MicroRNA‑643 regulates the expression of ZEB1 and inhibits tumorigenesis in osteosarcoma.

Osteosarcoma is among the most malignant types of tumor worldwide and has become a leading contributor to tumor incidence, particularly in adolescents. Resistance to conventional treatment and the complexity of osteosarcoma tumorigenesis has resulted in high mortality rates. MicroRNAs are a class of noncoding RNAs, which regulate numerous biological processes. However, the involvement of miR‑643 in osteosarcoma remains to be elucidated. In the present study, reverse transcription‑quantitative polymerase chain reaction, luciferase reporter assay, invasion assay, viability assay, western blot analysis and in vivo implantation were performed to analyze the action of miR‑643 in osteosarcoma. The results demonstrated that miR‑643 inhibited the progression of osteosarcoma and acted as a potential tumor suppressor. The expression of miR‑643 was downregulated in osteosarcoma tissues and cell lines. In addition, miR‑643 transfection significantly impaired the proliferation and invasion of osteosarcoma cells. The present study also identified Zinc finger E‑box‑binding homeobox 1 (ZEB1) as a direct target of miR‑643, and the ectopic expression of ZEB1 counteracted the effect of miR‑643 transfection. A significant inverse correlation was also found between the expression of miR‑643 and ZEB1. A low expression of miR‑643 or a high expression of ZEB1 was associated with poor patient survival rates. The results of the present study suggested that the decreased expression of miR‑643 may be involved in the mechanism underlying the development of osteosarcoma. The intricate interactions between miR‑643 and ZEB1 may serve as a potential therapeutic target in osteosarcoma oncogenesis.

Introduction

Osteosarcoma is one of the most malignant types of tumor and contributes to mortality rates among adolescents (1). It is reported that ~2/3 of cases of bone cancer are ascribed to osteosarcoma worldwide (2). Despite advances in cancer therapy, the survival rate of the majority of patients with osteosarcoma remains low (3). Previous studies have provided clues regarding the molecular mechanisms underlying the occurrence of osteosarcoma, however, the details remain to be fully elucidated (4). Therefore, further understanding of the mechanisms underlying tumor incidence and effective strategies for early diagnosis are urgently required.

MicroRNAs are a class of small noncoding RNAs of ~22 nucleotides, which can regulate gene expression by base-pairing with transcripts (5). Even the incomplete binding of microRNAs to targets can lead to degradation or translation inhibition (6). Different microRNA expression patterns can be found in different types of tumor, suggesting that microRNAs may be used as diagnostic markers or therapeutic targets (7). Several studies have suggested that microRNAs may be important in the dynamic modulation of carcinogenesis and may be used as biomarkers. For example, a previous study indicated that microRNA (miR)-539 may be a tumor suppressor in osteosarcoma and the inhibitory effect is mediated through matrix metallopeptidase-8 (8). miR-133a was found to suppress the progression and metastasis of osteosarcoma, the inhibitory function of which was mediated by insulin-like growth factor 1 receptor (9). In another previous study, Ge et al (10) showed that miR-497 can also inhibit the development of osteosarcoma by inducing cell cycle arrest and apoptosis, however, the exact target was not clearly defined. Certain microRNAs can promote the tumorigenesis of osteosarcoma. Ni et al (11) found that miR-301a is frequently upregulated in osteosarcoma and that aberrant expression of miR-301a promotes the proliferation of osteosarcoma cell lines. miR-17-5p has also shown diagnostic and prognostic significance in osteosarcoma, as the overexpression of miR-17-5p substantially promotes malignant phenotypes (12). Another report showed that miR-20a can advance osteosarcoma metastasis by targeting Fas and attenuating apoptotic pathways (13). However, the role of miR-643 in osteosarcoma remains to be fully elucidated.

In the present study, the role of miR-643 in osteosarcoma was investigated. It was shown that miR-643 was frequently downregulated in osteosarcoma tissues and cell lines. Introducing miR-643 mimics inhibited the proliferation and reduced the invasive capacity of MG-63 and Saos-2 cells. It was shown that Zinc finger E-box-binding homeobox 1 (ZEB1) may be a direct target of miR-643 using bioinformatics analysis. The overexpression of ZEB1 counteracted the effect of miR-643 and restored the malignant phenotypes of the osteosarcoma cells. Patients with higher expression levels of miR-643 had improved overall survival rates. miR-643 transfection also significantly inhibited solid tumor growth in vivo. Taken together, these results suggested a tumor suppressive role of miR-643 in osteosarcoma and suggested that miR-643 may be a putative target for targeted therapy and diagnosis.

