MicroRNA-136 functions as a tumor suppressor in osteosarcoma via regulating metadherin

Abstract

BACKGROUND:

Accumulating studies have reported the abnormal expression of microRNA-136 (miR-136) in numerous types of human cancer, and its involvement in cancer initiation and progression. However, there are no investigations of miR-136 in osteosarcoma (OS).

OBJECTIVE:

To explore the expression pattern, clinical significance and potential roles of miR-136 in OS.

METHODS:

miR-136 expression in clinical OS tissues and human OS cell lines were detected by qPCR, and its associations with clinicopathological characteristics of OS patients were statistically analyzed. Then, the effects of miR-136 on OS cell proliferation, migration and invasion were assessed in vitro. Its underling mechanisms were also investigated.

RESULTS:

miR-136 expression in OS tissues and cells were dramatically decreased compared with corresponding non-cancerous tissues and cells, respectively. Low miR-136 expression was significantly associated with aggressive clinical features, including the advanced clinical stage, the presence of lung and distant metastasis (all $P<$ 0.05). Additionally, enforced expression of miR-136 obviously inhibited the proliferation, migration and invasion of OS cells in vitro. Mechanistically, metadherin (MTDH) was predicted and verified as a target gene of miR-136. Further functional experiments indicated that the loss of MTDH abrogated the tumor suppressive roles of miR-136 in OS cells.

CONCLUSION:

Our findings provide the first evidence that the aberrant expression of miR-136 may be implicated into carcinogenesis and cancer progression of OS. Functionally, miR-136 may inhibit the proliferation, migration and invasion of OS cells via negatively regulating its target gene MTDH. Thus, miR-136-MTDH axis may be a potential therapeutic targets for the treatment of OS.

Keywords

Osteosarcoma microRNA-136 metadherin clinicopathologic characteristics proliferation migration invasion

1. Introduction

Osteosarcoma (OS) represents the third most common primary malignant bone tumor in childhood and adolescents [1]. It often occurs in the proximal tibia and humerus, as well as the metaphyseal regions of distal femur [2]. Typical symptoms and signs of this malignancy contain localized swelling, joint movement limitations, pain and trabecular bone destruction [3]. OS is characterized by the high degree of malignancy, as well as the increasing incidence of recurrence and metastasis [4]. Therapeutic strategies for OS therapy include surgical resection combined with radiotherapy and chemotherapy [5]. Despite many efforts have been made, the prognosis of OS is still unsatisfied. The five-year survival rate of OS patients is as low as 60–70% globally. What is more, the clinical outcome of patients with metastatic disease is usually far worse (less than 20%) [6]. Therefore, it is urgent to explore the underlying mechanisms of OS and identify novel biomarkers for improving the prognosis of OS patients.

Table 1
Associations of miR-136 expression with various clinicopathological characteristics of OS patients (qPCR analysis was performed to detect the expression levels of miR-136 in 100 OS tissues. The associations between miR-136 expression and various clinicopathological characteristics were assessed by the chi-square or Fisher’s exact tests)

Clinicopathological features	No. (%)	miR-136-low ( $n$ , %)	miR-136-high ( $n$ , %)	$P$
Gender
Male	78 (78.00)	42 (53.85)	36 (46.15)	0.36
Female	22 (22.00)	10 (45.45)	12 (54.55)
Age (years)
$<$ 20	70 (70.00)	36 (51.43)	34 (48.57)	0.82
$\geqslant$ 20	30 (30.00)	16 (53.33)	14 (46.67)
Anatomical location
Femur	56 (56.00)	27 (48.21)	29 (51.79)	0.41
Tibia	32 (32.00)	18 (56.25)	14 (43.75)
Humerus	8 (8.00)	5 (62.50)	3 (37.50)
Fibula	4 (4.00)	2 (50.00)	2 (50.00)
Clinical stage
IA–IB	6 (6.00)	0 (0)	6 (100.00)	0.008
IIA–IIB	26 (26.00)	4 (15.38)	22 (84.62)
III	68 (68.00)	48 (70.59)	20 (29.41)
Tumor size (cm)
$<$ 10	75 (75.00)	37 (49.33)	38 (50.67)	0.42
$\geqslant$ 10	25 (25.00)	15 (60.00)	10 (40.00)
Lung metastasis
Negative	58 (58.00)	18 (31.03)	40 (68.97)	0.02
Positive	42 (42.00)	34 (80.95)	8 (19.05)
Distant metastasis				0.03
Negative	60 (60.00)	22 (36.67)	38 (63.33)
Positive	40 (40.00)	30 (75.00)	10 (25.00)

Note: ‘NS’ refers to the difference without statistical significance.

