miR-138 decreases OS cell proliferation and cell cycle progression, while inducing apoptosis

As miR-138 was downregulated in OS, miR-138 mimics were transfected into Saos-2 and U2OS cells. After transfection, the expression of miR-138 was significantly increased when compared with that in the miR-NC group (Fig. 2A and B). The effect of miR-138 overexpression on the proliferation and apoptosis of Saos-2 and U2OS was then assessed. As presented in Fig. 2C and D, overexpression of miR-138 led to a significant decrease in cell proliferation. In addition, flow cytometric cell cycle analysis indicated that miR-138 upregulation caused an arrest at the G0/G1 stage (Fig. 2E and F). In addition, overexpression of miR-138 induced OS cell apoptosis (Fig. 2G and H). Therefore, overexpression of miR-138 decreased OS cell proliferation and cell cycle progression, while inducing cell apoptosis.

Figure 2.

Saos-2 and U2OS cells were transfected with miR-NC or miR-138 mimics. (A and B) Reverse transcription-quantitative polymerase chain reaction analysis was used to determine the expression of miR-138. (C and D) An MTT assay was used to determine the cell proliferation. Flow cytometry was used to determine (E and F) the cell cycle distribution and (G and H) the apoptotic rate. *P<0.05; **P<0.01 vs. miR-NC. miR-NC, scrambled miR mimics; miR, microRNA; PI, propidium iodide; FITC, fluorescein isothiocyanate; Q, quadrant; UL, upper left; LR, lower right.

TNFAIP8 is a direct target of miR-138 in OS cells

Bioinformatics analysis identified TNFAIP8 as a putative target of miR-138 (Fig. 3A). To confirm this predication, a fragment from the 3′-UTR of TNFAIP8 containing the miR-138 binding site and its mutated sequence were individually cloned into the pMirGLO reporter vector, generating WT-TNFAIP8-3′UTR and MT-TNFAIP8-3′UTR, respectively (Fig. 3B). A luciferase reporter gene assay then was performed using Saos-2 and U2OS cells. It was demonstrated that overexpression of miR-138 significantly reduced the luciferase activity in the WT-TNFAIP8-3′UTR group, while this effect was not present in the MT-TNFAIP8-3′UTR group (Fig. 3C and D). In addition, overexpression of miR-138 significantly reduced the protein expression of TNFAIP8 protein in SaOS-2 and U2OS cells (Fig. 3E and F).

Figure 3.

(A) Bioinformatics analysis identified TNFAIP8 as a putative target of miR-138. (B) The 3′-UTR of TNFAIP8 mRNA containing the miR-138 binding site and its mutated sequence were cloned into the pMirGLO reporter vector, generating WT-TNFAIP8-3′UTR and MT-TNFAIP8-3′UTR, respectively. (C and D) Overexpression of miR-138 significantly reduced the luciferase activity in the WT-TNFAIP8-3′UTR group of Saos-2 and U2OS cells, while this effect was eliminated in the MT-TNFAIP8-3′UTR group. (E and F) Western blot analysis was used to determine the protein levels of TNFAIP8 in Saos-2 and U2OS cells transfected with miR-NC or miR-138 mimics. **P<0.01 vs. miR-NC. UTR, untranslated region; miR-NC, scrambled miR mimics; miR, microRNA; WT, wild-type; MT, mutant; TNFAIP8, tumor necrosis factor-α-induced protein 8.

Knockdown of TNFAIP8 reduces OS cell proliferation, cell cycle progression and promotes apoptosis

The present study further assessed the role of TNFAIPT8 in the regulation of OS cell proliferation, cell cycle progression and apoptosis. NFAIP8-specific siRNA was used to transfect Saos-2 and U2OS cells, while NC siRNA was used as a control. After transfection, the protein expression of TNFAIP8 was significantly reduced in the TNFAIP8 siRNA group compared with that in the NC siRNA group (Fig. 4A and B). Further investigation revealed that knockdown of TNFAIP8 markedly decreased the proliferation and of Saos-2 and U2OS and caused cycle arrest in G0/G1 phase (Fig. 4C-F). In addition, an increased apoptotic rate of Saos-2 and U2OS cells was identified after silencing of TNFAIP8 (Fig. 4G and H). Accordingly, knockdown of TNFAIP8 reduced OS cell proliferation and cell cycle progression, and promoted apoptosis.

Figure 4.

Saos-2 and U2OS cells were transfected with NC siRNA or NFAIP8-specfic siRNA. (A and B) Western blot analysis was used to determine the protein levels of TNFAIP8. (C and D) An MTT assay was used to determine the cell proliferation. Flow cytometry was used to determine (E and F) the cell cycle distribution and (G and H) the apoptotic rate. **P<0.01 vs. NC siRNA. TNFAIP8, tumor necrosis factor-α-induced protein 8; siRNA, small interfering RNA; NC siRNA, negative control siRNA; miR, microRNA; PI, propidium iodide; FITC, fluorescein isothiocyanate; Q, quadrant; UL, upper left; LR, lower right.

miR-138 overexpression and TNFAIP8 silencing reduces matrix metalloproteinase (MMP)-2/−9-mediated OS cell invasion

The present study further assessed the potential role of miR-138 and TNFAIP8 in the regulation of OS cell invasion. Transwell assays demonstrated that the cell invasion was reduced in the miR-138 group compared with that in the miR-NC group (Fig. 5A and B). Similarly, knockdown of TNFAIP8 also reduced the invasive capacity of Saos-2 and U2OS cells (Fig. 5C and D). Furthermore, the protein expression of MMP-2 and −9, which are closely associated with tumor cell invasion, was assessed. Western blot analysis revealed that miR-138 overexpression as well as TNFAIP8 silencing reduced MMP2 and MMP9 protein levels (Fig. 5E-H). Therefore, miR-138 overexpression and TNFAIP8 silencing reduced MMP-2/−9-mediated OS cell invasion.