Materials and methods

Cell culture and human samples

The osteosarcoma cell lines used in the present study, including 143B, U-2OS, KHOS-312H, KHOS-240S, UMR-106, Saos-2, HOS, T1-73, MG-63 and Hs 890.T, and the normal cell line (hFOB) were purchased from The Shanghai Institute of Cell Biology (Shanghai, China). The 293T cell line was also obtained from The Shanghai Institute of Cell Biology. The cells were maintained in RPMI-1640 medium (Sigma; Merck Millipore, Darmstadt, Germany) supplemented by 2% fetal bovine serum (FBS; Sigma; Merck Millipore) streptomycin (100 µg/ml; Sigma; Merck Millipore) and penicillin (200 U/ml; Sigma; Merck Millipore) in 5% CO2 at 20°C. The surgically resected osteosarcoma specimens were acquired from patients at the Puai Hospital of Tongji Medical College (Wuhan, China) between June 2013 and August 2015. All patients signed formal consent forms. The experiments involving human specimens were reviewed and approved by the Ethics Committee of The Puai Hospital of Tongji Medical College (no. 2015B0013).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNAs were isolated from the osteosarcoma cell lines (MG-63 and Saos-2) and human samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In total, 2 µg total RNA in a final volume of 10 µl, containing 10 mM dNTP mix (Sigma; Merck Millipore) was used to generate complementary DNA. A total of 1 mg total RNA template was annealed with 1 ml (500 ng) random primer in a sterile RNase-free micro-centrifuge tube and heated at 70°C for 5 min. Then, a mix containing 5X RT buffer (4 ml), 100 U/µl reverse transcriptase (2 ml) and 50 U/µl RNase inhibitor (1 ml) was added (Sigma; Merck Millipore). The standard TaqMan protocol was used, as previously described (14). GAPDH was used as the control. The reactions were performed using the ABI PRISM® 7000 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The thermocycling conditions were as follows: 55°C for 2 min, 95°C for 10 min followed by 35 cycles of 95°C for 20 sec and 60°C for 1 min. The expression of miR-643 was calculated using the 2−ΔΔCq method (15). The procedure was repeated at least three times. The primer sequences were as follows: miR-643, sense 5′-GTTAGCGTGATAGCG-3′ and antisense 5′-CTGAGTAGCTGACGCTT-3′; GAPDH, sense 5′-GATGCAATTGCGCTGCATTGT-3′ and antisense, 5′-ATGAAACGTTACGTTGAT-3′.

Transfection of plasmids

The miR-643 mimics, miR-643 inhibitor, scramble and negative controls were all synthesized and purchased from Sigma (Shanghai, China); these were cloned into the pcDNA3.1 vector. The negative control, scramble and pcDNA-miR-643 vectors were then transfected into MG-63 and Saos-2 cells in 12-well plates using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following incubation for 48 h at 20°C, the culture medium was replaced with fresh medium. The expression levels of miR-643 were determined using RT-qPCR analysis.

Western blot analysis

Proteins were extracted using 1X radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and concentrations were determined by Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocols. Proteins were then subjected to 10% SDS-PAGE (100 µg/lane) and transferred onto polyvinylidene difluoride membranes (Sigma; Merck Millipore). Membranes were blocked with 3% fat-free milk for 1.5 h at 20°C and then probed with antibodies against ZEB1 (1:500; cat. no. 3396) and GAPDH (1:500; cat. no. 8884; both Cell Signaling Technology, Inc., Boston, MA, USA) at 4°C overnight. A peroxidase-conjugated secondary antibody was also used (cat. no. 7076; 1:1,000; Cell Signaling Technology, Inc.) for incubation at 20°C for 1 h. Chemiluminescence was used to visualize the results (GE Healthcare, Fairfield, CT, USA). The blots were quantified with ImageJ software v1.48 (National Institutes of Health, Bethesda, MD, USA).