MicroRNAs (miRNAs), a group of single stranded, non-coding and small RNAs with 18 $\sim$ 25 nucleotides in length, is extensively expressed in eukaryotic cells [7]. miRNAs regulate the expression of target genes negatively at a post-transcriptional level by binding to specific complimentary recognition sequences in the 3’-untranslated region (UTR) mRNA sequence of the corresponding target genes [8]. Functionally, growing evidences indicate that miRNAs play crucial roles in diverse biological processes, including development, cell growth, proliferation, apoptosis and cell cycle [9]. Under the pathological conditions, accumulating studies also reported that the molecular changes of miRNAs are often involved into carcinogenesis, progression and prognosis of many types of human cancers, including OS [10, 11]. On the basis of the functions of their target genes, miRNAs either function as oncogenes or tumor suppressors in different cancers [12, 13]. miR-136 was originally identified by cloning studies in mouse. In recent years, numerous investigations reported that the abnormal expression of miR-136 may be tightly associated with cancer initiation and progression [14, 15, 16, 17, 18]. For example, Zhao et al. [14] found that miR-136 function as an anti-oncogene and deficiency of miR-136 expression in ovarian cancer induced chemoresistance, which was consistent with the findings of Jeong et al. [15]; Yan et al. [16] revealed that miR-136 suppressed tumor invasion and metastasis by targeting RASAL2 in triple-negative breast cancer; Yuan et al. [17] indicated that miR-136 might act as a negative regulator in colon cancer progression; In contrast, Shen et al. [18] showed that miR-136 was upregulated in human non-small cell lung cancer and cell lines compared to their non-cancerous counterparts, and it also promoted Erk1/2 phosphorylation through targeting PPP2R2A in lung cancer cells. These previous data suggest that miR-136 may exert different roles in various types of human cancers. However, there are no studies of miR-136 in OS.

To explore the expression pattern, clinical significance and potential roles of miR-136 in OS, in the current study, expression levels of miR-136 in clinical OS tissues and human OS cell lines were detected by reverse transcription quantitative polymerase chain reaction (qPCR). Associations of miR-136 expression with clinicopathological characteristics of OS patients were statistically analyzed. Then, the effects of miR-136 on OS cell proliferation, migration and invasion were assessed in vitro. A putative target of miR-136, metadherin (MTDH), was obtained from miRTarBase, and was verified by luciferase activity assay. After that, the effect of MTDH loss on miR-136 downregulated in OS cells was further observed.

2. Materials and methods

2.1 Patients and tissue samples

Our study was performed in compliance to the National Research Council’s guide and approved by the Research Ethics Committee of Beijing Luhe Hospital Affiliated to Capital Medical University and Hangzhou Cancer Hospital. The written informed consent was obtained from all the patients with Osteosarcoma prior to collection of samples.

A total 100 histologically diagnosed OS and adjacent noncancerous bone tissues (located over 3 cm away from the tumor) were obtained from 100 patients with primary OS between April 2010 to March 2015 in the Department of Orthopedics, Beijing Luhe Hospital Affiliated to Capital Medical University and Department of Orthopedic Oncology, Hangzhou Cancer Hospital. Each pair of osteosarcoma and adjacent tissue was collected from the same patient after pathological verification. No patient received immunotherapy, radiotherapy or chemotherapy before surgery. Tissue specimens were immediately frozen in liquid nitrogen until further use. The clinicopathological features of these patients were summarized in Table 1.

2.2 Cell culture and transfection

Two OS cell lines MG-63 and U2OS, as well as human osteoblasts hFOB1.19 were obtained from the American Type Culture Collection (Hill, NJ, USA), and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 $\mu$ g/ml streptomycin. All cells were incubated at 37 ${}^{\circ}$ C in a humidified atmosphere with 5% CO ${}_{2}$ .