Figure 5.

Transwell assays were used to examine the invasion of Saos-2 and U2OS cells transfected with (A and B) miR-NC or miR-138 mimics and (C and D) NC siRNA or TNFAIP8-specific siRNA. **P<0.01 vs. miR-NC or NC siRNA. (E-H) Western blot analysis was used to determine the protein levels of MMP2 and MMP9 in Saos-2 and U2OS cells transfected with (E and F) miR-NC or miR-138 mimics or (G and H) NC siRNA or TNFAIP8-specific siRNA. Magnification, ×200. siRNA, small interfering RNA; NC siRNA, negative control siRNA; miR, microRNA; miR-NC, scrambled miR mimics; MMP, matrix metalloproteinase; TNFAIP8, tumor necrosis factor-α-induced protein 8.

Discussion

To date, the regulatory roles of miR-138 in OS and its progression as well as the underlying molecular mechanisms have remained elusive. The present study found that miR-138 was downregulated in OS tissues and cell lines. Overexpression of miR-138 reduced OS cell proliferation, cell cycle progression and invasion, while inducing cell apoptosis. TNFAIP8 was then identified as a target of miR-138. Knockdown of TNFAIP8 produced similar effects on the malignant phenotypes of OS cells to those of miR-138 overexpression.

miR-138 has been found to have suppressive effects on tumor cell proliferation and invasion in various cancer types (16,17,28). For instance, miR-138 inhibits the invasion of bladder cancer cells through inhibiting the protein expression of zinc finger E-box-binding homeobox 2 (17). Xu et al (28) reported that miR-138 suppressed the proliferation of oral squamous cell carcinoma cells by targeting Yes-associated protein 1. The present study identified that miR-138 was downregulated in OS tissues when compared with that in matched adjacent non-tumorous tissues. In addition, its expression in OS cell lines was also lower compared with that in a normal osteoblast cell line. Furthermore, overexpression of miR-138 inhibited the proliferation of OS cells through inducing cell cycle arrest at the G0/G1 stage. Similar findings were previously reported in nasopharyngeal carcinoma (29) and non-small cell lung cancer (30). In addition, the present study found that miR-138 induced OS cell apoptosis and inhibited cell invasion. Overexpression of miR-138 was also found to promote cell apoptosis in clear cell renal cell carcinoma (31), neuroblastoma (32) and gallbladder carcinoma (33). Yu et al (34) found that curcumin inhibited the proliferation and invasion of OS cells by upregulating the expression of miR-138, which was consistent with the present findings.

The present study further identified TNFAIP8 as a direct target of miR-138, and its expression was negatively regulated by miR-138 at the post-transcriptional level in OS cells. TNFAIP8, also known as NDEN or SCCS2, encodes an anti-apoptotic protein. A previous study found that TNFAIP8 is essential for the regulation of glucocorticoid-mediated apoptosis of thymocytes (35). Furthermore, TNFAIP8 was found to be an effector for Gα i) coupling to reduce cell death and induce cell transformation (36). TNFAIP8 was found to be frequently upregulated in human cancers, and to have an oncogenic role. For instance, high expression of TNFAIP8 was significantly associated with the tumor-nodes-metastasis stage, tumor depth, lymph node metastasis, distant metastasis, lymphatic invasion and venous invasion, as well as poor prognosis in esophageal squamous cell carcinoma patients (37). It also has effects on the response of prostate cancer to radiation and docetaxel as well as disease recurrence (38). In addition, TNFAIP8 was suggested to be a predictor of metastasis and a prognostic biomarker in patients with epithelial ovarian cancer (39). However, the role of TNFAIP8 in OS growth and metastasis and the underlying mechanisms has remained to be fully elucidated. The present study found that knockdown of TNFAIP8 inhibited OS cell proliferation and invasion, and induced cell apoptosis, and these effects were similar to those of miR-138 overexpression, which further suggested that the suppressive effects of miR-138 on the malignant phenotypes of OS cells were mediated, at least in part, through inhibition of TNFAIP8. Consistent with the present findings, Xing and Ren (26) also reported that inhibition of TNFAIP8 reduced OS cell viability and growth through inhibiting the cell cycle and inducing cell apoptosis in vitro and in vivo.

In conclusion, to the best of our knowledge, the present study was the first to suggest that miR-138 has an inhibitory role in OS cell proliferation and invasion through directly targeting TNFAIP8. The present study expanded the current understanding of the molecular mechanisms underlying OS growth and metastasis, and suggested the miR-138/TNFAIP8 interaction as a potential therapeutic target for OS.