Proliferation assay

A Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to measure the proliferation rates of cells. Following treatment for 24 h, the MG-63 and Saos-2 cells were re-suspended and seeded into a 96-well plate (106 cells per well) for 5 days. Subsequently, 10 µl MTT solution was added into each culture well at a final concentration of 10 mg/ml. The crystalline formazan was resolved in 100 µl sodium dodecyl sulfate (10%) solution for 1 day and the optical density at 490 nm was monitored using the Spectramax M5 microplate monitor according to the manufacturer's protocol (Molecular Devices LLC, Sunnyvale, CA, USA).

Transwell invasion assay

Transwell chambers were used to evaluate the invasion of cells (8 µm size; Sigma; Merck Millipore). The upper chamber was coated with Matrigel (Invitrogen; Thermo Fisher Scientific, Inc.) overnight. Subsequently, 1×105 cells transfected with pcDNA-miR-643, miR-643 inhibitor, scramble or negative controls were seeded into the upper chambers. The lower chambers were covered in RPMI-1640 medium with 5% FBS as chemoattractants. After 24 h at 20°C, the cells remaining on the top of the chamber were removed using cotton swabs. The cells, which had migrated into the lower chamber were fixed with 5% PFA and stained with 0.05% crystal violet. A Leica fluorescent microscope (DM-IRB; Leica Microsystems GmbH, Wetzlar, Germany) was used to quantify the results.

Immunohistochemistry

The sections of tissue specimens (5 µm) were incubated with ZEB1 antibodies (1:500) overnight at 4°C. The sections of the specimens were then treated with secondary antibody (1:1,000) and further incubated with streptavidin-horseradish peroxidase complex for 1 h at 20°C. The nuclei were stained with hematoxylin and the number of tumor cells was evaluated in each section. Diaminobenzidine was used as the dye (Sigma; Merck Millipore) and immunohistochemical staining was monitored using an optical light microscope (Olympus Corporation, Tokyo, Japan).

miR-643 target prediction

Algorithms were applied to predict target genes using the TargetScan v6.2 (www.genes.mit.edu/targetscan), DIANA-microT v5.0 (www.diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index) and miRDB v5.0 (www.mirdb.org) databases as described previously (15–17). Briefly, putative targets of miR-643 were ranked by z-scores and those ranked at the top with overlapping targets were selected for further verification.

Luciferase reporter assay

The 3′untranslated region (UTR) of ZEB1 with predicted miR-643 binding sites were amplified using PCR and inserted into pRL-TK luciferase reporter vectors (Sigma; Merck Millipore) to obtain the ZEB1 3′UTR wild-type (WT). The ZEB1 3′UTR mutant (MUT) was obtained using primers containing the mutant sequences. The recombinant plasmids were transfected into 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The pRL-TK plasmid containing Renilla luciferase was used as the control. Luciferase activities were quantified using a dual luciferase assay (Promega; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Statistical analysis

The results are presented as the mean ± standard deviation. Statistical significance was determined using Student's t-test with SPSS, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Spearman's correlation was used to measure the association between the expression of miR-643 and ZEB1. A Kaplan-Meier survival curve was analyzed using the log-rank test. Fisher's exact test was used to evaluate the correlation between miR-643 and different clinicopathological characteristics. All experiments were repeated at least three times.

Results

miR-643 is downregulated in osteosarcoma tissues and cell lines, and is correlated with survival rates

To quantify the intrinsic levels of miR-643, RT-qPCR analysis was performed. It was found that the expression of miR-643 was significantly decreased in 104 osteosarcoma tissue samples, compared with the normal adjacent tissues (Fig. 1A). In the well-established osteosarcoma cell lines, levels of miR-643 were also downregulated, compared with that in normal bone cells (Fig. 1B). In addition, low levels of miR-643 were correlated with poor survival rates (Fig. 1C). No significant correlations were found between the expression pattern of miR-643 and age or gender (Table I). However, the level of miR-643 was significantly associated with differentiation, tumor-necrosis-metastasis stage and metastasis (Table I). These results suggested that the lower level of miR-643 in the osteosarcoma cell lines and tissue specimens contributed to the malignancy of osteosarcoma. As the Saos-2 and MG-63 cells showed the most marked downregulated expression of miR-643 among the cells, these two cell lines for used for further analysis.