When the MG-63 and U2OS cells were grown in the logarithmic growth phase, the cells were cultured in a 24-well plate with 2 $\times$ 10 ${}^{5}$ cells per well overnight. miR-136 mimic, non-targeting control mimic (NC mimics), miR-136 inhibitor, control inhibitor (NC inhibitor), and MTDH-specific small interfering RNA (si-MTDH) were all synthesized by GenePharma Co., Ltd. (Shanghai, China). The sequences of miR-136 mimics: 5’-ACUCCAUUUGUUUUGAUGAUGGA-3’ (sense strand), 5’-UCCAUCAUCAAAACAAAUGG AGU-3’ (anti-sense strand); NC mimics: 5’-UUUGUA CUACACAAAAGUACUG-3’ (sense strand), and 5’-CAGUACUUUUGUGUAGUACAAA-3’ (anti-sensestrand). The siRNA oligos sequences targeting MTDH were: 5’-GGAGGAGGCUGGAAUGAAAdTdT-3’.Negative control siRNA: 5’-AACUCCUGCCUCCUU AUGUAUUUdTdT-3’. Cell transfection was performed by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Forty-eight hours after transfection, cells were verified and used for analysis.

2.3 RNA extraction and qPCR

Total RNA (including miRNA) was extracted from clinical tissues or cells by using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and purified with miRNeasy mini kit (Qiagen, Hilden, Germany). The RNA concentration was quantified on a NanoDrop 2000 (Thermo Scientific, USA) by O.D. 260/280 nm absorbance. Reverse transcription assay and real time PCR assay were performed using PrimeScript RT reagent Kit (TaKaRa, Dalian, China) and SYBR Premix Ex TaqII (Tli RNaseH Plus) kit (TaKaRa, Dalian, China) respectively. The miR-136 expression was normalized with U6 small RNA as internal control. The primer sequences used in this study were as following: miR-136, forward 5’-ACA CTC CAG CTG GGA CTC CAT TTG TTT TG-3’, reverse 5’-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TCC ATC AT-3’; U6, forward 5’-CTC GCT TCG GCA GCA CA-3’, reverse 5’-AAC GCT TCA CGA ATT TGC GT-3’. The relative expression was analyzed using 2 ${}^{-\Delta\Delta\rm CT}$ method. All experiments were conducted in triplicate.

2.4 Cell proliferation assay

The CCK-8 assay was performed to evaluate the cell proliferation of MG63 and U2OS cells transfected with miR-136/NC mimics or miR-136/NC inhibitor. At 48 h post-transfection, the cells were collected and seeded into 96-well plates at a density of 3,000 cells/well. Then, the cells were cultured at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ for 1, 2, 3 and 4 days, and the CCK-8 fluids with a final concentration of 10% were added into each well. After that, the cells were maintained at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ for 1 h. The absorbance was measured using a Microplate Reader (Bio-Rad, Hercules, CA, USA) at a 450 nm wavelength. All experiments were conducted in triplicate.

Figure 1.

Decreased expression of miR-136 in human osteosarcoma (OS) tissues and cells. (A) miR-136 expression was significantly decreased in OS tissues compared with matched normal tissues (OS vs. non-cancerous $=$ 1.43 $\pm$ 0.87 vs. 3.53 $\pm$ 1.92, $P<$ 0.001, $n=$ 100/group, paired Student’s t-test). (B) The expression levels of miR-136 in MG63 and U2OS cells were both markedly lower than those in human osteoblast hFOB1.19 cells (MG63 vs. hFOB1.19 $=$ 1.23 $\pm$ 0.85 vs. 2.45 $\pm$ 1.33, ** $P<$ 0.001; U2OS vs. hFOB1.19 $=$ 1.10 $\pm$ 0.83 vs. 2.45 $\pm$ 1.33, ** $P<$ 0.001, the experiments were conducted in triplicate, unpaired Student’s t test). qPCR analysis was performed to detect the expression levels of miR-136 in OS tissues and cell lines.

Figure 2.

miR-136 expression suppresses the cell proliferation, migration and invasion of OS cells in vitro. (A and B) Relative expression levels of miR-136 in blank, miR-136-mimic and NC-mimic-transfected MG63 and U2OS cells, respectively. (C and D) The cell viabilities of blank, miR-136-mimic and NC-mimic-transfected MG63 and U2OS cells, respectively. (E and F) The migrated and invasive capacities of blank, miR-136-mimic and NC-mimic-transfected MG63 and U2OS cells, respectively. ** $P<$ 0.01, comparison with blank; ${}^{\#\#}P<$ 0.01, comparison with NC-mimic group.