Figure 1.

miR-643 is downregulated in osteosarcoma cells and tissue specimens. (A) Results of reverse transcription-quantitative polymerase chain reaction analysis of 104 OS samples and paired normal adjacent tissues. (B) Expression of miR-643 in hFOB normal bone cells and 11 tumor cell lines. **P<0.01. (C) Kaplan-Meier survival curves for patients with OS. The plot was based on the expression of miR-643. The log-rank test was used (P=0.002). OS, osteosarcoma; miR, microRNA.

Table I.

Correlation between miR-643 and clinicopathological characteristics.

		Expression of miR-643
Feature	n	Low, n (%)	High, n (%)	P-value
Age (years)				0.357
<60	60	28 (46.7)	32 (53.3)
≥60	44	23 (52.3)	21 (47.7)
Gender				0.427
Male	49	25 (51.0)	24 (49.0)
Female	55	26 (47.3)	29 (52.7)
Differentiation				0.001
Well/moderate	60	21 (35.0)	39 (65.0)
Poor	44	30 (68.2)	14 (31.8)
Metastasis				0.005
Absent	59	22 (37.3)	37 (62.7)
Present	45	29 (64.4)	16 (35.6)
TNM stage				0.002
0/I	42	13 (31.0)	29 (69.0)
II/III/IV	62	38 (61.3)	24 (38.7)

miR, microRNA; TNM, tumor-node-metastasis.

miR-643 inhibits osteosarcoma cell proliferation and invasion

The present study examined whether miR-643 can affect the malignant phenotypes of osteosarcoma cells. The Saos-2 and MG-63 cells were either left untreated or were transfected with miR-643 mimics or scramble controls. Transfection with the miR-643 mimics significantly upregulated the intrinsic levels of miR-643 in the MG-63 and Saos-2 cells (Fig. 2A and B). It was also found that miR-643 transfection led to a marked decrease in the proliferation of MG-63 cells, compared with either the empty or scramble control cells (Fig. 2C). Qualitatively similar results were observed in the Saos-2 cells (Fig. 2D). It was also noted that transfection with miR-643 inhibitor reversed this effect and promoted proliferation (Fig. 2C and D). Accordingly, miR-643 transfection inhibited the invasion of MG-63 cells (Fig. 2E) and Saos-2 cells (Fig. 2F) and treatment with miR-643 inhibitor enhanced cell invasion (Fig. 2E and F). These results suggested that miR-643 inhibited the malignancy of osteosarcoma cells through inhibiting proliferation and invasion.

Figure 2.

miR-643 transfection inhibits osteosarcoma cell proliferation and invasion. (A) MG-63 or (B) Saos-2 cells were left untreated or transfected with scramble control, miR-643 mimics or miR-643 inhibitor. The expression was quantified using reverse transcription-quantitative polymerase chain reaction analysis. A 5-day proliferation assay for the (C) MG-63 and (D) Saos-2 cells transfected with scramble control, miR-643 mimics or miR-643 inhibitor or untreated. Transwell invasion assays for (E) MG-63 and (F) Saos-2 cells. Representative images are shown on the left (magnification, ×200) and quantification of invasive cell numbers is shown on the right. **P<0.01 vs. control; ##P<0.01 vs. scramble. miR, microRNA; O.D., optical density.

miR-643 targets ZEB1 in osteosarcoma

To identify possible targets of miR-643 in osteosarcoma, the present study used bioinformatics strategies. The online databases DIANA-microT, miRDB and TargetScan were used for target prediction. Overlapping results suggested ZEB1 may be a direct target. The base pairing between hsa-miR-643 and ZEB1 was determined (Fig. 3A). A luciferase reporter assay was then performed, which confirmed that the luciferase intensities were significantly downregulated in WT ZEB1 (Fig. 3B). Introducing mutations in the base pairing regions of ZEB1 showed minimal difference, compared with the control group (Fig. 3B). In addition, the miR-643 mimics decreased the mRNA levels of ZEB1 (Fig. 3C and D). Consistently, the protein expression of ZEB1 was downregulated with miR-643 transfection (Fig. 3E and F). These results suggested that ZEB1 may be a direct target of miR-643.

Figure 3.

ZEB1 is the direct target of miR-643. (A) Base pairing between miR-643 and ZEB1. The MUT ZEB1 plasmid is shown below. (B) Relative luciferase activities of 293T cells transfected with either WT or MUT ZEB1 plasmids. The error bars are derived from triplicate experiments. Quantification of mRNA expression of ZEB1 in (C) MG-63 and (D) Saos-2 cells transfected with empty, scramble control or miR-643 mimics. Western blot analysis was used to measure protein expression levels of ZEB1 in (E) MG-63 and (F) Saos-2 cells transfected with empty, scramble control or miR-643 mimics. Quantification of results is shown below. **P<0.01 vs. control; ##P<0.01 vs. scramble. miR mimics denote miR-643 mimics. miR, microRNA; ZEB1, Zinc finger E-box-binding homeobox 1; WT, wild-type; MUT, mutant.

Overexpression of ZEB1 can counteract the effect of miR-643 transfection in osteosarcoma cells

To further validate the direct target of ZEB1 by miR-643, rescue experiments were performed. pcDNA3.1 was used to overexpress ZEB1 in the MG-63 and Saos-2 cells. The results showed that pcDNA3.1-ZEB1 transfection significantly upregulated intrinsic levels of ZEB1, compared with those in the empty control (Fig. 4A and B). Consistently, ZEB1 transfection increased the proliferation of MG-63 cells compared with the cells in the empty control group (Fig. 4C). Similar results were obtained for the Saos-2 cells (Fig. 4D). It was also noted that the overexpression of ZEB1 markedly enhanced the invasive capacities of the MG-63 and Saos-2 cells (Fig. 4E). These results further confirmed that miR-643 mediated its tumor suppressive effect by targeting ZEB1.

Figure 4.

Restoration of the expression of ZEB1 can counteract the effect of miR-643. Expression of ZEB1 in (A) MG-63 and (B) Saos-2 cells transfected with empty control, pcDNA-ZEB1 or pcDNA-ZEB1+miR-643 mimics. Quantification is shown on the right. **P<0.01 vs. control. Proliferation assays for (C) MG-63 and (D) Saos-2 cells transfected with empty control, miR-643 mimics or pcDNA-ZEB1+miR-643 mimics. (E) Invasion assays for MG-63 (above) and Saos-2 (below) cells (magnification, ×200). The OS cells were either transfected with empty control, pcDNA-ZEB1 plasmids or pcDNA-ZEB1 plasmids+miR-643 mimics. The results were quantified and shown on the right. Invasion was normalized to the empty control groups. miR, microRNA; ZEB1, Zinc finger E-box-binding homeobox 1; O.D., optical density.

Lower expression levels of miR-643 are correlated with poor prognosis

To further confirm the role of miR-643, pairwise analysis was performed on the expression of miR-643 and ZEB1. It was found that the expression of miR-643 was inversely correlated with that of ZEB1 in the tissue specimens (Fig. 5A). In addition, the overall survival rate for the patients expressing a low level of ZEB1 was 52.1%, but was only 25.2% in the group with a high expression of ZEB1 (Fig. 5B). In tissue specimens with relatively higher expression levels of miR-643 (miR-643+), it was found that the ZEB1 staining was significantly lower, compared with that in specimens with low expression levels of miR-643 (miR-643-; Fig. 5C). The samples exhibiting relatively lower expression levels of miR-643 showed characteristics of metastatic osteosarcoma, compared with the normal adjacent tissues (Fig. 5C). These characteristics were less marked in osteosarcoma samples with higher expression levels of miR-643 (Fig. 5C). These results further suggested that the downregulation of miR-643 was correlated with higher expression levels of ZEB1 and poor prognosis.

Figure 5.

miR-643 correlates with poor prognosis and expression of ZEB1. (A) Inverse correlation between levels of miR-643 and expression of ZEB1 was observed in the 104 tissues specimens (P<0.0001). (B) Kaplan-Meier survival curves for patients with osteosarcoma based on expression of ZEB1 (P<0.001). (C) Immunohistochemical staining (magnification, ×400) for ZEB1 in NAT and osteosarcoma tissues. miR, microRNA; ZEB1, Zinc finger E-box-binding homeobox; miR-643-, low expression of miR-643; miR-643+, high expression of miR-643; NAT, normal adjacent tissue.