2.5 Cell migration and invasion assay

The transwell migration assay was performed to assess the migration abilities of MG63 and U2OS cells transfected with miR-136/NC mimics or miR-136/NC inhibitor. Following the transfection, the cells were harvested and resuspended in Eagle’s minimum essential medium. Cell suspension (100 $\mu$ l) was added to the top chamber of transwell (8 $\mu$ m pore size, Millipore, Billerica, MA, USA), and 400 $\mu$ l of medium containing 10% fetal bovine serum was added to the bottom chamber of well. At 12 h following the incubation, transwells were fixed in 4% paraformaldehyde and non-migrated cells in the upper side of the transwell membrane were removed completely.

Matrigel coated cell culture chambers (8 $\mu$ m pore size, Millipore, Billerica, MA, USA) were used to evaluate the invasion abilities of MG63 and U2OS cells transfected with miR-136/NC mimics or miR-136/NC inhibitor. The transfected cells were cultured in 24-well plates until confluent. At 48 h post-transfection, the transfected cells were resuspended in 200 $\mu$ l serum-free Eagle’s minimum essential medium and placed into the upper chamber of the insert with Matrigel. Bottom chambers were filled with conditioned medium. Following the incubation for 48 h, the cells remaining on the upper filter were scrubbed off.

The migrated or invasive MG63 and U2OS cells adherent to the lower surface of the filter were fixed with methanol, stained with 0.5% crystal violet for 20 min, photographed, and counted. The number of migrated or invasive MG63 and U2OS cells was counted at 200 $\times$ magnification from 10 different fields of each filter. All experiments were conducted in triplicate.

2.6 Western blot analysis

MTDH protein expression levels in MG63 and U2OS cells transfected with miR-136/NC-mimics, or siRNA-MTDH/siRNA-NC were detected by western blot analysis. Briefly, total protein in OS cells of different groups was extracted using 500 $\mu$ l of radio-immunoprecipitation assay buffer (RIPA buffer). Protein concentrations were quantified using the BCA protein assay kit (Pierce Chemical Company, Rockford, IL, USA). Then, equal amounts of protein extracts (10 $\mu$ g) were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass, USA). The primary antibody, a rabbit anti-human MTDH monoclonal antibody (1:500 dilution; Abcam, MA, USA) was used to detect the expression of MTDH protein. The relative expression of MTDH protein was normalized by GAPDH as a loading control. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Life Science, Amersham Biosciences Co., CA, USA) and analyzed densitometrically using Quantity One Image software (Bio-Rad, California, USA).

2.7 Dual luciferase reporter gene assay

According to the data of miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/, Release 6.0), MTDH was predicted as a putative target gene of miR-136. After that, dual luciferase reporter gene assay was performed to evaluate the binding efficiency between miR-136 and MTDH mRNA in OS cells. PGL3-MTDH 3’-UTR (5’-AACUUUUGGUAUAUGAUGGAGAA-3’)/ PGL3-MT DH 3’-UTR-mut (5’-AACUUUUGGUAU AUGAUGC UGAA-3’) luciferase reporter plasmid were purchased from Promega (Madison, WI). When MG63 and U2OS cells were grown on 70% confluence, the cells were cotransfected with miR-136 vector and PGL3-MTDH 3’-UTR/PGL3-MTDH 3’-UTR-mut luciferase reporter plasmid using Lipofectamine-2000 transfection reagent (Life Technologies Corporation, Carlsbad, USA). At 24 h post-transfection, cell lysates were prepared and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA) according to manufacturer’s instructions.

Figure 3.

MTDH is a direct target of miR-136 in OS cells. (A and B) Relative expression of MTDH protein in both MG63 and U2OS cells in different groups. ** $P<$ 0.01, comparison with blank; ${}^{\#\#}P<$ 0.01, comparison with NC-mimic group. (C) Complementary sequences between miR-136 and MTDH gene. (D and E) Luciferase activity in both MG63 and U2OS cells in different groups. ** $P<$ 0.01, comparison with the OS cells cotransfected with hsa-miR-136 vector and PGL3-vector; ${}^{\#\#}P<$ 0.01, comparison with the OS cells cotransfected with hsa-miR-136 vector and PGL3-MTDH 3’-UTR-mut.

Figure 4.