Discussion

Deregulated microRNA expression has been documented in several types of tumor and, to date, various microRNAs are involved in the malignancy of cancer (18). The effect of microRNA is primarily ascribed to the dynamic regulation of oncogenes and/or tumor suppressors (19). MicroRNAs can have diverse roles, possibly in a tumor type-specific manner (20). Therefore, identifying key microRNAs in tumorigenesis is important for elucidating the molecular mechanisms of tumor development and potential diagnoses.

In the present study, it was shown that miR-643 was involved in the tumorigenesis of osteosarcoma. miR-643 was frequently downregulated in osteosarcoma tissue specimens, compared with normal tissues. Qualitatively similar results were observed in osteosarcoma cell lines. miR-643 inhibited the malignant phenotypes of osteosarcoma, including proliferation and invasion. Using online databases, ZEB1 was identified as a direct target of miR-643 in osteosarcoma. The overexpression of ZEB1 significantly restored the effect of miR-643 mimic transfection. Notably, a reverse correlation was found between the expression of miR-643 and level of ZEB1, further consolidating the direct targeting effect. These results support the hypothesis that miR-643 can exert tumor suppressor-like behavior by targeting ZEB1.

Several studies have focused on the regulatory functions of microRNAs in osteosarcoma. However, few reports have shown the effect of miR-643 in osteosarcoma. A previous study using high-density lipoprotein (HDL)-microRNA profiling showed that miR-643 is involved in HDL-mediated transportation (21). However, transportation has not been found to associate miR-643 with tumorigenesis, particularly in osteosarcoma. A study by Sun et al (22) aimed to establish the association between miR-643, miR-18b-5p, miR-150 and 3940-5p with the clinicopathologic features of non-small cell lung cancer (NSCLC), the results of which showed no significant difference in the expression of miR-643 between tumorous and normal tissues. To date, there is limited information available for miR-643, particularly in osteosarcoma. The present study is the first, to the best of our knowledge, to report on a tumor suppressive role of miR-643 in osteosarcoma.

It is well known that the embryonic program of epithelial-mesenchymal transition (EMT) is reactivated during tumor development (23). The reactivation of EMT is characterized by loss of the expression of E-cadherin, which is largely mediated by ZEB1 (23). The zinc finger homeobox family of repressor ZEB1 is encoded by the TCF8 gene (24). The aberrant expression of ZEB1 has been associated with prostate carcinoma with a high Gleason score and contributes to metastasis (25). In renal clear cell carcinoma, ZEB1 has been implicated, the expression of which is regulated by hypoxia-inducible factor 1 (26). ZEB1 has also been shown to be involved in the malignant phenotypes of breast cancer (27). Screening has also shown an inverse correlation between the gene expression levels of TCF8 and chromodomain-helicase-DNA-binding protein 1 in breast cancer cells (23). A significant inverse correlation between ZEB1 and E-cadherin is also evident in NSCLC cells, including lung adenocarcinoma, in vivo (28). Therefore, ZEB1 contributes to the development of various tumors with epithelial origins. Several reports have associated microRNAs with the expression of ZEB1. For example, Li et al (29) showed that miR-455 can suppress the development of NSCLC via targeting ZEB1. In addition, miR-200b inhibits osteosarcoma by suppressing ZEB1 (30). miR-409-3p has also been shown to target ZEB1 in breast cancer, with downregulated miR-409-3p correlated with poor outcomes (31). The results of the present study revealed miR-643 also targets ZEB1 via its 5′ end. Therefore, ZEB1 may be a common target for microRNAs, and the significant association between microRNAs and ZEB1 suggests putative therapeutic strategies targeting this interaction. Whether the function of miR-643 can be extended to other types of cancer requires further examination. Future studies may assist in elucidating the hidden mechanisms in the complex regulatory network of microRNAs.

In conclusion, the present study indicated that miR-643 is a potent tumor suppressor in osteosarcoma. miR-643 directly targeted ZEB1 to induce tumor suppression. Lower expression levels of miR-643 or higher expression levels of ZEB1 were predictors of poor prognosis. As ZEB1 is critically involved in the EMT process, which promotes tumor metastasis, manipulating the intricate interaction between miR-643 and ZEB1 may be an effective and potential rationale for diagnosis.