Loss of MTDH abrogates the tumor suppressive roles of miR-136. (A and B) Relative expression of MTDH protein in both MG63 and U2OS cells in different groups. ** $P<$ 0.01, comparison with the OS cells transfected with hsa-miR-136 inhibitor; (C $\sim$ F) miR-136 knockdown significantly enhanced the cell proliferation, migration and invasion of OS cells, which were all distinctly suppressed by the loss of MTDH; g1 $\sim$ g4 refer to blank, miR-136-inhibitor, NC-inhibitor, and miR-136-inhibitor $+$ si-MTDH groups; ** $P<$ 0.01, comparison with the OS cells transfected with hsa-miR-136 inhibitor.

2.8 Statistical analysis

Our data analysis were performed using SPSS version 11.0 software for Windows (SPSS Inc, IL, USA). Data were showed as mean $\pm$ SD. The difference of miR-136 expression between OS and adjacent non-cancerous tissues was evaluated using paired Student’s $t$ -test. The associations between miR-136 expression and various clinicopathological characteristics were assessed by the chi-square or Fisher’s exact tests. The differences between cell groups with different treatment were evaluated by unpaired Student’s $t$ test or one-way ANOVA. Differences were considered significant when $P<$ 0.05.

3. Results

3.1 Decreased expression of miR-136 in human OS tissues and cells

To assess the potential clinical relevance of miR-136 to carcinogenesis of OS, we firstly detected the expression levels of miR-136 in OS tissues and cell lines using qPCR. As shown in Fig. 1A, miR-136 expression was significantly decreased in OS tissues compared with matched non-cancerous bone tissues (OS vs. non-cancerous $=$ 1.43 $\pm$ 0.87 vs. 3.53 $\pm$ 1.92, $P<$ 0.001, Fig. 1A). Consistently, the expression levels of miR-136 in MG63 and U2OS cells were both markedly lower than those in human osteoblast hFOB1.19 cells (MG63 vs. hFOB1.19 $=$ 1.23 $\pm$ 0.85 vs. 2.45 $\pm$ 1.33, $P<$ 0.001; U2OS vs. hFOB1.19 $=$ 1.10 $\pm$ 0.83 vs. 2.45 $\pm$ 1.33, $P<$ 0.001; Fig. 1B).

3.2 Decreased expression of miR-136 associates with aggressive progression of OS patients

To evaluate the associations of miR-136 expression with various clinicopathological characteristics of OS patients, all 100 patients were divided into low miR-136 expression ( $n=$ 52) and high miR-136 expression ( $n=$ 48) groups using the median value (1.33) of miR-136 expression as a cutoff value. As shown in Table 1, OS patients with lung metastasis showed lower miR-136 expression than those without ( $P=$ 0.02). The expression levels of miR-136 in OS patients with positive distant metastasis were decreased compared with negative patients ( $P=$ 0.03). Moreover, OS patients with advanced clinical stage more often had low miR-136 expression than those with early clinical stage ( $P=$ 0.008).

3.3 miR-136 expression suppresses the cell proliferation, migration and invasion of OS cells in vitro

To evaluate the potential significance of miR-136 in malignant phenotypes of human OS cells, we transfected miR-136 mimic and non-targeting control mimic (NC mimic) into MG63 and U2OS cells, respectively. Following the transfection, the expression levels of miR-136 in miR-136-mimic-transfected MG63 and U2OS cells were both significantly increased compared with that in NC mimic group (both $P<$ 0.001, Fig. 2A and B). The CCK-8 assay showed that the cell viabilities of two OS cell lines were both markedly decreased by miR-136 upregulation in comparison with NC mimic group (both $P<$ 0.01, Fig. 2C and D). The transwell migration and invasion assays further demonstrated that the enforced expression of miR-136 markedly suppressed the migrated and invasive capacities of OS cells (both $P<$ 0.05, Fig. 2E and F).

3.4 MTDH is a direct target of miR-136 in OS cells

According to miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/, Release 6.0), MTDH was a candidate target of miR-136. To validate this data, we firstly detected the expression level of MTDH protein in OS cells transfected with miR-136 mimics. As shown in Fig. 3A and B, enforced expression of miR-136 markedly reduced the expression of MTDH protein in both MG63 and U2OS cells (both $P<$ 0.01). Then, dual luciferase reporter gene assay was performed to verify the direct regulatory effect of miR-136 on MTDH expression by binding with the 3’-UTR of the gene. The complementary sequences between miR-136 and MTDH gene was shown in Fig. 3C. The luciferase activity in both MG63 and U2OS cells co-transfected with hsa-miR-136 vector and PGL3-MTDH 3’-UTR was dramatically lower than that with hsa-miR-136 vector and PGL3-MTDH 3’-UTR-mut (both $P<$ 0.01, Fig. 3D and E).

3.5 Loss of MTDH abrogates the tumor suppressive roles of miR-136

To validate whether the tumor suppressive roles of miR-136 in OS cells were associated with its regulation on MTDH, we transfected miR-136-inhibitor alone or combined with si-MTDH into two OS cell lines. As a result, the expression levels of MTDH protein was significantly reduced by the co-transfection of miR-136-inhibitor and si-MTDH in comparison with those in OS cells transfected with miR-136-inhibitor alone (both $P<$ 0.01, Fig. 4A and B). Functionally, the knockdown of miR-136 significantly enhanced the cell proliferation, migration and invasion of OS cells, which were all distinctly suppressed by the loss of MTDH (all $P<$ 0.05, Fig. 4C–F).

4. Discussion

miRNAs with multifaceted activities regulate the corresponding target gene expression and exhibit direct or indirect oncogenic or tumor suppressive properties in different types of cancers. In the current study, we observed the decreased expression of miR-136 in both clinical OS tissues and human OS cell lines for the first time. Importantly, low miR-136 expression was significantly associated with aggressive clinical features, including the advanced clinical stage, the presence of lung and distant metastasis. Moreover, we found that the enforced expression of miR-136 obviously inhibited the proliferation, migration and invasion of OS cells in vitro. Mechanistically, MTDH was predicted and verified as a target gene of miR-136, and further functional experiments indicated that the loss of MTDH abrogated the tumor suppressive roles of miR-136 in OS cells.

Recent studies has indicated that miR-136 may play tumor suppressive roles in breast cancer, ovarian cancer and colon cancer, or a oncogenic role in non-small cell lung cancer [14, 15, 16, 17, 18]. Here, our data suggested that miR-136 may function as an anti-oncogenic regulator in human OS. Since miRNAs exert their roles though directly regulating target genes, in this study, our results obtained from dual luciferase reporter gene assay suggest that the fragment at the 3’-UTR of the MTDH mRNA may be the complementary site for the miRNA-136 seed region. Thus, MTDH may be one of the direct targets of miR-136. MTDH, also known as Astrocyte elevated gene-1 (AEG-1) and Lysine-rich CEACAM1 co-isolated (LYRIC), has been indicated as a crucial oncogene with multifunctional roles in cancer development and progression via involved into various oncogenic signaling pathways, such as the ERK/mitogen-activated protein kinase and Wnt/ $\beta$ -catenin pathways, the Ha-Ras and PI3K/Akt pathways, the Aurora-A kinase signaling pathway and the nuclear factor- $\kappa$ B signaling pathway [19, 20]. Accumulating studies have reported the associations of MTDH with various human cancers, including OS [21, 22, 23, 24, 25]. Moreover, it also has been revealed the regulatory mechanisms of miR-136-MTDH axis in hepatocellular carcinoma [26] and gliomas [27, 28]. Zhao et al. [26] found that the hepatitis B viral X protein enabled to increase oncoprotein MTDH expression and to promote cell migration via reducing the expression of miR-136; Wu et al. [27] indicated that miR-136 might play a key role in temozolomide resistance by targeting MTDH in glioma cell line; Yang et al. [28] also observed that miR-136 could promote apoptosis of glioma cells by targeting MTDH. In line with these previous studies, our data demonstrated that the co-transfection of si-MTDH abrogated the effect of miR-136 knockdown on cell proliferation, migration, invasion of both two OS cells, implying an important roles of miR-136-MTDH axis in OS.

In conclusion, our findings provide the first evidence that the aberrant expression of miR-136 may be implicated into carcinogenesis and cancer progression of OS. Functionally, miR-136 may inhibit the proliferation, migration and invasion of OS cells via negatively regulating its target gene MTDH. Thus, miR-136-MTDH axis may be a potential therapeutic targets for the treatment of OS. Further studies are needed to validate these findings in vivo and clinical experiments.

Footnotes

Conflict of interest

None.

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